bluuebunny/crossref_metadata_2025 huggingface dataset:
INHIBITION OF DNA SYNTHESIS IN CHICK EMBRYO CULTURES BY DEPRIVATION OF EITHER SERUM OR ZINC
Authors: Rubin H. , Koide T.
Published: May 2004

The rate of DNA synthesis in cultures of chick embryo cells is proportional to the concentration of serum added. The concentration of serum required to stimulate DNA synthesis increases with cell population density and with the duration of culture after trypsinization. The increase of the serum requirement with population density is not caused by the depletion of serum constituents. The requirement of cells for external zinc in DNA synthesis also increases with population density and duration of culture. The kinetics of inhibition of DNA synthesis by deprivation of serum or zinc are similar. Serum deprivation, however, inhibits 2-deoxyglucose uptake and cell movement, but zinc deprivation does not. The deprivation of either serum or zinc inhibits RNA synthesis about twofold. Very low concentrations of actinomycin D prevent the resumption of RNA and DNA synthesis upon restoration of serum or zinc to deprived cultures.
CELL CONTACTS IN THE MOUSE MAMMARY GLAND
Authors: Pitelka Dorothy R. , Hamamoto Susan T. , Duafala Joan G. , Nemanic Michael K.
Published: May 2004

The nature and distribution of cell contacts have been examined in thin sections and freeze-fracture replicas of mammary gland samples from female C3H/Crgl mice at stages from birth through pregnancy, lactation, and postweaning involution. Epithelial cells of major mammary ducts at all stages examined are linked at their luminal borders by junctional complexes consisting of tight junctions, variable intermediate junctions, occasional small gap junctions, and one or more series of desmosomes. Scattered desmosomes and gap junctions link ductal epithelial and myoepithelial cells in all combinations; hemidesmosomes attach myoepithelial cells to the basal lamina. Freeze-fracture replicas confirm the erratic distribution of gap junctions and reveal a loose, irregular network of ridges comprising the continuous tight-junctional belts. Alveoli develop early in gestation and initially resemble ducts. Later, as alveoli and small ducts become actively secretory, they lose all desmosomes and most intermediate junctions, whereas tight and gap junctions persist, The tight-junctional network becomes compact and orderly, its undulating ridges oriented predominantly parallel to the luminal surface. It is suggested that these changes in junctional morphology, occurring in secretory cells around parturition, may be related to the greatly enhanced rate of movement of milk precursors and products through the lactating epithelium, or to the profound and recurrent changes in shape of secretory cells that occur in relation to myoepithelial cell contraction, or to both.
THE DIFFERENTIAL EFFECT OF ACTINOMYCIN D IN NORMAL AND VIRUS-TRANSFORMED CELLS
Authors: Williams J. G. , MacPherson I. A.
Published: May 2004

Actinomycin D (AMD) at concentrations up to 0,25 µg/ml shows a differential effect on cell RNA synthesis and on the replication of an influenza virus in normal and virally transformed cells, both functions being more resistant to AMD in the transformed cell. A possible explanation for these differences in AMD sensitivity is provided by the observation that isotopically labeled AMD is maintained at a lower concentration in transformed BHK 21/13 (BHK) cells. There is evidence that the decreased sensitivity of the transformed cells to AMD is a result of maintenance of a lower internal concentration of the drug, since a correlation exists for a number of polyoma virus-transformed clones between sensitivity to and uptake of AMD.
LOW RESISTANCE JUNCTIONS IN CRAYFISH
Authors: Peracchia Camillo
Published: May 2004

The ultrastructure of low resistance junctions between segments of lateral giant fibers in crayfish is studied in sections from specimens fixed either by conventional methods or by glutaraldehyde-H2O2 or by glutaraldehyde-lanthanum. Cross sections through junctions fixed by conventional glutaraldehyde display the usual trilaminar profile of two parallel membranes separated by a narrow gap. Most of the junctional regions appear covered by 500–800 Å vesicles which lie on both sides of the junction in rows adjacent to the membranes. Gross sections through junctions fixed by glutaraldehyde-H2O2 display, in regions containing vesicles, membranes with a beaded profile. The beads correspond to globules ∼125 Å in width and ∼170 Å in height arranged in a hexagonal pattern with a unit cell of ∼200 Å. The globules of one membrane match precisely with those of the adjacent membrane, and opposite globules seem to come in contact with each other at the center of the junction. The membrane of the vesicles also contains globules. Occasionally the globules of the vesicles seem to join with those of the junctional membranes, apparently forming intracellular junctions. Injunctions negatively stained by lanthanum the globules are seen organized into two arrangements. Areas containing globules in a hexagonal array with a unit cell of ∼200 Å (swollen pattern) are seen adjacent to areas in which the globules are more closely and disorderly packed (close packing), the minimum center-to-center distance between adjacent globules being ∼125 Å. At higher magnification each globule appears composed of six subunits arranged in a circle around a central region occupied by lanthanum (possibly a pit).
LOW RESISTANCE JUNCTIONS IN CRAYFISH
Authors: Peracchia Camillo
Published: May 2004

