bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after (125)I-α-bungarotoxin binding at mouse neuromuscular junctions
Authors: Fertuck HC , Salpeter MM
Published: May 2004

The distribution and quantitation of (125)I-α-bungarotoxin (α- BTX) binding sites and thus acetylcholine receptor (AChR) were determined in mouse sternomastoid muscle by electron microscope autoradiography. We found that a valid criterion for receptor saturation at the neuromuscular junction was the complete elimination of neurally evoked tetantic muscle contractions, since, when such a criterion was used for the endpoint of toxin incubation, α-BTX was bound to approximately 90 percent of total available endplate sites. When, without implying localization, the presynaptic axonal membrane was used as a convenient reference structure, the concentration of α-BTX relative to this membrane was determined to be 46,000 +/- 27 percent sites/μm(2). By testing various hypothetical models the distribution of developed grains was found to be consistent with the hypothesis that the main acetylcholine-receptive surface coincides with the electron-dense, thickened portion of the junctional fold membrane situated at the juxtaneuronal region of the folds and dipping approximately 2,200 A be approximately 30,500 +/- 27 percent sites/μm(2) of this postsynaptic dense membrane. There was a sharp gradient in α-BTX binding extrajunctionally, with the concentration decreasing to approximately 4 percent of the subsynaptic value within 1μm from the edge of an axonal terminal (bouton) to <1 percent within 3μm and to <0.2 percent beyond 7 μm from that terminal. Below 4,000 A (i.e. half-way from the top of the junctional folds) the concentration of α-BTX was also about 3 percent of the peak subsynaptic value. The binding density at the bottom of the junctional folds is this comparable to extrajunctional sarcolemma at equal distance from a nerve terminal. The molecular organization at the neuromuscular junction relative to its function is discussed.
Structural complexes in the squid giant axon membrane sensitive to ionic concentrations and cardiac glycosides
Authors: Villegas GM , Villegas J
Published: May 2004

Giant nerve fibers of squid Sepioteuthis sepiodea were incubated for 10 min in artificial sea water (ASW) under control conditions, in the absence of various ions, and in the presence of cardiac glycosides. The nerve fibers were fixed in OsO(4) and embedded in Epon, and structural complexes along the axolemma were studied. These complexes consist of a portion of axolemma exhibiting a three-layered substructure, an undercoating of a dense material (approximately 0.1μm in length and approximately 70-170 A in thickness), and a narrowing to disappearance of the axon-Schwann cell interspace. In the controls, the incidence of complexes per 1,000μm of axon perimeter was about 137. This number decreased to 10-25 percent when magnesium was not present in the incubating media, whatever the calcium concentration (88, 44, or 0 mM). In the presence of magnesium, the number and structural features of the complexes were preserved, though the number decreased to 65 percent when high calcium was simultaneously present. The complexes were also modified and decreased to 26-32 percent by incubating the nerves in solutions having low concentrations of sodium and potassium. The adding of 10(-5) M ouabain or strophanthoside to normal ASW incubating solution decreased them to 20-40 percent. Due to their sensitivity to changes in external ionic concentrations and to the presence of cardiac glycosides, the complexes are proposed to represent the structural correlate of specialized sites for active ion transport, although other factors may be involved.
Control of chemotaxis in Physarum polycephalum.
Authors: Durham A C , Ridgway E B
Published: May 2004

Plasmodia migrate towards those situations which increase the frequency of their alternations in streaming, and away from those which decrease the frequency. Therefore peristalsis-like waves in Physarum move in the direction opposite from the net movement of the organism. The mechanism is fundamentally related to other known types of chemotaxis.
Incorporation of polypeptides into thylakoid membranes of Chlamydomonas reinhardtii. Cyclic variations.
Authors: Bourguignon L Y , Palade G E
Published: May 2004

A purified fraction of unstacked thylakoid membranes (TMF1u) has been obtained from homogenates of Chlamydomonas reinhardtii (wild type 137+) by using repeated centrifugates in sucrose density gradients and low salt treatment. The contaminants of the fraction are reduced to a few mitochondria (approximately 3% of the total mitochondrial population), a few osmiophilic granules, and fragments of chloroplast envelopes. By SDS-polyacrylamide gel electrophoresis the polypeptide components of TMF1u were resolved into at least 30 bands. To determine the relative rates of assembly of newly synthesized polypeptides into thylakoid membranes, synchronized algal cells were doubly labeled in vivo with L-[14C] and L-[3H]arginine--used for long- and short-term labeling, respectively. TMF1u's were isolated from the labeled cells at selected time points during the cycle and the distribution of radioactivity was assayed in the gel electrophoretograms of their solubilized polypeptides. Incorporation of newly synthesized polypeptides into the bands of the gels was found to occur continuously but differentially throughout the cycle. Maximal rates of incorporation for the majority of the polypeptides were detected shortly after cell division (6D-7D; equivalent to early G1 phase). The rates of radioactive labeling decreased gradually to a low level at the end of the dark period and then rose slightly at the beginning of the next light period. The findings suggest that, in addition to the light/dark control postulated in the past, assembly of newly synthesized proteins into thylakoid membranes is activated by signals at work in the early G1 phase.
Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. III. Ribosomal RNA cistrons of the nucleus and leucoplast.
Authors: Siu C , Chiang K , Swift H
Published: May 2004

