bluuebunny/crossref_metadata_2025 huggingface dataset:
POSSIBILITIES FOR INTER- AND INTRACELLULAR TRANSLOCATION OF SOME ICOSAHEDRAL PLANT VIRUSES
Authors: de Zoeten G. A. , Gaard G.
Published: May 2004

Southern bean mosaic virus (SBMV) and Tomato ringspot virus (TomRV) were compared with regard to possible ways of inter- and intracellular translocation. The pore complexes in the nuclear membranes of nuclei in leaf palisade and mesophyll cells of several plant species commonly used in plant virus research were studied. The pore structure resembled that earlier described. The diameter of the pores was great enough to allow icosahedral plant viruses between 25 and 30 mµ wide to move through. SBMV occurred in noncrystalline form in nuclei of infected cells. Although this virus forms paracrystalline structures when partially purified, no virus crystals were seen in the cytoplasm of cells containing high concentrations of SBMV. It was established that this virus could move through nuclear pores. TomRV was found in infected leaf tissue in low concentrations. This virus showed a tendency to crystallize even when present in low concentrations. TomRV was observed only in the cytoplasm, not in nuclei. This virus was present in plasmodesmata, indicating the possibility of cell to cell translocation of whole particles through these structures.
RESOLUTION IN ELECTRON MICROSCOPE RADIOAUTOGRAPHY
Authors: Salpeter M. M. , Bachmann L. , Salpeter E. E.
Published: May 2004

An analysis of grain distributions around a radioactive line source (consisting of polystyrene-3H) showed that the shape of the distribution was independent of the factors that influence resolution, i.e. section and emulsion thickness, silver halide crystal, and developed grain size. These factors did effect the spread of the distribution, however, and thus the distance from the line source within which 50% of the total developed grains fell. We called this distance "half distance" (HD) and determined it for a variety of specimens. When grain distributions were normalized in units of HD, one could plot universal grain distributions for specimens with radioactive sources of various shapes. The use of HD and the universal curves in interpreting radioautograms is discussed.
OBSERVATIONS ON THE RELATIONSHIP OF THE GOLGI APPARATUS TO WALL FORMATION IN THE MARINE CHRYSOPHYCEAN ALGA, PLEUROCHRYSIS SCHERFFELII PRINGSHEIM
Authors: Brown R. M.
Published: May 2004

The role of the Golgi apparatus in wall formation of vegetative cells of a marine chrysophyte, Pleurochrysis scherffelii, is described. Wall fragments are synthesized within the cisternae of the Golgi apparatus. A single Golgi apparatus is always located at the cell periphery, and the distended cisternae are oriented toward the cell surface. A highly-ordered body found near the inflated cisternae is associated with spherical, membrane-bounded bodies which may be involved in the progressive degeneration of cisternal membranes which release wall fragments. Protoplast movement has been detected by time-lapse cinephotomicrography and is correlated at the ultrastructural level with change in positions of the Golgi cisternae. Wall-synthesizing capacity is greatest during transverse wall formation. Senescent cells lack a Golgi apparatus with inflated cisternae. In addition, wall fragments are not present in the Golgi cisternae at this stage. Zoosporogenesis results in a temporary loss of the wall-forming capacity of the Golgi apparatus; this activity then resumes with the formation of a different morphological entity, the scale. Preliminary quantitative measurements of the turnover capacity of the Golgi apparatus have been made. From these data it has been determined that between 41 and 82 Golgi generations are required to synthesize the cell wall of an actively growing cell; this estimate indicates that approximately one cisterna is produced every 2 min, provided the cell generation time is 3 days. The time-lapse cinephotomicrographic data confirm that the rate of production of Golgi cisternae is at least one cisterna every 2 min.
PLASMA MEMBRANES OF THE RAT LIVER
Authors: Song C. S. , Rubin W. , Rifkind A. B. , Kappas A.
Published: May 2004

A method is described for the rapid isolation of a plasma membrane fraction containing a high concentration of intact bile canaliculi from the rat liver. Isolated bile canaliculi retain most of the ultrastructural features exhibited in the intact liver cell. The final fraction contains 5'-nucleotidase activity at approximately the same concentration as that in previous preparations of plasma membranes. In the presence of 0.01 M Mg++, 5'-nucleotidase exhibits a double pH optimum at pH values of 7.5 and 9.5. The activities of glucose-6-phosphatase and alkaline phosphatase are present in low amounts. Cytochrome P-450 is not detectable. Na+-K+-activation of ATPase is observed to the extent of 20–36% in about half of the assays. The availability of a method for preparation of intact bile canaliculi should prove useful for studying the biochemical events associated with the transport of bile constituents into canaliculi.
ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS
Authors: Howell S. L. , Fink C. J. , Lacy P. E.
Published: May 2004

A method has been devised for the isolation of a secretory granule fraction from isolated rat islets of Langerhans. The islets were homogenized in buffered sucrose, and the homogenate was separated into nuclear, mitochondrial, secretory granule, and microsomal fractions by differential centrifugation. The secretory granule fraction was purified by differential centrifugation in discontinuous sucrose density gradients. A greater degree of purification could be achieved by the use of two successive gradients of this type, although the final yield was greatly reduced. Biochemical and morphological characterization of the fractions was obtained; the secretory granule fraction contained both insulin and glucagon. The limiting membranes of the granules remained intact and the general appearance of the granules was similar to that seen within the whole islet cells.
THE REGULATION OF RNA SYNTHESIS AND PROCESSING IN THE NUCLEOLUS DURING INHIBITION OF PROTEIN SYNTHESIS
Authors: Willems Margherita , Penman Maria , Penman Sheldon
Published: May 2004