Low resistance junctions between axons of crayfish ganglia are studied by freeze-fracture and negative staining. In freeze-fracture, fracture planes that go through a junctional membrane expose two faces, both internal, called face A and face B. Face A belongs to the internal membrane leaflet and faces the gap. Face B belongs to the external membrane leaflet and faces the axoplasm. Face A displays pits, 60–100 Å in diameter, arranged in a hexagonal array with a unit cell of ∼200 Å. An ∼25 Å bump is frequently seen at the center of each pit. Some pits are occupied by a globule ∼125 Å in diameter, which displays a central depression ∼25 Å in size. Face B contains globules also arranged in a fairly regular hexagonal pattern. The center-to-center distance between adjacent globules is most frequently ∼200 Å; however, occasionally certain globules are seen separated by a distance as short as ∼125 Å. The top surface of the globules occasionally displays a starlike profile and seems to contain a central depression ∼25 Å in diameter. In negatively stained preparations of membranes from the nerve cord, two types of membranes are seen containing a fairly regular pattern. In one, globules ∼95 Å in diameter form a hexagonal close packing with a unit cell of ∼95 Å. In the other, globules of the same size are organized in a larger hexagonal array with a unit cell of ∼155 Å (swollen arrangement). Some of the globules forming the swollen arrangement are seen containing six subunits. The six subunits form a hexagon which is skewed with respect to the main rows of hexagons in such a way that the subunits lie on rows which make an angle of ∼37° with the main rows.
ELECTRON MICROSCOPE AUTORADIOGRAPHIC DETECTION OF SITES OF PROTEIN SYNTHESIS IN THE RABBIT RETINA MÜLLER CELLS
Authors: Magalhães M. M. , Coimbra Antonio
Published: May 2004

Rabbit retinas were incubated in medium containing 500 µCi of [3H]leucine for 3 min, and transferred to medium without isotope for another 7, 17, 37, 57, and 117 min. Retinal pieces were fixed in paraformaldehyde and osmium tetroxide and embedded in Epon. Thin sections were autoradiographed with Ilford L4 emulsion, and a quantitative study of silver grain distribution per Müller cell portion, and per Müller cell organelle, was carried out. Grain density per unit area was high over the middle cell portion at each incubation interval. Silver grains were numerous over background cytoplasm (which comprised free ribosomes) but their percentage was constant at all times and their relative concentration low. Silver grains were numerous and highly concentrated, at pulse incubation, over the rough endoplasmic reticulum (RER) and then decreased sharply, but this decline coincided with an increase over the Golgi complex, peaking at 20 min. Another peak appeared over the cell periphery at 60 min. These findings suggest the simultaneous synthesis of two types of proteins in Müller cells; structural proteins in background cytoplasm and proteins of secretory type in the RER.
A NEW METHOD FOR ANALYZING ELECTRON MICROSCOPE AUTORADIOGRAPHS USING HYPOTHETICAL GRAIN DISTRIBUTIONS
Authors: Blackett N. M. , Parry D. M.
Published: May 2004

A new method for the analysis of electron microscope autoradiographs is described which has two advantages over other methods. This method provides estimates for the activity in different regions of the section and takes into account the actual geometrical shape and position of different regions within the section. Use is made of the measured distribution of grains about a thin line source to construct a hypothetical distribution of autoradiographic grains which is then compared with the actual distribution of grains. Different activities are then ascribed to various regions until a satisfactory agreement with the autoradiographic grains is obtained using the χ2 significance. In the next paper, an example of the use of this method is given for the distribution of grains in the nucleus of erythroid bone marrow cells labeled with radioactive iron, 55Fe.
ULTRASTRUCTURAL ORGANIZATION OF CHLOROPLAST THYLAKOIDS OF THE GREEN ALGA "OOCYSTIS MARSSONII"
Authors: Pendland Jacquelyn C. , Aldrich H. C.
Published: May 2004

Intact cells of "Oocystis marssonii" were thin sectioned and freeze-etched, using conventional and double-recovery techniques. Thylakoids extend the length of the single chloroplast and occur in stacks of three to five. The peripheral thylakoids in a stack often alternate between adjacent stacks. Interpretation of double-recovery results suggests that membranes in unstacked regions are asymmetrical, with one face smooth and the matching face covered with closely packed 85–90 Å diameter particles. Adjacent membranes in stacked regions evidently share 170 Å diameter particles, and either membrane in a stacked region may fracture. The two fracture planes thus made possible may expose nearly entire 170 Å particles or only the upper portion of such particles, creating in the latter case images of 125–135 Å diameter particles. Fracture planes in all cases appear to occur through the interior of the membrane, in the plane between the hydrophobic ends of the lipid bilayer proposed in numerous membrane models.
ENHANCEMENT OF SENDAI VIRUS-MEDIATED CELL FUSION BY CUPRIC IONS
Authors: Wainberg M. A. , Howe C. , Godman G. C.
Published: May 2004

The effect of divalent cations on cell fusion by concentrated Sendai virus, inactivated by beta-propiolactone, was investigated using Vero and mouse L-929 cells in monolayers. With both cell lines, which are normally resistant to exogenous viral fusion, Cu2+ in sublethal concentrations was found to promote polykaryon formation to a marked degree. The simultaneous presence of Cu2+ and virus was required for this effect, which was thought to be related to the cytotoxic action of Cu2+ on the cell membrane. Accordingly, under standard conditions and in the absence of virus, leakage of isotopically labeled intracellular protein was shown to bear a quantitative relationship to Cu2+ concentration. Concomitant changes in the membrane were seen electron microscopically to consist of loss of microvilli and the appearance of numerous vesicles on, or adjacent to, the membrane. The relationship of enhanced fusibility to these toxic changes was not further elucidated. The fusion-promoting effect of Cu2+ far exceeded that of Ca2+; and other cations tested had no effect.
GROWTH INHIBITION OF MURINE TUMOR CELLS, IN VITRO, BY PUROMYCIN, [6N]O2'-DIBUTYRYL 3',5'-ADENOSINE MONOPHOSPHATE, OR ADENOSINE
Authors: Thomas D. B. , Medley Gay , Lingwood C. A.
Published: May 2004