The colorless alga Polytoma obtusum has been found to possess leucoplasts, and two kinds of ribosomes with sedimentation values of 73S and 79S. The ribosomal RNA (rRNA) of the 73S but not the 79S ribosomes was shown to hybridize with the leucoplast DNA (rho - 1.682 g/ml). Nuclear DNA of Polytoma (rho = 1.711) showed specific hybridization with rRNA from the 79S ribosomes. Saturation hybridization indicated that only one copy of the rRNA cistrons was present per leucoplast genome, with an average buoyant density of rho = 1.700. On the other hand, about 750 copies of the cytoplasmic rRNA cistrons were present per nuclear genome with a density of rho = 1.709. Heterologous hybridization studies with Chlamydomonas reinhardtii rRNAs showed an estimated 80% homology between the two cytoplasmic rRNAs, but only a 50% homology between chloroplast and leucoplast rRNAs of the two species. We conclude that the leucoplasts of Polytoma derive from chloroplasts of a Chlamydomonas-like ancestor, but that the leucoplast rRNA cistrons have diverged in evolution more extensively than the cistrons for cytoplasmic rRNA.
Effect of cytochalasin B on the adhesion of mouse peritoneal macrophages.
Authors: Helantjaris T G , Lombardi P S , Glasgow L A
Published: May 2004

The adhesion of normal mouse macrophages to glass surfaces was reduced by nontoxic levels (1-50 mug/ml) of cytochalasin B in combination with a centrifugal force (1,000-8,000 g). Macrophages nonspecifically activated by Corynebacterium acnes were also detached by this treatment, but less effectively. The effects of cytochalasin B treatment on these cells were shown to be reversible. After detachment, the cells reattached to glass, appeared morphologically normal, and behaved like untreated cells as judged by adhesion, acid phosphatase levels, and phagocytosis. The effect of cytochalasin B on several parameters of phagocytosis by normal macrophages was also examined. The results demonstrate that cytochalasin B can be used to detach macrophages from surfaces and suggest a functional relationship between phagocytosis and macrophage adhesion to surfaces. Furthermore, the effect of cytochalasin B on adhesion of phagocytic cells provides a probe for further investigation of the adhesion of cells to surfaces.
Asymmetry of spermiation and sperm surface charge patterns over the giant acrosome in the musk shrew Suncus murinus.
Authors: Cooper G W , Bedford J M
Published: May 2004

Spermatozoa of the shrew Suncus murinus, a mammal with abdominal testes, exhibit four unusual features: a giant acrosome; a dorsoventral asymmetry of their spermiation; a dorsoventral asymmetry of their head surface character; and also apparent surface maturity as they enter the epididymis. A Sertoli cell-periacrosomal cisternal complex envelops the giant acrosome during spermatid maturation. Spermiation is heraled by asymmetrical disorganization of the subplasmalemmal components of this complex and is completed by retraction of the Sertoli cell from the ventral and then the dorsal face of the spermatid head. This sequence or release is correlated with an asynchronous acquisition of negative surface charges on the spermatid head-demonstrable on glutaraldehyde-stabilized cells by the binding at pH 1.8 of positively charged colloidal particles of ferric oxide. Mature epididymal spermatozoa exhibit an asymmetry in the patterns of distribution of bound colloid over the dorsal vs. ventral surfaces of the sperm head, as well as regional differences between the tail midpiece and principal piece. Surface distributions of anionic residues and lectin (Con A)-binding sites characteristic of mature Suncus spermatozoa are demonstrable within the testis, unlike the situation in most nannals where distinct modifications of the sperm surface occur during epididymal passage.
Visualization of stimulated nerve endings by preferential calcium accumulation of mitochondria.
Authors: Parducz A , Joó F
Published: May 2004

Calcium was detected by X-ray microanalysis in the mitochondria of electrically stimulated nerve endings. The phenomenon described here offers a simple means for identifying the stimulated nerve endings in the electron microscope and appears to be a promising new method for following spontaneous and drug-stimulated translocation of calcium in relation to the regulation of neurotransmitter release.
Changes in the structure of neuromuscular junctions caused by variations in osmotic pressure.
Authors: Clark A W
Published: May 2004

Neuromuscular junctions of the frog, Rana pipiens, were examined for structural modifications produced by exposure to increased and reduced osmotic pressure (pi). Preparations exposed to increased pi for varying lengths of time were fixed with either OSO4-Veronal with and without calcium, glutaraldehyde-phosphate, or glutaraldehyde-formaldehyde-phosphate as primary fixatives. The greatest difference between the fixatives was seen in preparations exposed to increased pi for 5 min, corresponding to the time when miniature endplate potential frequency is highest. The 5-min OSO4 calcium-free preparations appeared comparatively normal, while those fixed with OSO4 and 2 mM CaCl2 or aldehyde-phosphate had wide infoldings of the presynaptic membrane and a reduced number of synaptic vesicles. Aldehyde-phosphate had the same effect on mouse diaphragm. Another series of frog preparations were conditioned to elevated pi and then returned to normal Ringer's for varying times before fixation in OSO4-phosphate. Preparations fixed 2 min after their return to normal Ringer's showed marked disruption of the presynaptic membrane as well as apparently rupturing vesicles. If fixed after 10 min, terminals were depleted of vesicles although the presynaptic membrane had returned to its normal position and appearance.
Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. II. Membrane marker enzymes and membrane ultrastructure.
Authors: Rosano T G , Lee S K , Jones D H
Published: May 2004