The effect of protein synthesis inhibition by cycloheximide on nucleolar RNA synthesis and processing has been studied in HeLa cells. Synthesis of 45S RNA precursor falls rapidly after administration of the drug. However, the nucleolar content of 45S RNA remains relatively constant for at least 1 hr because the time required for cleavage of the precursor molecule into its products is lengthened after treatment with cycloheximide. The efficiency of transformation of 45S RNA to 32S RNA remains constant with approximately one molecule of the 32S RNA produced for each cleavage of a molecule of 45S RNA. However, shortly after the cessation of protein synthesis the formation of 18S RNA becomes abortive. The amount of 32S RNA present in the nucleolus remains relatively constant. After long periods of protein synthesis inhibition the 28S RNA continues to be synthesized and exported to the cytoplasm but at a greatly reduced rate. When the protein synthesis inhibitor is removed, a prompt, although partial, recovery in the synthesis rate of 45S RNA occurs. The various aspects of RNA synthesis regulation and processing are discussed.
PANETH AND GOBLET CELL RENEWAL IN MOUSE DUODENAL CRYPTS
Authors: Troughton W. David , Trier Jerry S.
Published: May 2004

Proliferation of Paneth and goblet cells of mouse duodenal crypts was studied by high resolution light microscope radioautography. In one group of mice, blood levels of thymidine-3H were sustained for up to 12 hr by repeated injections of isotope to facilitate identification of proliferating cells. In these animals, many goblet cell nuclei incorporated thymidine-3H whereas only 1 of 6261 tabulated Paneth cells was labeled. Cells intermediate in structure between undifferentiated and goblet cells and between undifferentiated and Paneth cells were identified and their light and electron microscopic features are described. A significant number of these "intermediate" cells incorporated thymidine-3H into their nuclei. Another group of mice received a single injection of thymidine-3H. These animals were killed 4 hr to 29 days after isotope administration. Goblet cells and intermediate cells with labeled nuclei were identified 4 hr after thymidine-3H but could not be seen after 15 days. In contrast, Paneth cells with labeled nuclei were not observed until 24 hr after thymidine-3H but were still present at 29 days, long after labeled undifferentiated, goblet, and intermediate cells had disappeared. We conclude that differentiated Paneth cells in mouse duodenum do not normally proliferate, but, instead, arise by differentiation from undifferentiated crypt cells or from intermediate cells. Moreover, once formed, Paneth cells persist in crypts for a prolonged period. In contrast, intermediate cells and crypt goblet cells proliferate actively and are less stable cell populations than differentiated Paneth cells. The precise function of the intermediate cells is not known, but they may represent transition forms between undifferentiated cells and the more matrure secretory cells. Damage of crypt epithelial cells, thought to be due to radiation effects, was evident in both groups of mice.
REFORMATION OF NUCLEOLI AFTER ETHIONE-INDUCED FRAGMENTATION IN THE ABSENCE OF SIGNIFICANT PROTEIN SYNTHESIS
Authors: Shinozuka Hisashi , Farber Emmanuel
Published: May 2004

The rat liver nucleolus, after fragmentation induced by ethionine treatment, has been found to undergo complete reformation by adenine in the presence of a dose of cycloheximide sufficient to cause inhibition of protein synthesis by 90–95%. In contrast, actinomycin D given along with adenine was followed by the appearance of a small compact mass containing only the fibrillar component with no evident granules. This structure resembled pseudonucleoli seen in the anucleolate mutant of Xenopus laevis or in certain early stages of amphibian oocytes. Actinomycin D administered 2 hr after adenine induced a segregation of the fibrillar and granular components of nucleoli similar to that induced in the normal nucleolus. The implications of these findings in relation to nucleolar organization are briefly discussed.
VESICLES ASSOCIATED WITH CALCIFICATION IN THE MATRIX OF EPIPHYSEAL CARTILAGE
Authors: Anderson H. Clarke
Published: May 2004

Vesicles have been identified within the cartilage matrix of the upper tibial epiphyseal plate of normal mice. They were seen at all levels within the plate and usually did not appear to be in contact with cartilage cells. Vesicles were concentrated within the matrix of the longitudinal septa from the proliferative zone downward. They varied considerably in size (∼300 A to ∼1 µ) and in shape. They were bounded by unit membranes, and contained materials of varying density including, rarely, ribosomes. A close association was demonstrated between matrix vesicles and calcification: in the lower hypertrophic and calcifying zones of the epiphysis, vesicles were found in juxtaposition to needle-like structures removed by demineralization with ethylenediaminetetraacetate and identified by electron diffraction as hydroxyapatite and/or fluorapatite crystal structure—the former being indistinguishable from the latter for most cases in which electron diffraction methods are employed. Decalcification also revealed electron-opaque, partially membrane-bounded structures within previously calcified cartilage of the epiphyseal plate and underlying metaphysis which corresponded in size and distribution to matrix vesicles. It is suggested that matrix vesicles are derived from cells and that they may play a role in initiating calcification at the epiphysis.
THE HUMAN CHROMOSOME
Authors: Abuelo J. G. , Moore Dorothy E.
Published: May 2004