The cytostatic effects of puromycm, [6N]O2'-dibutyryl 3',5'-adenosine monophosphate, and adenosine on asynchronous and synchronous cultures of the murine mastocytoma, P815Y, have been studied. Cell growth was arrested after a minimum of one further division. A model is proposed for the inhibition of cell division in which the periods of inhibition and growth arrest are separated in time by one cell cycle.
SUPPRESSION OF PIGMENTATION IN MOUSE MELANOMA CELLS BY 5-BROMODEOXYURIDINE
Authors: Wrathall Jean R. , Oliver Constance , Silagi Selma , Essner Edward
Published: May 2004

Low concentrations (1–3 µg/ml) of 5-bromodeoxyuridine (BrdU) reversibly suppress pigmentation in a highly pigmented clone (B559) of cultured B16 mouse melanoma cells. We have found that unpigmented cells (clone C3471), derived by long-term culture of B559 cells in 1 µg of BrdU/ml, were completely amelanotic with no biochemically or cytochemically detectable tyrosinase activity or ultrastructural evidence of premelanosomes. The process by which pigmentation is suppressed was studied in B559 cells during a 7-day period of growth with BrdU (3 µg/ml). Assays of tyrosinase activity showed that activity was reduced after 1 day and decreased progressively, approaching zero by 7 days. A quantitatively minor part of this reduction was directly attributable to the appearance of a dialyzable inhibitor of tyrosinase activity. Acrylamide gel electrophoresis revealed two bands of activity corresponding in Rx values to the T1 and T2 forms of soluble tyrosinase. Both were progressively reduced during growth with BrdU but one form (T1) was consistently affected earlier than the other (T2). Ultrastructural-cytochemical studies also showed an early effect on the localization of tyrosinase reaction product. At day 3, reaction product was no longer present in Golgi saccules and Golgi-associated smooth surfaced tubules, but was still seen within premelanosomes, compound melanosomes, and occasional Golgi-associated vesicles. By 7 days tyrosinase reaction product was usually not demonstrable. The number of premelanosomes was progressively decreased during growth with BrdU. Premelanosomes became concentrated in the juxtanuclear region and at day 3 many were contained within abnormally large and numerous compound melanosomes. Premelanosomes and compound melanosomes were rarely seen at 7 days, by which time the cultures were nearly amelanotic. The coordinated suppression of melanogenesis by BrdU may provide a useful model in which to study the normal regulation of this process.
SUBCELLULAR MORPHOMETRIC AND BIOCHEMICAL ANALYSES OF DEVELOPING RAT HEPATOCYTES
Authors: Herzfeld Annemarie , Federman Micheline , Greengard Olga
Published: May 2004

Livers of rats between the 16th gestational and 100th postnatal day of age were subjected to quantitative biochemical and electron microscope, morphometric analyses. The amount of total mitochondrial protein per gram of liver remained at 34% of the adult level throughout the last 4 days of gestation but this was the period of rapid rise in the levels of cytochrome c oxidase, aspartate aminotransferase, and glutamate dehydrogenase in mitochondria; the nuclear fraction also acquired some glutamate dehydrogenase but lost most of it during postnatal development. During early postnatal life the amount of mitochondrial protein rose in parallel with the levels of cytochrome c oxidase and glutamate dehydrogenase but the upsurges of glutaminase and, later, of ornithine aminotransferase were accompanied by relatively little change in total mitochondrial protein. The surface area of rough endoplasmic reticulum per unit volume of hepatocyte cytoplasm (SvRER) did not change significantly throughout the period of development studied. From the 16th day of gestation to term the surface area of smooth ER (SvSER), the volume occupied by mitochondria (VvMT) and their number (NvMT) remained at 30, 66, and 45% of their adult values, respectively. VvMT and NvMT attained their maximal levels by the 2nd postnatal day and SvSER between days 2 and 12. Mitochondria of adult liver are thus smaller and contain more protein per unit volume than do those of fetal liver. After the 12th postnatal day, hepatocytes treble their size; they acquire more cytoplasm with additional enzymes but without further change in organelle concentration. The data reveal several distinct phases in the differentiation of hepatocytes. Each phase can be characterized by the extent to which the quantity and composition of various subcellular compartments evolve.
THE EFFECT OF POLY-L-LYSINE ON THE UPTAKE OF REOVIRUS DOUBLE-STRANDED RNA IN MACROPHAGES IN VITRO
Authors: Seljelid Rolf , Silverstein Samuel C. , Cohn Zanvil A.
Published: May 2004

The effect of polycations on cultured mouse peitoneal macrophages has been examined. Polycations, at concentrations greater than 5 µg/ml, are toxic for macrophages) as measured by failure of the cells to exclude vital dyes. At toxic concentrations polycations bind in large amounts to nuclei and endoplasmic reticulum, while at nontoxic levels polycations bind selectively to the cell surface. Nontoxic concentrations of polycations stimulate binding of reovirus double-stranded (ds) RNA to the macrophages by forming polycation-dsRNA complexes either in the medium or at the cell surface. These complexes enter the cell in endocytic vacuoles and are concentrated in secondary lysosomes. Despite exposure to the acid hydrolases within this cell compartment, the dsRNA and the polycation (poly-L-lysine) are conserved in a macromolecular form within the vacuolar system. The mechanism(s) by which the uptake of infectious nucleic acids and the induction of interferon by dsRNA are stimulated by polycations are discussed.
ULTRASTRUCTURAL LOCALIZATION OF PHOSPHOLIPID SYNTHESIS IN THE RAT TRIGEMINAL NERVE DURING MYELINATION
Authors: Benes Francine , Higgins Joan A. , Barrnett Russell J.
Published: May 2004