The activity of cytochrome oxidase (an inner mitochondrial membrane marker) in mouse mammary gland homogenates was found to increase five- to sixfold from late pregnancy to day 8 of lactation, while that of monoamine oxidase (an outer membrane marker) increased only about 25%. The specific activity of cytochrome oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased fivefold. This reflects the fact that both cytochrome oxidase and mitochondrial protein are increasing at a much greater rate than is monoamine oxidase activity. Mixing experiments preclude the possibility that the release or removal of an inhibitor or stimulator produces the changes in enzymatic activity. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. In each preparation the pattern was the same with little change in the ratio until late pregnancy; and then a three- to fourfold increase occurred and the values reached a maximum by day 8 of lactation. These experiments were interpreted as demonstrating that the observed enzymatic changes are reflective of alterations in the mitochondria of the mammary parenchymal cell population. Electron micrographs of mid-pregnant and mid-lactating mammary parenchymal cells in situ were prepared, and distinct changes in the mitochondrial morphology noted. The most significant and obvious change is the large increase in the number of inner membrane cristae and an increase in matrix density in the lactating gland cell. Therefore, both enzymatic and morphological studies support the concept of an expansion of the mitochondrial inner membrane during presecretory differentiation in the mouse mammary parenchymal cell.
Coated-vesicle shells, particle/chain material, and tubulin in brain synaptosomes. An electron microscope and biochemical study.
Authors: Kadota T , Kadota K , Gray E G
Published: May 2004

Coated vesicles (CVs), plain synaptic vesicles (PSVs), and nonvesicular flocculent material were isolated from synaptosomes and examined with goniometry and high-resolution electron microscopy after either negative staining or various biochemical procedures. The flocculent material (i.e. the presynaptic matrix material except CV shells) is largely composed of particulate or elongated (chainlike) structures; some of this material (here referred to as particle/chain material) is attached to PSVs. The results obtained were: (a) the proteinaceous properties of the CV coat (also referred to as CV shell) and the particle/chain material were demonstrated with chymotrypsin; (b) the CV shell, studied with various negative-staining techniques, differs from the particle/chain material since it has no 3-4-nm globular subunits and reacts differently to alkaline pH; (c) the particle/chain material consists of aggregates of 3-4-nm globular subunits, four of which yield 8-10-nm fine particles; and these particles can be further aggregated into chains 8-10 nm wide and up to 30-60 nm long showing a "hollow" core; (d) vinblastine sulfate induced ringlike or helical crystalloid precipitates closely resembling the vinblastine-induced microtubule crystals reported in the literature, but vinblastine had no effect on either the CV shell material or the particle/chain material.
Effects of nuclease and protease digestion on the ultrastructure of Paramecium basal bodies.
Authors: Dippell R V
Published: May 2004

The action of deoxyribonuclease, ribonuclease, perchloric acid, and pronase on the fine structure of basal bodies of sectioned Paramecium was observed as part of a more extensive autoradiographic electron microscope analysis directed toward the problem of basal body DNA. DNase was found to have no detectable effect on basal body fine structure. Pronase first solubilized the linkers and C tubules of the triplets, then attacked the protein portion of the axosome, a localized portion of the ciliary axoneme adjacent to the distal end of the basal body, the rim fiber, and newly described lumen spiral complex. Prolonged pronase treatment disrupted the remaining microtubular elements, basal body plates, and cartwheel. RNase removed material from the axosome and the lumen complex, a conspicuous structure occupying the central portion of the basal body and consisting of a twisted or looped 90-A diam fiber or, more probably, pair of fibers, in association with large, dense granules. The apparent removal of both RNA and protein from this basal body structure by either of the two corresponding enzymes suggests an unusual organization of the two components. Observations from this and other laboratories suggest that the basal body RNA is single stranded. Its function is unknown but alternatives are discussed.
The germinal vesicle nucleus of Xenopus laevis oocytes as a selective storage receptacle for proteins.
Authors: Merriam R W , Hill R J
Published: May 2004

The amorphous nucleoplasm of the germinal vesicle nucleus of Xenopus laevis oocytes has been selectively extracted under conditions which leave the nuclear formed elements morphologically intact. The nucleoplasm contains about 97% of the total nuclear proteins and on SDS-polyacrylamide gels some 68 polypeptides can be distinguished. On the basis of solubility differences, the nucleoplasmic proteins can be classified into two categories. The first consists of soluble or easily solubilized proteins which comprise about 34 polypeptides making up 87% of the nucleoplasm. A few of these proteins show electrophoretic mobilities similar to those of soluble proteins of the cytoplasm, but most are unique to the nucleus. The residual 13% of the nucleoplasmic proteins are tightly bound to a nucleoplasmic gel and can be extracted only by solubilizing the gel. The solubility characteristics of the proteinaceous gel suggest a complex held together by salt, nonpolar, hydrogen, and possibly disulfide bonding. Some 34 polypeptides can be distinguished in this gel fraction, including prominent and highly enriched polypeptides of about 115,000 and 46,000 daltons. The relatively soluble fraction of the nucleoplasm does not contain informofers and contains little or no nucleic acid. Evidence is presented that if histones are present in the germinal vesicle, they can comprise no more than about 8% of the total protein. The possibility is discussed that the unique polypeptides of the nucleoplasm may be sequestered there by selective adsorption to or in the nuclear gel.
Hormonal regulation of gap junction differentiation.
Authors: Decker R S
Published: May 2004