Human lymphocytes were grown in short-term tissue culture and were arrested in metaphase with Colcemid. Their chromosomes were prepared by the Langmuir trough-critical point drying technique and were examined under the electron microscope. In addition, some chromosomes were digested with trypsin, Pronase, or DNase. The chromosomes consist entirely of tightly packed, 240 ± 50-A chromatin fibers. Trypsin and Pronase treatments induce relaxation of fiber packing and reveal certain underlying fiber arrangements. Furthermore, trypsin treatment demonstrates that the chromatin fiber has a 25–50 A trypsin-resistant core surrounded by a trypsin-sensitive sheath. DNase digestion suggests that this core contains DNA.
EFFECT OF THE RUBY LASER MICROBEAM ON MITOCHONDRIA OF KB CELLS SUPRAVITALLY STAINED BY PINACYANOL
Authors: Tanaka Yasukazu
Published: May 2004

With pinacyanol as the supravital stain, a preferential effect on mitochondria of KB cells was achieved by the irradiation with the ruby laser beam. The observation confirmed the results of other workers using janus green B in the same experimental system. The preferential effect on mitochondria was noted in the area extending 8–10 µ beyond the nonpreferential damage of 4–5 µ in diameter. The opaque material associated with mitochondria possibly represented coagulated protein. The effect involved cristae mitochondriales without severe disarrangement of their structure. The opaque material could be interpreted as the result of direct interaction between mitochondria and the laser beam, even though the mitochondria were noted outside of the previously estimated focal spot size of about 3 µ Within the thickness of 2–4 µ of monolayered cells, larger areas of damage can be accounted for by divergence of the beam which is focused by a microscope objective of very short focal length. A threshold of biologic effectiveness is probably also involved.
AN ELECTRON MICROSCOPIC CYTOCHEMICAL STUDY OF MACROPHAGES DURING UTERINE INVOLUTION
Authors: Brandes David , Anton Elsa
Published: May 2004

Acid phosphatase was localized at the fine structural level in rat endometrial phagocytes during the period of postpartum involution. These cells showed intense phagocytotic and pinocytotic activities, which were accompanied by the development of abundant lysosomes. Phagosomes acquired their enzymatic complement by fusion with lysosomes; the same appeared to be true in the case of pinocytotic vesicles, but, because of the small size of these vesicles, this point could not be established with certainty. Digestion within some phagolysosomes led to the formation of electron-lucent vacuoles containing solubilized products. Other phagolysosomes showed accumulation of lipid residues in the form of droplets and myelin figures, and the structures acquired the appearance of residual bodies. In many macrophages, overfeeding led to the formation of unusually large numbers of phagolysosomes, which occupied almost the entire cytoplasm with exclusion of other cell organelles. In these cells the presence of abundant lead deposits, apparently free in the cytoplasm suggested an intracytoplasmic release of hydrolases.
EFFECTS OF ACRIFLAVINE ON THE MITOCHONDRIA AND KINETOPLAST OF CRITHIDIA FASCICULATA
Authors: Hill George C. , Anderson Winston A.
Published: May 2004

The effects of acriflavine on the fine structure and function of the mitochondria and the kinetoplast in Crithidia fasciculata have been investigated. A mitochondrial fraction was prepared by differential centrifugation of cells broken by grinding with neutral alumina. Isolated mitochondria or intact cells revealed by spectrophotometric measurements the presence of cytochromes a + a3, b, c555 and o. After cells were grown in acriflavine for 3–4 days, the fine structure of the mitochondria and their cytochrome content were affected. Cells grown in 5.0 µM acriflavine had a threefold decrease in cytochrome a + a3 and decreased respiratory activity. The mitochondrial preparation from these cells had a fivefold decrease in cytochrome a + a3 and a less but significant decrease of other cytochromes present. There was also a decrease in the mitochondrial enzyme activities of NADH, succinic and L-α-glycerophosphate oxidases, and succinic and L-α-glycerophosphate dehydrogenases. Dyskinetoplastic cells could be demonstrated after growth in 1.0 µM acriflavine. At 5 µM, 80–90% of the cells were dyskinetoplastic. The kinetoplastic DNA was condensed, nonfibrillar, and did not incorporate thymidine-3H. The mitochondria in these cells had few cristae and were shorter and more swollen than the controls. Acriflavine may induce the fine structure effects we have observed and may affect the formation of the mitochondria in C. fasciculata.
PATHS OF TRANSTUBULAR WATER FLOW IN ISOLATED RENAL COLLECTING TUBULES
Authors: Grantham Jared J. , Ganote Charles E. , Burg Maurice B. , Orloff Jack
Published: May 2004

The cells of perfused rabbit collecting tubules swell and the intercellular spaces widen during osmotic flow of water from lumen to bath induced by antidiuretic hormone (ADH). Ouabain had no influence on these changes. In the absence of net water flow intercellular width was unaffected when tubules were swollen in hypotonic external media. Therefore, during ADH-induced flow widening of intercellular spaces is not a consequence of osmotic swelling of a closed intercellular compartment containing trapped solutes, but rather is due to flow of solution through the channel. Direct evidence of intercellular flow was obtained. Nonperfused tubules swollen in hypotonic media were reimmersed in isotonic solution with resultant entry of water into intercellular spaces. The widened spaces gradually collapsed completely. Spaces enlarged in this manner could be emptied more rapidly by increasing the transtubular hydrostatic pressure difference. In electron micrographs a path of exit of sufficient width to accommodate the observed rate of fluid flow was seen at the base of the intercellular channel. It is concluded that the intercellular spaces communicate with the external extracellular fluid and that water, having entered the cells across the luminal plasma membrane in response in ADH, leaves the cells by osmosis across both the lateral and basilar surface membranes.
DYNAMIC CHANGES IN THE ULTRASTRUCTURE OF THE ACINAR CELL OF THE RAT PAROTID GLAND DURING THE SECRETORY CYCLE
Authors: Amsterdam Avraham , Ohad Itzhak , Schramm Michael
Published: May 2004