A method for the ultrastructural localization of acyltransferase enzymes involved in phospholipid metabolism has been applied to the developing rat trigeminal nerve. Determination of acyltransferase levels in the nerve indicated that a peak of activity occurs at the 8th day after birth with gradual declines of activity up to 15 days. Morphological surveys and determinations of cholesterol levels suggested that heavy myelin formation occurs in the nerve during this latter period. Fixed nerves incubated in a medium for localization of acyltransferases indicated deposition of reaction product associated with Golgi cisternae, intracellular smooth vesicles, and the plasma membrane of the Schwann cell in the incipient stages of myelin formation. Golgi-derived vesicles appeared to move toward the Schwann cell surface and fuse with the plasma membrane. Activity continued to be detectable in the plasma membrane of the internal mesaxon as long as cytoplasm was evident and mature myelin membrane was not yet formed. Cells in which myelin formation appeared advanced showed little or no enzyme marker. Consistent with cytochemical observations were biochemical determinations of acyltransferases which showed high levels of the enzymes in microsomes, while no activity could be detected in the myelin fraction. Acyltransferase reaction product was also observed in the Golgi apparatus of ganglion cell bodies, axoplasmic smooth vesicles, and the axolemma. Localization of acyltransferase enzymes in Schwann cells, ganglion cell bodies, and axons during development of the nerve is discussed in relation to membrane biogenesis in the nervous system.
LOCALIZATION OF Na+, K+-ATPASE AND OTHER ENZYMES IN TELEOST PSEUDOBRANCH
Authors: Dendy Leslie A. , Philpott Charles W. , Deter Russell L.
Published: May 2004

The pseudobranch of the pinfish Lagodon rhomboides is an unusually homogeneous and structurally simple tissue, well suited to cell fractionation studies. Its principal cell type, closely related to the chloride cells of teleost gill, is characterized by numerous mitochondria in close association with abundant tubular invaginations of the plasma membrane. Other cytoplasmic organelles are rarely encountered. In broken fresh pseudobranch cells negatively stained with ammonium molybdate, a 40 Å particulate layer was observed on the intracellular surface of the tubular plasma membrane fragments. Nuclear (N), mitochondrial-light mitochondrial (M+L), and microsomal (P) fractions, obtained by differential centrifugation, were characterized by examination of fixed, embedded pellets and unfixed preparations negatively stained with ammonium molybdate and potassium phosphotung-state. Mitochondria, in orthodox configuration and retaining their outer membranes, were observed in M+L and N. Significant amounts of tubular, sheetlike, or vesicular membrane fragments were observed in all three fractions. Many such fragments, when negatively stained, showed the 40 Å particulate surface layer characteristic of plasma membrane invaginations, and in some cases 20-Å projections could be resolved on the opposite (extracellular) surface. Since these morphological observations, together with previously presented biochemical data, suggest a plasma membrane localization of Na+, K+-ATPase, the possible association of the enzyme with membrane projections is discussed.
VISUALIZATION OF FREEZING DAMAGE
Authors: Bank Harvey , Mazur Peter
Published: May 2004

Freeze-cleaving can be used as a direct probe to examine the ultrastructural alterations of biological material due to freezing. We examined the thesis that at least two factors, which are oppositely dependent upon cooling velocity, determine the survival of cells subjected to freezing. According to this thesis, when cells are cooled at rates exceeding a critical velocity, a decrease in viability is caused by the presence of intracellular ice; but cells cooled at rates less than this critical velocity do not contain appreciable amounts of intracellular ice and are killed by prolonged exposure to a solution that is altered by the presence of ice. As a test of this hypothesis, we examined freeze-fractured replicas of the yeast Saccharomyces cerevisiae after suspensions had been cooled at rates ranging from 1.8 to 75,000°C/min. Some of the frozen samples were cleaved and replicated immediately in order to minimize artifacts due to sample handling. Other samples were deeply etched or were rewarmed to -20°C and recooled before replication. Yeast cells cooled at or above the rate necessary to preserve maximal viability (∼7°C/min) contained intracellular ice, whereas cells cooled below this rate showed no evidence of intracellular ice.
AUTORADIOGRAPHIC LOCALIZATION OF NEW RNA SYNTHESIS IN HYPERTROPHYING SKELETAL MUSCLE
Authors: Jablecki Charles K. , Heuser John E. , Kaufman Seymour
Published: May 2004