Thin-section, tracer, and freeze-cleave experiments on hypophysectomized Rana pipiens larvae reveal that gap junctions form between differentiating ependymoglial cells in response to thyroid hormone. These junctions assemble in large particle-free areas of the plasma membrane known as formation plaques. Between 20 and 40 h after hormone application, formation plaque area increases approximately 26-fold while gap junction area rises about 20-fold. The differentiation of these junctions requires the synthesis of new protein and probably RNA as well. On the basis of inhibitor experiments, it can be reported that formation plaques develop at about 16-20 h after hormone treatment and stages in the construction of gap junctions appear 4-8 h later. These studies suggest that gap junction subunits are synthesized and inserted into formation plaque membrane during the differentiation of the anuran ependymoglial cells.
The Fine Anatomy of the Optic Nerve of Anurans—An Electron Microscope Study
Authors: Maturana Humberto R.
Published: May 2004

In the optic nerve of Anurans numerous myelinated and unmyelinated axons appear under the electron microscope as compact bundles that are closely bounded by one or several glial cells. In these bundles the unmyelinated fibers (0.15 to 0.6 µ in diameter) are many times more numerous than the myelinated fibers, and are separated from each other, from the bounding glial cells, or from adjacent myelin sheaths, by an extracellular gap that is 90 to 250 A wide. This intercellular space is continuous with the extracellular space in the periphery of the nerve through the numerous mesaxons and cell boundaries which reach the surface. Numerous desmosomes reinforce the attachments of adjacent glial membranes.

The myelinated axons do not follow any preferential course and, like the unmyelinated ones, have a sinuous path, continuously shifting their relative position and passing from one bundle to another. At the nodes of Ranvier they behave entirely like unmyelinated axons in their relations to the surrounding cells. At the internodes they lie between the unmyelinated axons without showing an obvious myelogenic connection with the surrounding glial cells. In the absence of connective tissue separating individual myelinated fibers and with each glial cell simultaneously related to many axons, this myelogenic connection is highly distorted by other passing fibers and is very difficult to demonstrate. However, the mode of ending of the myelin layers at the nodes of Ranvier and the spiral disposition of the myelin layers indicate that myelination of these fibers occurs by a process similar to that of peripheral nerves.

There are no incisures of Schmidt-Lantermann in the optic myelinated fibers.
Electron Microscopy of the Centrifuged Sea Urchin Egg, with a Note on the Structure of the Ground Cytoplasm
Authors: Gross Paul R. , Philpott Delbert E. , Nass Sylvan
Published: May 2004

Centrifuged, unfertilized eggs of the sea urchin, Arbacia punctulata, have been studied with the electron microscope. Subcellular particles were stratified by centrifuging living cells, known to be normally fertilizable, for five minutes at 3,000 g. The layered subcellular particles, including cortical granules, 16 mµ RNP particles, pigment, yolk, mitochondria, and oil droplets, possess characteristic ultrastructural features by which they may be identified in situ. The clear zone contains 16 mµ particles, most of them freely dispersed, scattered mitochondria, and a few composite structures made up of annulate lamellae in parallel layers or in association with dense, spherical aggregates of the RNP particles. Free 16 mµ particles are found, in addition, throughout the cell, in the interstices between the stratified larger particles. They show a tendency to form ramifying aggregates resulting from certain types of injury to the cell. A few vesicular structures, found mainly in the clear zone, have attached RNP particles, and appear to be related to the ER of tissue cells. Other vesicles, bounded by smooth membranes, are found throughout the cell. These are extremely variable in size, number, and distribution; their total number appears to depend upon conditions of fixation. It is suggested that limited formation of such structures is a normal property of the ground cytoplasm in this cell, but that fixed cells with very large numbers of smooth surfaced vesicles have produced the latter as a response to chemical injury. A model of the ground cytoplasm is proposed whose aim is to reconcile the rheological behavior of the living cell with the ultrastructural features observed.
The Relative Concentration of Solids in the Nucleolus, Nucleus, and Cytoplasm of the Developing Nerve Cell of the Chick
Authors: Merriam R. W. , Koch William E.
Published: May 2004

Growing and differentiating nerve cells of the fifth cranial ganglion of the chick embryo were studied by several means. During the period of 70 hours to 11 days of incubation (Hamburger-Hamilton stages 19 to 37) average cell mass increased more than 4.5 times while cells changed from relatively undifferentiated neuroblasts to morphologically characteristic nerve cells with long processes. By making simplifying assumptions about thickness of nucleus and nucleolus, relative to cytoplasmic thickness, it was possible to calculate solute concentration of nucleus and nucleolus relative to that of the cytoplasm from measurements of optical retardations through living cells. Differences in relative solute concentration were observed in nucleolus, cytoplasm, and nucleoplasm in the approximate ratio 1.2:1.0:0.8, respectively. The ratio remained essentially constant during the growth period examined despite the fact that the cell components grow at markedly different rates. This suggests that solid concentrations are physical characteristics of nucleus, nucleolus, and cytoplasm which are maintained even during rapid growth and differentiation.