Synchronization of the secretory cycle in vivo was obtained by injecting isoprenaline as an inducer of secretion. A quantitative correlation between enzyme release, its subsequent reaccumulation, and the sequence of ultrastructural changes was found. At the ultrastructural level secretion was paralleled by depletion of zymogen granules through fusion of the granule membrane with the lumen membrane and discharge of the content. Each zymogen granule membrane, once connected with the lumen, acted as a lumen membrane. Fusion was thus sequential and resulted in a dramatic enlargement of the lumen space. During the entire process the passage between the lumen and the intercellular space remained blocked by the tight junctions, as shown by their impenetrability to ferritin. Reduction of the lumen size following enzyme discharge seemed to be achieved by withdrawal of lumen membrane in the form of small smooth vesicles which appeared mostly in the apical part of the cell. At the same time, the cell retracted towards the lumen, the whole process being completed within 2 hr from onset of secretion. Disappearance of the smooth vesicle followed, concomitant with formation of many condensing vacuoles and appearance of mature zymogen granules. The fate of the zymogen granule membrane, including its fusion with the lumen membrane, resorption in the form of small smooth vesicles, and its eventual reutilization mediated by the Golgi system, is discussed.
THE FINE STRUCTURE OF ACANTHAMOEBA CASTELLANII (NEFF STRAIN)
Authors: Bowers Blair , Korn Edward D.
Published: May 2004

Encysting cells of Acanthamoeba castellanii, Neff strain, have been examined with the electron microscope. The wall structure and cytoplasmic changes during encystment are described. The cyst wall is composed of two major layers: a laminar, fibrous exocyst with a variable amount of matrix material, and an endocyst of fine fibrils in a granular matrix. The two layers are normally separated by a space except where they form opercula in the center of ostioles (exits for excysting amebae). An additional amorphous layer is probably present between the wall and the protoplast in the mature cyst. Early in encystment the Golgi complex is enlarged and contains a densely staining material that appears to contribute to wall formation. Vacuoles containing cytoplasmic debris (autolysosomes) are present in encysting cells and the contents of some of the vacuoles are deposited in the developing cyst wall. Lamellate bodies develop in the mitochondria and appear in the cytoplasm. Several changes are associated with the mitochondrial intracristate granule. The nucleus releases small buds into the cytoplasm, and the nucleolus decreases to less than half its original volume. The cytoplasm increases in electron density and its volume is reduced by about 80%. The water expulsion vesicle is the only cellular compartment without dense content in the mature cyst. The volume fractions of lipid droplets, Golgi complex, mitochondria, digestive vacuoles, and autolysosomes have been determined at different stages of encystment by stereological analysis of electron micrographs. By chemical analyses, dry weight, protein, phospholipid, and glycogen are lower and neutral lipid is higher in the mature cyst than in the trophozoite.
ENZYMATIC AND CHROMOSOMAL CHARACTERIZATION OF HELA VARIANTS
Authors: Bottomley R. H. , Trainer A. L. , Griffin M. J.
Published: May 2004

Seven strains of HeLa cells have been characterized by the number of chromosomes and the activity of the enzymes alkaline phosphatase, glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, and lactic dehydrogenase. All seven strains were found to differ as to chromosome numbers and enzyme levels despite the fact that two strains were called HeLa and three were called HeLa S3. Three strains were found to have a stemline in which greater than 60% of the cells demonstrated a single chromosome number, and this characteristic was stable for at least 6 months. A nomenclature for these clones has been suggested by the use of the stemline chromosome number as a subscript following HeLa. These three clones were, therefore, designated HeLa65, HeLa71, and HeLa75. Karyotypes were made of the stemlines of these clones and were compared with enzyme levels. Alkaline phosphatase showed the greatest variation from cell line to cell line with a 200-fold difference in levels, whereas glucose-6-phosphate dehydrogenase showed variation in activity over a 12-fold range, lactic dehydrogenase over an 8-fold range, and 6-phosphogluconic dehydrogenase over a 2-fold range. It is suggested that human cell strains can be used for biochemical studies if they are cloned and if the clones are relatively stable at least with respect to modal chromosome number and karyotype.
MITOTIC AND NONMITOTIC MULTIPLE-LAYERED PERFUSION CULTURES
Authors: Kruse Paul F. , Whittle Wilbur , Miedema Ed
Published: May 2004