Work-induced growth of rat soleus muscle is accompanied by an early increase in new RNA synthesis. To determine the cell type(s) responsible for the increased RNA synthesis, we compared light autoradiographs of control and hypertrophying muscles from rats injected with tritiated uridine 12, 24, and 48 h after inducing hypertrophy. There was an increased number of silver grains over autoradiographs of hypertrophied muscle. This increase occurred over connective tissue cells; there was no increase in the number of silver grains over the muscle fibers. Quantitative studies demonstrated that between 70 and 80% of the radioactivity in the muscle that survived fixation and washing was in RNA. Pretreatment of the animals with actinomycin D reduced in parallel both the radioactivity in RNA and the number of silver grains over autoradiographs. Proliferation of the connective tissue in hypertrophying muscle was evident in light micrographs, and electron micrographs identified the proliferating cells as enlarged fibroblasts and macrophages; the connective tissue cells remained after hypertrophy was completed. Thus, proliferating connective tissue cells are the major site of the increase in new RNA synthesis during acute work-induced growth of skeletal muscle. It is suggested that in the analysis of physiological adaptations of muscle, the connective tissue cells deserve consideration as a site of significant molecular activity.
CHANGES IN SURFACE MORPHOLOGY OF CHINESE HAMSTER OVARY CELLS DURING THE CELL CYCLE
Authors: Porter Keith , Prescott David , Frye Jearl
Published: May 2004

Synchronized populations of Chinese hamster ovary (CHO) cells in confluent culture have been examined by scanning electron microscopy and their surface changes noted as the cells progress through the cycle. During G1 it is characteristic for cells to show large numbers of microvilli, blebs, and ruffles. Except for the ruffles, these tend to diminish in prominence during S and the cells become relatively smooth as they spread thinly over the substrate. During G2 microvilli increase in number and the cells thicken in anticipation of rounding up for mitosis. It appears that the changes observed here reflect the changing capacity of CHO cells during the cycle to respond to contact with other cells in the population, because, as noted in the succeeding paper (Rubin and Everhart), CHO cells in sparse nonconfluent cultures do not show the same wide range of changes during the cell cycle. Normal, nontransformed cells of equivalent type in confluent culture are essentially devoid of microvilli, blebs, and ruffles. The relation of these surface configurations to the internal structure of the cell is discussed.
THE EFFECT OF CELL-TO-CELL CONTACT ON THE SURFACE MORPHOLOGY OF CHINESE HAMSTER OVARY CELLS
Authors: Rubin R. W. , Everhart L. P.
Published: May 2004

In the previous report (Porter et al., in this issue) morphological changes in Chinese hamster ovary (CHO) cells during the cell cycle were described. In this report we describe the role of intercellular contact on these changes. We find that intercellular contact is required for cells to exhibit the morphologies Porter et al. described for S and G2. When cells are synchronized by mitotic selection and plated onto cover slips at very low density such that no intercellular contact occurs, the cells remain in a G1 configuration (rounded and highly blebbed through G1, S, and G2). This G1 morphology is also observed in nonsynchronized log phase cells plated at low densities and allowed to grow for several generations. The addition of conditioned medium from confluent cultures does not induce low density cells to change morphology during the cell cycle. These results indicate that extensive intercellular contact is required for the complete expression of the morphological changes associated with the cell cycle (as described by Porter et al.). It is concluded that although classic contact inhibition of movement and of growth may be absent in this transformed cell line, some contact-dependent response persists.
TWO ACTIONS OF CYCLIC AMP ON MELANOSOME MOVEMENT IN FROG SKIN
Authors: Magun Bruce
Published: May 2004

Photomicrography and reflectance microphotometry were used to monitor melanosome movement in frog skin melanocytes in vitro in response to hormonal stimulation and cytochalasin B (CB). Melanocyte-stimulating hormone (MSH), theophylline, and dibutyryl cyclic AMP (DiBcAMP) induced melanosome dispersion (darkening) which was promptly arrested by cytochalasin B in concentrations of 5–20 µg/ml. Melanosome aggregation (skin lightening) occurred only after removal of the darkening agent (MSH, theophylline, or DiBcAMP) and proceeded in the presence or absence of CB. When CB was added to darkened skins, they did not lighten and melanosomes remained in the dispersed state. Use of CB has permitted the dissection of cyclic AMP-mediated melanosome dispersion into two distinct events. The first, induction of melanosome dispersion, is CB sensitive. The second action of intracellular cyclic AMP involves an uncoupling of the centripetal motive force, and is CB insensitive. In the latter process, production of cyclic AMP appears to produce the same result as application of microtubule-disrupting agents.
SELECTIVE DEPOSITION OF LANTHANUM IN MAMMALIAN CARDIAC CELL MEMBRANES
Authors: Martinez-Palomo A. , Benitez D. , Alanis J.
Published: May 2004

Perfusion of beating false tendons of the dog heart with ionic lanthanum produced drastic but reversible modifications of the excitability and the transmembrane action potential of Purkinje cells. Ultrastructural examination of these cells revealed the appearance of a fine extracellular precipitate detectable on unstained sections. In addition, specimens perfused with La+++ showed a striking increase in the contrast of the sarcolemma, particularly in gap junctions and in pinocytic vesicles. La+++ deposits were restricted to the cytoplasmic leaflets of the sarcolemma; no precipitates were found at the plasma membrane of fibroblasts, endothelial and smooth muscle cells, or unmyelinated nerve fibers present in the same specimens. A selective deposition of La+++ was also observed in the sarcolemma of atrial and ventricular cells of dog, rabbit, and cat hearts, as well as in the membrane of the transverse tubular system of ventricular cells. Both the electrophysiological effects and the ultrastructural membrane deposits produced by La+++ disappeared when the specimens were subsequently perfused with phosphate-containing tyrode solution. These results tend to demonstrate that a distinctive feature of the sarcolemma of mammalian cardiac cells is the presence of regions with a high surface density of binding sites for polyvalent cations.
NUCLEAR MEMBRANE FUSION IN FERTILIZED LYTECHINUS VARIEGATUS EGGS
Authors: Aronson John F.
Published: May 2004

