bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the synthesis and intracellular transport of lipoprotein particles in rat liver.
Authors: Glaumann H , Bergstrand A , Ericsson J L
Published: May 2004

Lipoprotein particles (d less than 1.03 g/ml) were isolated from rough and smooth microsomes and from the Golgi apparatus of rat liver, and were characterized chemically and morphologically. The rough endoplasmic reticulum (ER) particles were rich in protein (50%) and contained phospholipids (PLP) and triglycerides (TG) in smaller amounts, whereas the lipoprotein particles emanating from the smooth ER, and especially the Golgi apparatus, were rich in TG and PLP, resembling very low density lipoproteins (VLDL) of serum. The difference in chemical composition among the particles was associated with change in size both in situ and in isolated lipoprotein fractions. The rough ER particles were 200-800 A in diameter (mean similar to 420 A); the smooth er particles 200-900 A (mean similar to 520 A); the Golgi particles 350-950 A (mean similar to 580A); and serum VLDL 300-800 A (mean similar to 450 A). Generally, lipoprotein particles were rare in the rough ER, frequent but diffusely dispersed in smooth ER, and occurring mainly in clusters in "secretory vesicles" of the Golgi complex. They were seldom observed in the cisternal compartments of the Golgi complex. At short intervals (less than 15 min), intravenously injected radioactive glycerol was preferentially channelled into TG, whereas at later time points the majority of the isotope was recovered in the PLP. Three TG pools were distinguished: (a) a cytoplasmic pool with a slow turnover rate; (b) a membrane-associated TG pool; and (c) a pool corresponding to the TG moiety of lipoprotein particles, which showed the highest initial rate of labeling and fastest turnover. When, after pulse labeling, the appearance of incorporation of radioactive glycerol into TG or PLP of isolated lipoproteins was followed from one subcellular fraction to the other, a sequence of labeling was noted. During the first interval, TG from both rough and smooth microsomal lipoproteins displayed a high rate of labeling with peak value at 6 min, followed by a quick fall-off, while the Golgi lipoproteins reached maximal level at 10-20 min after administration. There was an interval of 10-15 min before the appearance of labeled VLDL in serum. It is concluded that the assembly of the apoproteins and lipid moieties into lipoprotein particles-presumed to be precursors of liver VLDL-begins in the rough ER and continues in the smooth ER. Also, there is a parallel change in chemical composition and size of the lipoprotein particles as they make their way through the ER and the Golgi apparatus. Some remodeling of the particles may take place in the Golgi apparatus before discharge into the circulation.
Electrophoretic analysis of liver and testis histones of the frog Rana pipiens.
Authors: Alder D , Gorovsky M A
Published: May 2004

Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3.
Cell surface changes and Rous sarcoma virus gene expression in synchronized cells.
Authors: Hale A H , Winkelhake J L , Weber M J
Published: May 2004

We have investigated whether cell surface changes associated with growth control and malignant transformation are linked to the cell cycle. Chicken embryo cells synchronized by double thymidine block were examined for cell-cycle-dependent alterations in membrane function (measured by transport of 2-deoxyglucose, uridine, thymidine, and mannitol), in cell surface morphology (examined by scanning electron microscopy), and in the ability of tumor virus gene expression to induce a transformation-specific change in membrane function. We reach the following conclusions: (a) The high rate of 2-deoxyglucose transport seen in transformed cells and the low rates of 2-deoxyglucose and uridine transport characteristic of density-inhibited cells do not occur in normal growing cells as they traverse the cell cycle. (b) Although there are cell cycle-dependent changes in surface morphology, they are not reflected in corresponding changes in membrane function. (c) Tumor virus gene expression can alter cell membrane function at any stage in the cell cycle and without progression through the cell cycle.
The basal apparatus. Mass isolation from the molluscan ciliated gill epithelium and a preliminary characterization of striated rootlets.
Authors: Stephens R E
Published: May 2004

The basal apparatus, consisting of an array of interconnected basal bodies bearing bifurcating striated rootlets encompassing a nucleus, has been isolated from hypertonically deciliated columnar gill epithelial cells of the bay scallop Aequipecten irradians through gentle lysis with Triton X-100. The rootlets, 8-10 mum in length, were not easily preserved with conventional electron microscope fixatives, suggesting that the extent of their contribution to cellular architecture has been somewhat underestimated, even though Englemann described many of the structural details of the basal apparatus in 1880. The striated rootlets were soluble at high but not at low pH, in 2 M solutions of sodium azide and potassium thiocyanate but not sodium or potassium chloride, in 1% deoxycholate but not digitonin, and in the denaturing solvents 6 M guanidine-HC1, 8 M urea, and 1% sodium dodecylsulfate at 100 degrees C. The protein found consistently when rootlets were solubilized migrated on SDS-polyacrylamide gels as a closely spaced doublet with apparent molecular weights of 230,000 and 250,000 daltons. This unique protein, distinct from tropocollagen or various muscle components, has been named ankyrin because of the rootlet's anchor-like function in the cell.
Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins.
Authors: Bonner W M
Published: May 2004