Cell types in addition to those previously described (Kruse et al. 1963. J. Nat. Cancer Inst. 31:109; Kruse and Miedema. 1965. J. Cell Biol. 27:273) were found to form multiple-layered cultures by perfusion-culture technique. Dense populations containing 43 x 106 embryonic rat muscle (NF-ER) cells, 23 x 106 diploid human tonsillar (NF-JAM) cells, 77 x 106 human pleural effusion isolate (RPMI 2650) cells, 35 x 106 embryonic diploid human lung (Flow 2000) cells, 21 x 106 bovine lung (FB4BM) cells, 108 x 106 bat lung (Tb1Lu) cells, and 81 x 106 SV-40 virus-transformed embryonic diploid human lung (WI-38VA13A) cells were obtained in 6–14 days from dilute inocula in T-60 or T-75 flasks; these were equivalent to about 4, 3, 3, 4, 2, 4, and eight monolayers, respectively. Perfusion of an NF-ER culture for 6 wk with medium plus 10% whole calf serum yielded a cell density equivalent to 12 monolayers (140 x 106 cells per T-75 flask). This culture exhibited random labeling of nuclei from bottom to top after pulsing for 90 min with thymidine-3H. Medium plus 0.1% serum maintained NF-JAM cultures at constant viable cell numbers with virtual absence of thymidine-3H labeling. Similar results were obtained with WI-38 cultures, but WI-38VA13A cells continued active DNA synthesis and mitosis in medium with 0.1% serum to form 16–20 layers of cells (191–239 x 106 cells per T-75 flask) in 27 days. WI-38VA13A cells ceased proliferation and became nonviable rapidly in serumless medium.
A FINE STRUCTURAL ANALYSIS OF CLEAVAGE INDUCTION AND FURROWING IN THE EGGS OF ARBACIA PUNCTULATA
Authors: Tilney Lewis G. , Marsland Douglas
Published: May 2004

A fine structural study has been carried out on the various formed elements present before, during, and after the first cleavage division, not only in normally developing Arbacia eggs, but also in eggs which have been induced to cleave prematurely by high-pressure centrifugation. The aim has been to ascertain whether or not any of the morphologically identifiable components may be involved in initiating the furrowing process. Also, attention has been given to the fine structure of the cytoplasmic cortex, particulary in the walls of the furrow, in the hope of reaching a better understanding of the mechanics of cleavage. The annulate lamellae and the membranous envelope of the nucleus are the only formed elements which disappear shortly before cleavage, not only in eggs undergoing normal division, but also in eggs which have been induced to cleave ahead of schedule by high-pressure, high-force centrifugation. Therefore, it is suggested as a tentative hypothesis that materials liberated upon disintegration of the nuclear membrane and the annulate lamellae play an essential role in initiating and effecting the furrowing reaction, especially since the stratification of these elements in experimentally induced eggs corresponds to the position of the developing furrow. Another of the membranous elements in the egg, the Golgi complex, shows considerable modification as a result of high-pressure centrifugation, but these structures do not undergo disintegration. Rather, they become curled into rounded bodies. The vacuole population is not greatly affected by inducing treatments. During cleavage, both naturally occurring and experimentally induced, a considerable number of 50 A filaments appear in the denser cytoplasmic cortex, but only in the walls of the furrow. These filaments are similar to those which have been demonstrated in a number of contractile cells. Accordingly, it is suggested that this fibrillar system may be actively involved in the development of the cleavage force.
ION-INDUCED ULTRASTRUCTURAL TRANSFORMATIONS IN ISOLATED MITOCHONDRIA
Authors: Hackenbrock Charles R. , Caplan Arnold I.
Published: May 2004

The energized uptake of low levels of Ca2+ in the presence and absence of phosphate by isolated rat liver mitochondria, and the perturbation effected by this activity on ultrastructural and metabolic parameters of mitochondria have been investigated. In the presence of phosphate, low levels of Ca2+ are taken up by mitochondria and result in various degrees of ultrastructural expansion of the inner mitochondrial compartment. This indicates that low levels of Ca2+ in the presence of phosphate, are accumulated in an osmotically active form into the water phase of the inner compartment. The first clearly observable quantitative increase in the volume of the inner compartment occurs after the accumulation of 100 nmoles Ca2+/mg protein. An accumulation of 150–200 nmoles Ca2+/mg protein, which is equivalent to the osmolar concentration of endogenous K+, is required to effect a doubling of the volume of the inner compartment. This degree of osmotic perturbation occurs as mitochondria transform from a condensed to an orthodox conformation. The osmotically induced orthodox conformation differs from the mechanochemically induced orthodox conformation previously described, in that its development is concomitant with a marked decrease in acceptor control and oxidative phosphorylation efficiency and it fails to transform to a condensed conformation in response to addition of ADP. In the absence of added phosphate, a maximum of 190 nmoles Ca2+/mg protein was found to be taken up by mitochondria (state 6). Ca2+ is apparently bound under state 6 conditions since the uptake does not effect an ultrastructural expansion of the inner compartment. Phosphate added after state 6 Ca2+ binding, however, results in an immediate ultrastructural expansion of the inner compartment. The addition of phosphate to mitochondria in the absence of exogenous Ca2- fails to effect an osmotic ultrastructural transformation. Under state 6 conditions, the binding of between 40 and 190 nmoles Ca2+/mg protein results in the formation of dense matrix inclusions which appear to be composed of tightly packed, concentrically oriented membranes. Under conditions in which the bound Ca2+ is subsequently released, there is a concomitant loss in the density of these matrix inclusions, leaving behind morphologically distinct membrane whorls in the mitochondrial matrix.
THE SITE OF ORIGIN OF ELECTRICAL RESPONSES IN VISUAL CELLS OF THE LEECH, HIRUDO MEDICINALIS
Authors: Lasansky A. , Fuortes M. G. F.
Published: May 2004