Fusion of apposed nuclear envelopes is frequently seen at telophase during postmitotic reorganization of the nucleus, but only rarely at other times in the cell cycle. We attempted to define an experimental system for studying changes in the nuclear envelope related to the cell cycle by varying the time of pronuclear apposition in fertilized Lytechinus variegatus eggs. This approach was based on the assumption that the period from fertilization to metaphase of the first cleavage division corresponds to the period from telophase to metaphase in the generalized cell cycle. The experimental approach used was to block the movement of the pronuclei with Colcemid and then to release this block at varying times after insemination by photochemically inactivating the Colcemid. The results show that apposed pronuclear envelopes can fuse from soon after insemination until the anticipated time of prometaphase. Fusion occurred in about 3 min as scored by light microscopy and this time did not vary significantly with the time after insemination. The potential for nuclear fusion is not restricted to pronuclei alone since diploid nuclei in binucleate cells could be fused using centrifugation in solutions of Colcemid to bring the nuclei into apposition. It is suggested that the potential for nuclear fusion is not necessarily related to the cell cycle and that modification of the nuclear envelope, possibly by association with chromatin or other fibrous material restricts nuclear fusion in most multinucleated cells.
POLARIZED INTERCELLULAR BRIDGES IN OVARIAN FOLLICLES OF THE CECROPIA MOTH
Authors: Woodruff Richard I. , Telfer William H.
Published: May 2004

Fluorescein-labeled rabbit serum globulin was injected into vitellogenic oocytes of the cecropia moth. Though the label spread throughout the ooplasm in less than 30 min, it was unable even after 2 h to cross the complex of intercellular bridges connecting the oocyte to its seven nurse cells. After injection into a single nurse cell, fluorescence was detected in the oocyte adjacent to the bridge complex within 3 min and had spread throughout the ooplasm in 30 min. Here also, the cell bodies of the six uninjected nurse cells remained nonfluorescent. Four of the nurse cells are not bridged directly to the oocyte but only through the apical ends of their siblings. Unidirectional movement must therefore occur in the apical cytoplasm of the nurse cells, as well as in the intercellular bridges. The nurse cells of healthy follicles had an intracellular electrical potential -40 mV relative to blood or dissecting solution, while oocytes measured -30 mV. A mV difference was also detected by direct comparison between a ground electrode in one cell and a recording electrode in the other. Three conditions were found in which the 10 mV difference was reduced or reversed in polarity. In all three cases fluorescent globulin was able in some degree to cross the bridges from the oocyte to the nurse cells.
EFFECTS OF THALLIUM SALTS ON NEURONAL MITOCHONDRIA IN ORGANOTYPIC CORD-GANGLIA-MUSCLE COMBINATION CULTURES
Authors: Spencer Peter S. , Peterson Edith R. , Madrid A. Ricardo , Raine Cedric S.
Published: May 2004

A functionally coupled organotypic complex of cultured dorsal root ganglia, spinal cord peripheral nerve, and muscle has been employed in an experimental approach to the investigation of the neurotoxic effects of thallium. Selected cultures, grown for up to 12 wk in vitro, were exposed to thallous salts for periods ranging up to 4 days. Cytopathic effects were first detected after 2 h of exposure with the appearance of considerably enlarged mitochondria in axons of peripheral nerve fibers. With time, the matrix space of these mitochondria became progressively swollen, transforming the organelle into an axonal vacuole bounded by the original outer mitochondrial membrane. Coalescence of adjacent axonal vacuoles produced massive internal axon compartments, the membranes of which were shown by electron microprobe mass spectrometry to have an affinity for thallium. Other axoplasmic components were displaced within a distended but intact axolemma. The resultant fiber swelling caused myelin retraction from nodes of Ranvier but no degeneration. Impulses could still propagate along the nerve fibers throughout the time course of the experiment. Comparable, but less severe changes were seen in dorsal root ganglion neurons and in central nerve fibers. Other cell types showed no mitochondrial change. It is uncertain how these findings relate to the neurotoxic effects of thallium in vivo, but a sensitivity of the nerve cell and especially its axon to thallous salts is indicated.
NITROUS OXIDE: EFFECTS ON THE MITOTIC APPARATUS AND CHROMOSOME MOVEMENT IN HELA CELLS
Authors: Brinkley B. R. , Rao Potu N.
Published: May 2004

When HeLa cells were grown in the presence of nitrous oxide (N2O) under pressure (80 lb/in2) mitosis was inhibited and the chromosomes displayed a typical colchicine metaphase (c-metaphase) configuration when examined by light microscopy. When the cells were returned to a 37°C incubator, mitosis was resumed and the cells entered G1 synchronously. Ultrastructural studies of N2O-blocked cells revealed a bipolar spindle with centriole pairs at each pole. Both chromosomal and interpolar (pole-to-pole) microtubules were also present. Thus, N2O, unlike most c-mitotic agents, appeared to have little or no effect upon spindle microtubule assembly. However, the failure of chromo somes to become properly aligned onto the metaphase plate indicated an impairment in normal prometaphase movement. The alignment of spindle microtubules was frequently atypical with some chromosomal microtubules extending from kinetochores to the poles, while others extended out at acute angles from the spindle axis. These ultrastructural studies indicated that N2O blocked cells at a stage in mitosis more advanced than that produced by Colcemid or other c-mitotic agents. Like Colcemid, however, prolonged arrest in mitosis with N2O led to an increased incidence of multipolar spindles.
LOCALIZATION OF THE ENZYMES OF KETOGENESIS IN RAT LIVER MITOCHONDRIA
Authors: Chapman M. John , Miller Leonard R. , Ontko Joseph A.
Published: May 2004