By cytochemical means it was demonstrated that mass increase in the nucleus is not associated with increase in deoxyribonucleic acid. Both ribonucleic acid and protein are in greater concentration in nucleolus and cytoplasm than in the nucleoplasm. Electron microscopy shows interruptions in the nuclear envelope as well as an approximately even distribution of electron density in nucleus and cytoplasm. It is pointed out that consistent differences in solid concentration can exist on either side of the nuclear envelope even though it contains "pores." Implications of these data are discussed.
The Rejoining Time of Chromatid Breaks Induced by Gamma Radiation in Vicia faba Root Tips at 3 °C
Authors: Savage J. R. K. , Neary G. J. , Evans H. J.
Published: May 2004

The observation was made previously that the reduction in radiosensitivity in Vicia faba (as measured by postirradiation root growth) by prolonging the exposure time from about 10 minutes to 24 hours is much less marked at 3°C. than at 19°C. If chromosome damage is mainly responsible for the reduced root growth, this observation might be explained by a smaller drop in the "two-hit" aberration component, resulting from an increased time for which breaks are available for rejoining at 3°C.

This hypothesis was tested by comparing chromatid aberration frequencies in root meristem cells produced by 105 rads of 60Co γ rays, given at dose rates of 19.4 and 0.073 rads per minute. Beans were maintained in aerated water at 2°C. prior to and during irradiation, and at this temperature the rate of development of cells was such that the two different exposure times both occupied a period during which the cell sensitivity was approximately constant. Immediately subsequent to irradiation, the roots were returned to 19°C. and examined cytologically.

All chromatid aberrations were less frequent after low dose rate treatment, but only the chromatid interchange reduction was significant.

The average time for which breaks are available for reunion, calculated from Lea's G function, was found to be 12 hours (95 per cent C.L. 6 to 24 hours).
The Morphology and Behavior of Living Exoerythrocytic Stages of Plasmodium gallinaceum and P. fallax and Their Host Cells
Authors: Huff Clay G. , Pipkin Alan C. , Weathersby A. B. , Jensen Dinniemaud V.
Published: May 2004

The morphology and behavior of living exoerythrocytic stages of Plasmodium gallinaceum and P. fallax were studied by the use of tissue cultures, phase contrast microscopy, and time-lapse cinephotomicrography. The morphology of exoerythrocytic stages of these two species was essentially that previously observed in fixed, stained material, with the following exceptions: (1) the presence of a filament on one end of the merozoite, (2) the absence of clefts in the cytoplasm of the large schizonts, and (3) the absence of a vacuole-like space around the parasite.

The following behavior was observed either directly or in time-lapse sequences: (1) emergence of merozoites from mature schizonts, (2) progressive motility of free merozoites, (3) entry of merozoites, both actively and passively, into host cells, (4) nuclear division in the parasite, (5) the various stages of schizogony, including final production of merozoites, (6) massive infection of host cells, and (7) phagocytosis of merozoites and attempted phagocytosis of mature schizonts by macrophages.

Exoerythrocytic stages of P. fallax differed from those of P. gallinaceum in that the merozoites of the former were (1) somewhat more curved in shape and (2) present in fewer numbers in mature schizonts.

The use of tissue culture, phase contrast microscopy, and time-lapse cinephotomicrography promises to solve many of the remaining problems concerning exoerythrocytic stages of malarial parasites and their interrelationships with host cells.
Isolation of Cytoplasmic Pituitary Granules with Gonadotropic Activity
Authors: Hartley Marshall W. , McShan W. H. , Ris Hans
Published: May 2004

A fraction isolated from the anterior pituitary glands of rats castrate for 8 weeks contained essentially a single cytoplasmic constituent with which the major portion of the gonadotropic hormone activity was associated. The glands were homogenized in an 0.25 M sucrose + 7.3 per cent polyvinylpyrrolidone (PVP) solution and fractionated by differential centrifugation to give a heterogeneous small granule fraction which contained almost all the gonadotropic hormone activity. The active supernatant containing this small granule fraction was separated into layers by isopycnic gradient centrifugation on a continuous 6 to 45 per cent sucrose + 17.5 per cent "diodrast" + 5 x 10-4 M "versene" gradient at 100,000 g for 2 hours. Three layers were obtained and the pellet from the active bottom layer was sectioned, examined with the electron microscope, and found to contain 200 mµ granules, mitochondria, ergastoplasm, and other cellular debris. This layer was fractionated further by isopycnic and differential centrifugation to obtain a pellet which contained the major portion of the gonadotropic hormone activity. Because of the heterogeneity of this fraction, due to the contamination of the 200 mµ granules with mitochondria and other cellular debris, the active layer and the resuspended active pellet, obtained by centrifuging this layer first at 17,000 g then diluting the supernatant and centrifuging at 30,000 g for 1 hour, were filtered through Millipore HA paper with a pore size of 0.45 µ. The cytoplasmic material containing the gonadotropic hormone activity passed through the filter paper and this activity was recovered in the pellets obtained by centrifuging at 100,000 g for 1 hour. These active pellets consisted almost entirely of 200 mµ granules with a minimum amount of contamination, and they contained the major portion of the gonadotropic hormone activity with practically none remaining in the supernatant fraction. These results are discussed in view of their importance to the cytology of the pituitary gland.
The Intracellular Localization of Hormonal Activity in Transplantable Thyrotropin-Secreting Pituitary Tumors in Mice
Authors: Kamat Virendra B. , Wallach Donald F. Hoelzl , Crigler John F. , Ladman Aaron J.
Published: May 2004

Mouse pituitary tumors secreting almost exclusively thyroid stimulating hormone have been characterized electron microscopically.