In leech visual cells the presumed light-absorbing structures are microvilli arising from the membrane of what would seem to be a large intracellular vacuole. This vacuole, however, is an extracellular compartment, since it communicates with the intercellular spaces through narrow channels. Therefore, the membrane of the microvilli is—as in other invertebrate visual cells—a part of the cell membrane. Visual responses recorded with an electrode within the vacuole were compared with the intracellular recordings. Following illumination the vacuole becomes negative with respect to the outside fluid, while the cells are depolarized. This finding indicates that inward current penetrates the cell through the microvillar membrane. It is concluded, therefore, that the electrical response (receptor potential) originates as a result of changes in the properties of the light-absorbing membrane.
CHOLESTEROL REQUIREMENT OF PRIMARY DIPLOID HUMAN FIBROBLASTS
Authors: Holmes Richard , Helms Judy , Mercer Gretchen
Published: May 2004

Primary cultures of fibroblast-like cells were obtained from skin and articular cartilage of human donors in the age bracket of 1 to 15 years. For growth these cultures required 1 mg/liter of cholesterol added to Medium A2 plus acetyl choline, Na pyruvate, and D-galactosamine HCl (APG) containing 10% lipoprotein-free human serum. Established cell lines did not require cholesterol for growth. Eagle's medium could be used in place of Medium A2 plus APG with the same results. Desmosterol could replace cholesterol but lansterol or 7 dehydrocholesterol could not. Other cholesterol precursors were tested and found to be inactive. With the proviso that cholesterol precursors entered the cell and had to be converted to cholesterol to function, it was concluded that the particular primaries studied lacked a functional enzyme system to reduce the double bond at carbon 7.
RIBOSOME PRECURSOR PARTICLES IN NUCLEOLI
Authors: Liau Ming C. , Perry Robert P.
Published: May 2004

Ribonucleoprotein (RNP) particles containing the precursors of ribosomal RNA were extracted from L cell nucleoli and analyzed under conditions comparable to those used in the characterization of cytoplasmic ribosomes. Using nucleoli from cells suitably labeled with 3H-uridine, we detected three basic RNP components, sedimenting at approximately 62S, 78S, and 110S in sucrose gradients containing magnesium. A fourth particle, sedimenting at about 95S, appears to be a dimer of the 62S and 78S components. When centrifuged in gradients containing EDTA, the 62S, 78S, and 110S particles sediment at about 55S, 65S, and 80S, respectively. RNA was extracted from RNP particles which were prepared by two cycles of zonal centrifugation. The 62S particles yielded 32S RNA and a detectable amount of 28S RNA, the 78S structures, 32S RNA and possibly some 36S RNA, and the 110S particles, a mixture of 45S, 36S, and 32S RNA's. When cells were pulsed briefly and further incubated in the presence of actinomycin D, there was a gradual shift of radioactivity from heavier to lighter particles. This observation is consistent with the scheme of maturation: 110S → 78S → 62S. The principal buoyant densities in cesium chloride of the 110S, 78S, and 62S particles are 1.465, 1.490, and 1.545, respectively. These densities are all significantly lower than 1.570, which is characteristic of the mature large subunit of cytoplasmic ribosomes, suggesting that the precursor particles have a relatively higher ratio of protein to RNA, and that ribosome maturation involves, in addition to decrease in the size of the RNA molecules, a progressive decrease in the proportion of associated protein.
CELL KILLING BY ACTINOMYCIN D IN RELATION TO THE GROWTH CYCLE OF CHINESE HAMSTER CELLS
Authors: Elkind M. M. , Kano E. , Sutton-Gilbert H.
Published: May 2004

Using Chinese hamster cells in culture, we have measured the effectiveness of actinomycin D to suppress division as a function of the position, or age, of a cell in its growth cycle. Cells were first exposed to millimolar concentrations of hydroxyurea in order to produce a synchronized population just before the onset of DNA synthesis. Thereafter, the survival response after 30 min exposures to actinomycin D was measured. Cells become resistant as they enter the S phase and then sensitive again in the latter part of S. When they reach G2 (or G2-mitosis) they are maximally resistant; at 1.0 µg/ml, for example, the survival in G2 is 30-fold greater than it is in G1. These results, plus measurements reported earlier on the interaction of damage in S cells due to actinomycin D and X-irradiation, suggest that the age-response pattern of the toxic effects of this drug probably reflects both the functional capacity of DNA-actinomycin complexes and the ability of this antibiotic to penetrate chromatin and bind to DNA.
CHLOROPLASTS AS FUNCTIONAL ORGANELLES IN ANIMAL TISSUES
Authors: Trench Robert K. , Greene Richard W. , Bystrom Barbara G.
Published: May 2004

The marine gastropod molluscs Tridachia crispata, Tridachiella diomedea, and Placobranchus ianthobapsus (Sacoglossa, Opisthobranchia) possess free functional chloroplasts within the cells of the digestive diverticula, as determined by observations on ultrastructure, pigment analyses, and experiments on photosynthetic capacity. In the light, the chloroplasts incorporate H14CO3- in situ. Reduced radiocarbon is translocated to various chloroplast-free tissues in the animals. The slugs feed on siphonaceous algae from which the chloroplasts are derived. Pigments from the slugs and from known siphonaceous algae, when separated chromatographically and compared, showed similar components. Absorption spectra of extracts of slugs and algae were very similar. The larvae of the slugs are pigment-free up to the post-veliger stage, suggesting that chloroplasts are acquired de novo. with each new generation.
A SYMMETRICAL, EXTRACELLULAR FIBRIL
Authors: Bruns Romaine R.
Published: May 2004