The localization of the enzymes of ketogenesis in isolated rat liver mitochondria has been investigated. Mitochondrial subfractions were isolated after disruption of this subcellular organelle by (a) hypotonic lysis in water, which permitted the ultracentrifugal separation of the soluble and membranous compartments of the mitochondrion, or by (b) a procedure involving swelling, contraction, and ultrasonic treatment, which permitted the isolation from discontinuous sucrose gradients of subfractions rich in intermembrane space protein, outer membrane, and inner membrane-matrix particles. Two membrane subfractions were invariably present as distinct bands at the lower interface of the discontinuous gradient. The upper of these two bands was found to be a highly purified preparation of outer mitochondrial membrane. Subfractions rich in matrix and in inner membrane were isolated from inner membrane-matrix particles after hypotonic treatment. The content of the various mitochondrial compartments in all subfractions was assessed from their enzymic and electron microscopic characteristics. The ketogenic activity of each subfraction was determined by measuring its capacity to form ketone bodies from acetyl CoA. The activity of this process was markedly enhanced by dithiothreitol. These measurements of ketone body formation, together with assays of individual enzymes of the ketogenic pathway, show that thiolase, HMGCoA synthase, and HMGCoA cleavage enzyme are localized in the matrix of the inner membrane-matrix particles. The rates of ketone body formation indicate that the HMGCoA synthase is the rate-limiting enzyme of the pathway in subfractions of high matrix content. Studies with sodium chloride indicate that a large portion of the HMGCoA synthase, which remains present in membrane subfractions derived from water-treated mitochondria, is bound by ionic interaction to component(s) of the membrane.
GANGLIOSIDE PATTERNS AND PHENOTYPIC CHARACTERISTICS IN A NORMAL VARIANT AND A TRANSFORMED BACK VARIANT OF A SIMIAN VIRUS 40-INDUCED HAMSTER TUMOR CELL LINE
Authors: Nigam Vijai N. , Lallier R. , Brailovsky C.
Published: May 2004

Ganglioside patterns of a cloned Simian virus 40- (SV40) induced hamster tumor cell (Cl2TSV5-S), its normal variant (Cl2TSV5-R) which are Cl2TSV5-S gradually adapted to grow in the presence of 2 µg/ml actinomycin D and exhibit certain normal phenotypic characteristics, and its back variant (Cl2TSV5-RR), which are Cl2TSV5-R cells grown in the absence of actinomycin D for more than 60 passages and which exhibit greater phenotypic similarity to Cl2TSV5-S cells, have been analyzed. All three cell lines contain N(acetylneuraminyl) galactosylglycosyl ceramide (hematoside, GM3), N-acetylgalactosaminyl (N-acetylneuraminyl) galactosylglucosyl ceramide (GM2), and a higher ganglioside tentatively identified as disialohematoside. However, Cl2TSV5-R have more GM2 than Cl2TSV5-S whereas Cl2TSV5-RR contain an intermediate amount of GM2. The amount of GM2 is correlative with the activity of UDP-N-acetylgalactosamine: hematoside N-acetylgalactosaminyl transferase in the extract of the three cell lines and with their agglutination by wheat germ agglutinin.
FURTHER CHARACTERIZATION OF THE F1-HISTONE PHOSPHOKINASE OF METAPHASE-ARRESTED ANIMAL CELLS
Authors: Lake Robert S.
Published: May 2004

Exponentially growing Chinese hamster cells are found to contain two major phosphokinase activities with specificity for the phosphorylation of F1 (lysine-rich) histone. These two activities, designated KI and KII, were extracted with 0.35 M NaCl and fractionated in 0.2 M NaCl by Sephadex G-200 gel filtration. KI, which is similar to the ubiquitous cyclic 3',5'-adenosine monophosphate (cAMP)-dependent phosphokinase, differs from KII by several criteria. KII is mol wt 90,000, cAMP independent, rapidly turned over in vivo, low Km for ATP, and phosphorylates F1 histone at several unique sites. Comparative examination of metaphase-arrested (M) and counterpart interphase (I) cells for these two activities reveals that KII is responsible for the overall high activity in M-arrested cells. Pulse labeling of cells with 32P during traverse of the G2-M phase of the cell cycle reveals an in vivo tryptic-phosphopeptide pattern in whole unfractionated F1 which is unique to M cells. Seven major phosphopeptides derived by in vitro phosphorylation of F1 with the KII enzyme correspond to these M cell-specific phosphorylation sites observed in vivo. It is suggested that KII activity predominates during the G2-M transition and that F1 is its natural in vivo substrate.
SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE
Authors: Ley Kenneth D. , Murphy Marilyn M.
Published: May 2004

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([3H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([3H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [3H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G1 because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.
FRACTURE FACES OF ZONULAE OCCLUDENTES FROM "TIGHT" AND "LEAKY" EPITHELIA
Authors: Claude Philippa , Goodenough Daniel A.
Published: May 2004