Tumors of known thyrotropin content were separated into nuclear, mitochondrial, microsomal, and soluble fractions by differential centrifugation. The hormonal activity of these fractions was correlated with that of the total homogenates and with their nitrogen and phosphorus content. Essentially all the thyrotropin of the homogenate was recovered in a particulate fraction sedimenting between 20,000 and 40,000 g. This fraction contained the RNA granules and membranous components typical of microsomal pellets, but also showed the presence of small dense bodies surrounded by smooth membranes. These bodies were also visible within the endoplasmic reticulum of intact cells, and it is postulated that these bodies may represent the sites of intracellular elaboration and/or storage of TSH.

Thyrotropin is tightly associated with microsomal particles but can be brought into solution by treatment with alkaline media, deoxycholate, and certain organic solvents.
Absorption Microscopy of Enzymatically Treated Cell-Free Chloroplasts
Authors: Greenblatt C. L. , Olson R. A. , Engel E. K.
Published: May 2004

Monochromatic light microscopy at 435 mµ shows in Euglena gracilis, the distribution of chlorophyll and the general orientation and geometry of chloroplasts in vivo. In addition it discloses, in swelling chloroplasts, a lamellar pigmented structure. Changes in this structure are observed in extruded swollen chloroplasts treated with lipolytic or proteolytic enzymes. Lipolytic enzymes produce an increase in the number of visible lamellae while proteolytic enzymes disrupt the lamellar array.

Correlation of chloroplast swelling behavior and the effects of enzymatic degradation with current electron microscope observations support the following: (1) the pigment lamellae observed in vivo consist of component laminae; (2) the lamellae are separated by sites of swelling; and (3) the integrity of the lamellar structure is primarily dependent upon the intact state of the protein.
Permeability of Rat Liver Microsomes to Sucrose and Carboxypoly-glucose in vitro
Authors: Share Leonard , Hansrote Ronald W.
Published: May 2004

A study was made of the permeability of the microsomes to C14-sucrose and to C14-carboxypolyglucose, a branch-chained glucose polymer with a molecular weight of approximately 50,000. It was concluded that the microsomal membranes are permeable to sucrose on the basis of the following evidence: the volume of distribution of C14-sucrose was 84 per cent of the total microsomal pellet water; the sucrose unavailable volume, the per cent dry weights of the microsomal pellets, and the optical density of microsomal suspensions were independent of the concentration of sucrose in the suspending medium. It is suggested that the microsomal water which is unavailable to sucrose may be bound to protein and/or ribonucleic acid of the microsomes. The volume of distribution of C14-carboxypolyglucose was 44 per cent of the total pellet water, and it is considered that the microsomal membranes may be impermeable to this compound. Pretreatment with ribonuclease resulted in small increases in the volumes of distribution of both C14-sucrose and C14-carboxypolyglucose.
Studies on the Fine Structure of Microorganisms
Authors: Hashimoto Tadayo , Gerhardt Philipp , Conti S. F. , Naylor H. B.
Published: May 2004

The fine structure of cells of Saccharomyces cerevisiae engaged in the formation of ascospores was studied in electron micrographs of ultrathin sections. Although the mode of the first reduction division could not be clearly determined, the second nuclear division appeared to proceed in a manner similar to that observed previously during vegetative division. That is, division by constriction of the existing nucleus occurs without dissolution of the nuclear membrane and without involvement of discrete chromosomes. Variously shaped areas of low electron density were discerned within the nucleoplasm; these had not been previously seen in the vegetative nucleus. The significance of this nuclear differentiation and its possible similarity to nuclear structures reported in bacteria and an imperfect fungus are discussed.

The cytoplasmic membrane appears first in the developing ascospore. The formation of an outer coat and an inner coat then follows. The cytoplasmic vacuole was observed not to be incorporated into the spore. An unusual intracytoplasmic membrane was observed in the spore and appeared to be at least temporarily continuous with the nuclear membrane.
Electron Microscopic Observations on Mitochondria of Rat Testes Fixed in Potassium Permanganate
Authors: Zebrun William , Mollenhauer Hilton H.
Published: May 2004

A morphological investigation of mitochondria within the seminal epithelial cells of rat testes fixed in potassium permanganate reveals differences in electron opacity between the internal mitochondrial membranes of spermatogenic cells and those of Sertoli cells. Some interpretations of the apparent density differences are briefly discussed. It is concluded that the different effects of permanganate fixation upon the mitochondria of these cell types may reflect a significant structural difference between them.
Egg Membrane Lytic Activity of Sperm Extract and its Significance in Relation to Sperm Entry in Hydroides hexagonus (Annelida)
Authors: Colwin Arthur L. , Colwin Laura Hunter
Published: May 2004

Previous electron microscope studies indicated that the individual spermatozoön of Hydroides hexagonus forms a hole in the vitelline membrane by means of lysis. Other observations established that the hole is real, being visible in living material during sperm entry.