Symmetrical, extracellular fibrils, which are related to the "special fibrils" of the dermis described by Palade and Farquhar, have been found along the outer surface of the basement membrane covering the notochord in the tail of Rana catesbeiana (bullfrog) tadpoles. The fibrils are ∼7,500 A long and occur singly or in clusters. The single fibrils are characterized by a symmetrical transverse band pattern and by attachment at both ends to the basement membrane. The clusters are various complex configurations which seemingly represent symmetrical fibrils in different states of aggregation. Symmetrical fibrils also occur in the skin of the tadpole tail and in the skin of the toad, Bufo marinus. It is proposed that a narrow, symmetrical fibril is the fundamental "special fibril."
DEVELOPMENT OF THE NEUROMUSCULAR JUNCTION
Authors: Lentz Thomas L.
Published: May 2004

Development of the neuromuscular junction on differentiating muscle was investigated in the regenerating limb of the newt Triturus. Motor end-plate formation begins when vesicle-filled axon terminations approach differentiating muscle cells that have reached the stage of a multinucleate cell containing myofibrils. Slight ridges or elevations occur on the muscle surface, and there is an increase in density of the cytoplasm immediately beneath the plasma membrane of the elevation. The axon becomes more closely approximated to the muscle cell and comes to lie in a shallow depression or gutter on the surface of the muscle. The surface ridges increase in length and constrict at their bases to form junctional folds. In the axon terminal, focal accumulations of vesicles are found where the axon contour projects slightly opposite the secondary synaptic clefts. Cholinesterase activity in the developing junctions was demonstrated by the thiolacetic acid-lead nitrate method. Enzymatic activity is not found on intercellular nerve fibers or the muscle surface prior to close approximation of axon endings and muscle. Eserine- and DFP-sensitive activity appears concurrently with morphological differentiation. Activity occurs in membranous tubulovesicles in the sarcoplasm subjacent to the neuromuscular junction and in association with the sarcolemma. The largest reaction deposits occur at the tips of the emerging junctional folds. Smaller and less numerous localizations occur on the axon membrane and within the axoplasm. It is concluded from these studies that the nerve endings have an inductive effect on both the morphological and chemical specializations of the neuromuscular junction.
THE ISOLATION OF LYSOSOMES FROM EHRLICH ASCITES TUMOR CELLS FOLLOWING PRETREATMENT OF MICE WITH TRITON WR-1339
Authors: Horvat Agnes , Baxandall Jane , Touster Oscar
Published: May 2004

A method is described for obtaining highly purified lysosomes from Ehrlich ascites tumo cells grown in mice injected with Triton WR-1339. The isolated particles show a high specific activity for aryl sulfatase, representing an 80–90-fold purification over the homogenate, and a 15–18% yield of the total enzyme activity. Mitochondrial and microsomal marker enzymes are present in negligible amounts (0.2% of the activity of the homogenate). The biochemical evidence for a rather high degree of homogeneity of the fraction is supported by the electron microscopic examination of the purified lysosomes. The intracellular localizations of N-acetyl-ß-glucosaminidase, NADH-cytochrome c reductase and NADPH-cytochrome c reductase in Ehrlich ascites cells are also reported, the first two being present in highest concentration in the combined mitochondrial-lysosomal fraction and the third in the microsomal fraction.
THE USE OF BEEF LIVER CATALASE AS A PROTEIN TRACER FOR ELECTRON MICROSCOPY
Authors: Venkatachalam M. A. , Fahimi H. Dariush
Published: May 2004

Beef liver catalase was injected intravenously into mice, and its distribution in the kidney, myocardium, and liver was studied with the electron microscope. A specific and relatively sensitive method was developed for its ultrastructural localization, based on the peroxidatic activity of catalase and employing a modified Graham and Karnovsky incubation medium. The main features of the medium were a higher concentration of diaminobenzidine, barium peroxide as the source of peroxide, and pH of 8.5. Ultrastructurally, the enzyme was seen to permeate the endothelial fenestrae and basement membranes of tubular and glomerular capillaries of the kidney. The urinary space and tubular lumina contained no reaction product. In the myocardial capillaries, the tracer filled the pinocytotic vesicles but did not diffuse across the intercellular clefts of the endothelium. In liver, uptake of catalase was seen both in hepatocytes and in Kupffer cells.
SARCOPLASMIC RETICULUM OF AN UNUSUALLY FAST-ACTING CRUSTACEAN MUSCLE
Authors: Rosenbluth Jack
Published: May 2004

The fast-acting, synchronous "remotor" muscle of the lobster second antenna was examined by light and electron microscopy and was found to have a more profuse sarcoplasmic reticulum (SR) than any other muscle known. Myofibrils are widely separated from one another and occupy only about one-fourth of the volume of the muscle; most of the remaining volume is taken up by the SR, which resembles the smooth-surfaced reticulum of steroid-secreting cells. Dense granules (0.03–0.1 µ in diameter) are scattered through the reticulum. T-tubules penetrate into the fibers and form dyads along the A bands of myofibrils; however, ferritin-labeling experiments show that the volume of the T-system is very small compared with that of the SR. Myofibrils are ∼0.5 µ x 1.0 µ in cross section and consist of thick filaments, which appear tubular except at the M region, and thin filaments, which are situated midway between neighboring thick filaments. The ratio of thin to thick filaments is 3:1. The extreme development of the SR in this muscle is discussed in relation to the exceedingly short duration of the contraction-relaxation cycle.
BETA GRANULE FORMATION IN ISOLATED ISLETS OF LANGERHANS
Authors: Howell S. L. , Kostianovsky M. , Lacy P. E.
Published: May 2004