Epithelia vary with respect to transepithelial permeability. In those that are considered "leaky", a large fraction of the passive transepithelial flux appears to follow the paracellular route, passing across the zonulae occludentes and moving down the intercellular clefts. In "tight" epithelia, the resistance of the paracellular pathway to passive flux is greatly increased. To see whether differences in the morphology of the zonula occludens could contribute to this variability in leakiness among epithelia, replicas of zonulae occludentes in freeze-fractured material from a variety of tight and leaky epithelia were examined. The junctions appear as a branching and anastomosing network of strands or grooves on the A and B membrane fracture faces, respectively. It was found that the zonula occludens from a "very leaky" epithelium, the proximal convoluted tubule of the mouse kidney, is extremely shallow in the apical-basal direction, consisting in most places of only one junctional strand. In contrast, the "very tight" frog urinary bladder exhibits a zonula occludens that is relatively deep (>0.5 µm) in the apical-basal direction, and consists of five or more interconnected junctional strands interposed between luminal and lateral membrane surfaces. Epithelia of intermediate permeabilities exhibited junctions with intermediate or variable morphology. Toad urinary bladder, mouse stomach, jejunum, and distal tubule, rabbit gallbladder, and Necturus kidney and gallbladder were also examined, and the morphological data from these epithelia were compared to physiological data from the literature.
COMPLEMENTARY CHROMATIC ADAPTATION IN A FILAMENTOUS BLUE-GREEN ALGA
Authors: Bennett Allen , Bogorad Lawrence
Published: May 2004

Fluorescent and red light environments generate greatly different patterns of pigmentation and morphology in Fremyella diplosiphon. Most strikingly, red-illuminated cultures contain no measurable C-phycoerythrin and have a mean filament length about 10 times shorter than fluorescent-illuminated cultures. C-phycoerythrin behaves as a photoinducible constituent of this alga. Spectrophotometric and immunochemical procedures were devised so that C-phycoerythrin metabolism could be studied quantitatively with [14C]-phenylalanine pulse-chased cultures. Transfer of red-illuminated cultures to fluorescent light initiates C-phycoerythrin production by essentially de novo synthesis. C-phycoerythrin is not degraded to any significant extent in cultures continuously illuminated with fluorescent light. Transfer of fluorescent-illuminated cultures to red light causes an abrupt cessation of C-phycoerythrin synthesis. The C-phycoerythrin content of cultures adapting to red light decreases and subsequently becomes constant. Loss of C-phycoerythrin is not brought about by metabolic degradation, but rather by a decrease in mean filament length which is effected by transcellular breakage. In this experimental system, light influences intracellular C-phycoerythrin levels by regulating the rate of synthesis of the chromoprotein.
LIGHT AND ELECTRON MICROSCOPE LOCALIZATION OF BINDING SITES OF ANTIBODIES AGAINST OVINE LUTEINIZING HORMONE AND ITS TWO SUBUNITS IN RAT ADENOHYPOPHYSIS USING PEROXIDASE-LABELED ANTIBODY TECHNIQUE
Authors: Tougard C. , Kerdelhue B. , Tixier-Vidal A. , Jutisz M.
Published: May 2004

The binding sites of antisera generated in the guinea pig against ovine luteinizing hormone (oLH) and its two subunits (oLHα and oLHß) have been localized in rat anterior pituitaries taken from normal or castrated males and from ovariectomized females with the peroxidase-labeled antibody method, using light and electron microscopy. With the light microscope, the cells positive with antiserum to ovine luteinizing hormone (A-oLH) were violet after the Alcian blue-periodic acid-Schiff (AB-PAS) staining; they were also positive for A-oLHα and for A-oLHß and, from castrated males, they displayed an increased affinity for A-oLHß. Another cell type which was blue after the AB-PAS method reacted with the A-oLHα only; these cells, presumably thyrotropic cells, were retracted after castration and, besides their affinity for A-oLHα, acquired an affinity for A-oLHß. As seen through the electron microscope, two cell types were positive for A-oLH, A-oLHß, and A-oLHα and may be identified as luteinizing hormone-secreting cells. Type A cells were characterized by two classes of rounded, secretory granules. Type B cells were smaller and contained only small secretory granules. 1 mo after the rats were castrated the type A cells were hypertrophied and vacuolized. In both cases the secretory granules were the main sites of the antigenicity with the three antisera. A positive reaction was also found in the cytoplasm, particularly in hypertrophied cells from ovariectomized females and with A-oLHß. The cisternae of the rough endoplasmic reticulum were usually negative, except in highly degranulated cells from ovariectomized females and with A-oLHß.
BIOCHEMICAL CHANGES DURING GROWTH AND ENCYSTMENT OF THE CELLULAR SLIME MOLD POLYSPHONDYLIUM PALLIDUM
Authors: Githens S. , Karnovsky M. L.
Published: May 2004

The growth of the cellular slime mold, Polysphondylium pallidum, was studied on a semidefined medium in shaken suspension. When the medium contained large quantities of particulate material, growth was more rapid and the cellular size and protein content were smaller than when growth occurred on a medium containing less particulate material. The cellular levels of DNA, RNA, and protein; of lysosomal enzymes (acid phosphatase, acid proteinase); and of peroxisomal enzymes (catalase) were assayed during growth and the subsequent stationary phase that led eventually to encystment. Only DNA remained at a constant cellular level. Encystment of exponentially growing cells could also be initiated by washing them and introducing them into a soluble peptone medium. The rate of encystment was proportional to the osmolarity of this medium. The encystment process was followed with respect to the cellular levels of DNA, RNA, protein, carbohydrates, acid phosphatase, acid ß-N-Ac-glucosaminidase, and catalase. The most dramatic change occurred in the cellular cellulose content, which increased by at least an order of magnitude by the time encystment was morphologically complete. It was concluded that the encystment of this slime mold in suspension exhibits a number of biochemical similarities to the development of this and other cellular slime molds on a surface.