During the present investigation sea water extracts from frozen-thawed sperm were tested for lytic effect on the membrane. In normal living eggs the membrane appears as a single thick envelope, but in electron micrographs of sections it is seen to consist of a narrow outer border layer, a wide principal or middle layer, and a narrow inner border layer. After immersion in sperm extract the outer border layer elevates but does not dissolve, the middle layer liquefies and disappears, and the inner border layer seems not to change. This is interpreted as lysis of the middle layer. The extract exerted the same effect on fertilized and unfertilized eggs.

In electron micrographs the sections treated with extract greatly resemble that part of the membrane which has been penetrated by the individual spermatozoön. It is concluded that the individual spermatozoön, too, exerts a lytic effect. Together, the present and two earlier studies are considered clearly to demonstrate that in Hydroides the individual spermatozoön does indeed make an entry hole in the egg membrane by applying lytic material to that part of the membrane in its own vicinity.
Electron Microscope Studies of Nuclear Extrusions in Pancreatic Acinar Cells of the Rat
Authors: Clark Wallace H.
Published: May 2004

This paper describes "blebs" protruding from the surface of the nucleus into the cytoplasm. The "blebs" are separated from the cytoplasm by 2 membranes which are continuous with the outer and inner nuclear membranes. The "blebs" contain 3 structurally distinct substances. Two of these substances (ß and γ substances) are similar to extranucleolar karyoplasm and nucleolar material. The other substance (α substance) is present in every "bleb," but it cannot be readily compared to a recognizable nuclear structure. Cytoplasmic vesicles are described that are apparently different from the Golgi vesicles or the vesicular component of the ergastoplasm. It is suggested that these vesicles may be of nuclear "bleb" origin.

A dark karyoplasmic zone extending from the region of the nucleolus into the nuclear "bleb" is shown. This zone may be similar in some respects to the preformed pathway ("Leitbahn") described by Altmann (3) and Hertl (28) and could reflect movement of nuclear material from the nucleolar region into the cytoplasm.

The "blebs" are thought to be homologous to structures described by many light microscopists, but they are considerably larger than the nuclear "blebs" described previously by electron microscopists.
An Effective Method of Preparing Sections of Bacillus polymyxa Sporangia and Spores for Electron Microscopy
Authors: Holbert Pauline E.
Published: May 2004

Bacillus polymyxa sporangia and spores were prepared for examination in the electron microscope by methods whose critical features were apparently: judicious use of vacuum, to encourage complete penetration of the embedding medium; the use of epoxy resins as embedding media; and cutting of the thin sections with a diamond knife. Electron micrographs of material prepared in this manner exhibit undeformed sporangial sections. Some of the structures revealed have been shown before, though perhaps less distinctly; other structures are revealed here for the first time. While this single study does not pretend to elucidate all the complexities of sporulation in bacteria, these and similar images should make this possible, and some mention of the preparatory techniques that lead to them seems advisable at this time.
Studies on the Function of Intracellular Ribonucleases
Authors: Roth Jay S.
Published: May 2004

To determine the possible significance of in vivo or in vitro enzyme action in ribonucleoprotein systems, rat liver microsomes and ribonucleoprotein particles (RNP) prepared from them by deoxycholate treatment were incubated for 1 hour at 37°C. with crystalline pancreatic ribonuclease (RNase) or various RNase-free crystalline proteolytic enzymes. The extent of the degradation of the RNA of the microsomes and RNP was determined and the protein degradation estimated in both cases. With either microsomes or RNP, RNase (0.5 to 1.0 mg. per ml.) degraded from 75 to 95 per cent of the RNA, with little protein breakdown being apparent when microsomes were used but with significant protein degradation in the RNP. When microsomes were treated with proteolytic enzymes approximately 40 to 50 per cent of the original microsomal protein became nonsedimentable while at the same time 60 to 80 per cent of the RNA was also found to be non-sedimentable. Of the non-sedimentable RNA, approximately one-third was in the form of acid-precipitable RNA while the remainder was in the form of acid-soluble nucleotides. When RNP was treated with proteolytic enzymes, about 95 per cent of the RNA could no longer be sedimented. About half of this appeared as acid-precipitable RNA and half as acid-soluble nucleotides. Both microsomes and RNP contained significant RNase activity with RNP exhibiting about 10 times the specific activity of microsomes. Some of the characteristics of this RNase activity were determined and the results with proteolytic enzymes interpreted in light of this activity.
Asynchronous Duplication of Chromosomes in Cultured Cells of Chinese Hamster
Authors: Taylor J. Herbert
Published: May 2004

Chromosome duplication (DNA synthesis) was studied in cultured cells of Chinese hamsters by means of autoradiography following thymidine-H3 incorporation. The technique used was to expose an asynchronously dividing population of rapidly growing cells for a 10 minute interval to a medium with thymidine-H3. Cells were then transferred to a medium with excess unlabeled thymidine. The population was sampled at intervals thereafter and studies made of the frequency of labeled interphases and division figures, and the patterns of labeling of specific chromosomes.