The distribution of radioautographic grains over organelles within the beta cells of rat islets of Langerhans was investigated at various times after pulse labeling of the isolated islets with tritium-labeled amino acids. Ten minutes after the start of labeling most of the grains were situated over the endoplasmic reticulum and cytoplasm; by contrast, 60 min from the start of labeling the majority of the grains were associated with the beta granules. At 20, 30, and 45 minutes after pulse labeling the proportion of grains associated with the Golgi complex was increased two- to three-fold over the 10- or 60-minute values. The distribution of radioautographic grains over granules in the intact cells did not suggest that the electron-lucent type of secretory granules were precursors of the electron-opaque granules. Furthermore, studies of the pattern of grains over granules isolated by centrifugation 60 min after pulse labeling showed no preferential labeling of the electron-lucent type of granule. It is concluded that labeled amino acids are incorporated initially in the endoplasmic reticulum, and that the label subsequently appears in the beta granules. The Golgi complex participates either in the formation of the beta granule or in the translocation of the granule through the cytoplasm of the cell.
RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS
Authors: Hendler Richard W. , Burgess Amelia H. , Scharff Raymond
Published: May 2004

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates: nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and α-ketoglutarate. Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide. Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble. It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E. coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme. The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control.
THE DISTRIBUTION OF THE WATER-SOLUBLE INORGANIC ORTHOPHOSPHATE IONS WITHIN THE CELL: ACCUMULATION IN THE NUCLEUS
Authors: Libanati Cesar M. , Tandler Carlos J.
Published: May 2004

Lead acetate treatment of unfixed cells immobilizes the intracellular water-soluble, inorganic orthophosphate ions as microcrystalline lead hydroxyapatite precipitates (see reference 1). These precipitates have been analyzed with the electron microprobe. A much higher concentration of phosphorus has been found in the nucleoli of maize root tip cells fixed in lead acetate-glutaraldehyde (organic phosphorus plus inorganic orthophosphate), as compared to the nucleoli of roots fixed in glutaraldehyde alone (organic phosphorus). The concentration of the inorganic orthophosphate pool in these nucleoli is three to five times as high as the concentration of the macromolecular organic phosphate. Since nearly all of the latter is in RNA, the concentration of inorganic phosphate in the nucleolus is calculated to be roughly 0.5–0.8 M. About 30%—and up to 50%—of the total cellular inorganic phosphate is accumulated in the nucleolus since the mean concentration per cell is about 10-2 M. In the extranucleolar part of the nucleus the mean concentration was estimated by densitometry to be roughly six times less than in the nucleolus (⩽ 0.1 M), and appears more concentrated in the nucleoplasm than in the condensed chromatin. While there is no direct evidence for the concentration in the cytoplasm, it certainly must be much lower than the mean cellular level (i.e., < 10-2 M) since the nucleus is about 10% of the total cell volume. The implications of this compartmentation in the intact cell are discussed in connection with (A) the availability of orthophosphate ions for the cytoplasm in those processes in which these ions affect the rate of enzymatic reactions, and (B) protein nucleic acid interactions within the nucleus and nucleolus.
CHOLINE METABOLISM AND MEMBRANE FORMATION IN RAT HEPATOMA CELLS GROWN IN SUSPENSION CULTURE
Authors: Plagemann Peter G. W.
Published: May 2004

The incorporation of methyl-labeled choline into phosphorylcholine and phosphatidylcholine of cellular membranes by Novikoff rat hepatoma cells (line N1S1-67) during growth in suspension culture was investigated. Upon initiation of a fresh culture at 105 cells/ml, the rate of synthesis of phosphorylcholine by the cells was four to five times greater than that of the synthesis of phosphatidylcholine. While the rate of synthesis of the latter remained relatively constant, the rate of phosphorylation of choline decreased progressively during the course of the growth cycle of the culture to 10–20% of the initial rate when the culture reached stationary phase at 3 x 106 cells/ml. The decrease in phosphorylcholine synthesis during the growth cycle was not due to depletion of choline in the medium or a decrease in its concentration, but was correlated with a decrease in choline kinase activity of the cells as measured in cell-free extracts. Newly synthesized phosphatidylcholine was detectable in cells only as an integral part of cellular membranes. Its distribution among various cytoplasmic membrane structures separated by isopycnic centrifugation in sucrose density gradients remained relatively constant during the growth cycle. About 50% was associated with the mitochondria, and the remainder with plasma membrane fragments and other membranous structures with mean densities of about 1.15 and 1.13 g/cm3, respectively. However, the density of the mitochondria increased from about 1.167 g/cm3 in early exponential phase cells to about 1.190 g/cm3 in stationary phase cells. The finding that the density of the entire propulation of mitochondria changed simultaneously and progressively is in agreement with the view that mitochondria grow by addition of phospholipids and structural proteins and increase in number by division.