The average generation time during these experiments was about 14 hours. DNA synthesis occurred during an interval of about 6 hours and stopped 2 to 3 hours before metaphase. After metaphase the chromosomes usually begin duplication again within 5 to 6 hours.

Grain counting, to estimate the amount of tritium incorporated after a short contact with thymidine-H3 and at intervals after transfer to a medium with excess unlabeled thymidine, indicated that the intracellular pool of labeled precursors was diluted within less than a minute so that further labeling would not be detected. The chromosomes labeled during the contact period retained their precise pattern of labeling through another duplication cycle and no turnover of DNA or loss of tritium was detectable.

Five or 6 chromosomes of the complement have segments typically late in duplication. Two of these are the X and Y chromosomes. The long arm of the X chromosome and the whole Y chromosome are duplicated in the last half of the interval of DNA synthesis. The short arm of the X chromosome in a male strain is duplicated in the first half of the interval. In another strain (female), one X chromosome had the same timing, but the other one was all duplicated in the last half of the period of DNA synthesis. The DNA in the short arms of 2 medium sized chromosomes, as well as most of the DNA in 1 or 2 of the smallest chromosomes of the complement was replicated late.

The study has led to the hypothesis that various chromosomes or parts of chromosomes have a genetically controlled sequence in duplication which may have some functional significance.
The Fine Structure of Streptomyces coelicolor
Authors: Glauert Audrey M. , Hopwood David A.
Published: May 2004

Colonies and spore suspensions of Streptomyces coelicolor were fixed by the method of Kellenberger, Ryter, and Séchaud (1958) and embedded in methacrylate or araldite. Thin sections were cut with an A. F. Huxley microtome and examined in a Siemens' Elmiskop I.

At all stages of development the hyphae of Streptomyces coelicolor have an extensive membranous component in the cytoplasm. The membranes are continuous with the plasma membrane and have a variety of configurations at different places in the hyphae. Tubular structures, vesicles, and parallel stacks of membranes are seen. In some areas concentric layers of membranes form whorled structures which are particularly frequent in the region of developing cross-walls and within maturing spores. In the spores membranous structures often lie embedded in the nuclear material. In disintegrating hyphae the intracytoplasmic membranes round off into small vesicles and remain when the rest of the cytoplasmic structure has gone.

In the absence of typical mitochondria and other cytoplasmic membranous structures it is possible that the membranous component of the cytoplasm of Streptomyces coelicolor may perform the functions of the endoplasmic reticulum and/or the mitochondria of higher cells.
The Relationship of Internal Conductance and Membrane Capacity to Mitochondrial Volume
Authors: Pauly Helmut , Packer Lester
Published: May 2004

A study was made of the effect of mitochondrial size on the electrical properties of the membrane and the internal conductivity of mitochondria. The dielectric constant and electrical conductivity of suspensions of guinea pig heart mitochondria were examined in the frequency range 5 x 105 to 2.5 x 108 C.P.S.

Membrane capacity was calculated to be 1.1 to 1.3 µf./cm.2 and was virtually the same in mitochondria whose surface area was made to vary by a factor of 4 by osmotic means. This finding suggested that some mechanism must exist for the transfer of mitochondrial material into membrane structure during fluctuations in mitochondrial size.

The electrical capacity of the membrane was unaffected by a 33-fold change in potassium chloride concentration.

The internal conductance of swollen mitochondria was 2 to 3 times lower than that of the external medium. In shrunken mitochondria the internal conductance was virtually independent of the conductivity of the external medium.

These results were discussed in relation to current concepts of mitochondrial structure.
Is there "Metabolic" DNA in the Mouse Seminal Vesicle?
Authors: Gall Joseph G. , Johnson William W.
Published: May 2004

This study was designed to answer the question: Is H3-thymidine uptake by nuclei of the mouse seminal vesicle evidence for DNA synthesis and mitosis, or does it signify some "metabolic" function of DNA unrelated to chromosome duplication?

Mice were given an intraperitoneal injection of H3-thymidine. Six hours later Feulgen squashes of the seminal vesicle epithelium were made and covered with autoradiographic stripping film. The silver grains above labeled nuclei were counted, and the Feulgen dye contents of these same nuclei were determined photometrically after removal of the grains from the emulsion. Unlabeled nuclei were also measured.

The dye contents of non-radioactive nuclei form a unimodal distribution, indicating that polyploidy is absent from this tissue. The radioactive nuclei fall into two groups. In the first, the average dye content is the same as that of the cold nuclei (2C). In the second, the values range from 2C to 4C.

In the 2C to 4C group the grain count is proportional to the dye content, showing that incorporation is correlated with synthesis.

The radioactive 2C nuclei arose by mitosis during the course of the experiment. This is shown by the following facts: (1) They frequently occur in pairs. (2) They average smaller than unlabeled 2C nuclei. (3) Their average grain count is approximately half that of the 4C nuclei. (4) Labeled division figures are found. (5) A mitotic rate estimated from the number of labeled 2C nuclei accords reasonably well with one based on the number of observed mitoses.

Since the incorporation of thymidine accompanies DNA synthesis and precedes mitosis, there is no reason to postulate a special "metabolic" DNA in this tissue.