A technique is presented which enables one to measure the extent to which a protein enters and accumulates in the nucleus of the frog oocyte. In this method, the protein, labeled with 125-I, is microinjected into the oocyte. After incubation, the oocyte is manually enucleated and the radioactivity in the nucleus and cytoplasm is determined. Using this technique, proteins lighter than 20,000 daltons were found to enter the nucleus and completely equilibrate between the nucleus and cytoplasm within 24 h. The entry of proteins heavier than 69,000 daltons was severely hindered. Histones and histone fractions entered as quickly as other small proteins, but, in contrast to these proteins, they accumulated in the nucleus to different extents, depending on the total amount of histone injected into the oocyte and the identity of the histone. Evidence is presented that histone fractions compete with each other for accumulation in the nucleus.
Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells.
Authors: Hubbard A L , Cohn Z A
Published: May 2004

The fate of the L-cell plasma membrane proteins labeled by enzymatic iodination was studied. The disappearance of label from growing cells exhibits a biphasic behavior, with 5-20% lost rapidly (t1/2 similar to 2 h) and 80-90% lost relatively slowly (t1/2 similar to 25-33 h). The loss is temperature dependent and serum independent, and is accompanied by the appearance of 51% (125-I)monoiodotyrosine (MIT) in the medium by 47 h. A variable amount (1-14%) of acid-insoluble label can be recovered in the medium over 47 h. Sodium dodecyl sulfate (SDS)-polyacrylamide gel labeling patterns from cells cultured up to 48 h after iodination reveal no change in the relative distribution of radioactivity, indicating similar rates of degradation for most of the labeled membrane proteins. The fate of the labeled membrane proteins was studied at various times after phagocytosis of nondigestible polystyrene particles. Iodinated L cells phagocytose sufficient 1.1 mum latex beads in 60 min to interiorize 15-30% of the total cell surface area. Electron microscope autoradiography confirmed that labeled membrane is internalized during phagocytosis. The latex-containing phagocytic vacuoles are isolated by flotation in a discontinuous sucrose gradient. 15-30% of the total incorporated label and a comparable percentage of alkaline phosphodiesterase I activity (PDase, a plasma membrane enzyme marker) are recovered in the phagocytic vacuole fraction. Lysosomal enzyme activities are found in the latex vacuole fraction, indicating formation of phagolysosomes. SDS gel analyses reveal that all of the radioactive proteins initially present on the intact cell's surface are interiorized to the same relative extent. Incorporated label and PDase activity disappear much more rapidly from the phagolysosomes than from the whole cell. In the phagolysosomal compartment, greater than 70% of the TCA-precipitable labeled proteins and all of the PDase activity are lost rapidly (t1/2 equals 1-2 h) but similar 30% of the labeled proteins in this compartment are degraded with a 17-20 h half-life. The slowly degraded label is due to specific long-lived polypeptides, of 85,000 and 8,000-15,000 daltons, which remain in the phagolysosomal membrane up to 40 h after phagocytosis.
Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate.
Authors: Silva P P , Martínez-Palomo A , Gonzalez-Robles A
Published: May 2004

Treatment of living entamoeba histolytica cells with low concentrations of concanavalin A (con A) and peroxidase results in redistribution of the plasma membrane con A receptors to one pole of the cell where a morphologically distinct region--the uroid--is formed. Capping of con A receptors is not accompanied by parallel accumulation of ruthenium red-stainable components. In capped cells, the pattern of distribution of acidic sites ionized at pH 1.8 (labeled by colloidal iron) at the outer surface and of membrane particles (integral membrane components revealed by freeze-fracture) is not altered over the uroid region. Cytochemistry of substrate-attached microexudate located in regions adjacent to E. histolytica cells demonstrates the presence of con A binding sites and ruthenium red- and alcian blue-stainable components and the absent of colloidal iron binding sites. In a previous report we demonstrated that glycerol-induced aggregation of the plasma membrane particles is accompanied by a discontinuous distribution of colloidal iron binding sites, while con A receptors and acidic sites ionized at pH 4.0 remain uniformly distributed over the cell surface. Taken together, our experiments show that, in E. histolytica cells, peripheral membrane components may move independently of integral components and, also, that certain surface determinants may redistribute independently of others. These results point to the complexity of the membrane structure-cell surface relationship in E. histolytica plasma membranes relative to the membrane of the erythrocyte ghost where integral components (the membrane-intercalated particles) contain all antigens, receptors, and anionic sites labeled so far. We conclude that fluidity of integral membrane components (integral membrane fluidity) cannot be inferred from the demonstration of the mobility of surface components nor, conversely, can the fluidity of peripheral membrane components (peripheral membrane fluidity) be assumed from demonstration of the mobility of integral membrane components.
Reinitiation of DNA synthesis in senescent human fibroblasts upon fusion with cells of unlimited growth potential.
Authors: Norwood T H , Pendergrass W R , Martin G M
Published: May 2004

Postreplicative, "senescent" human fibroblasts were fused to HeLa or to SV-40 transformed human fibroblasts with Sendai virus. DNA synthesis was reinitiated in senescent nuclei in a high proportion of the heterodikaryons. The [3H]thymidine labeling index of senescent fibroblast nuclei in heteropolykaryons was a function of the ratio of HeLa to senescent nuclei.
Synthesis and turnover of ribulose biphosphate carboxylase and of its subunits during the cell cycle of Chlamydomonas reinhardtii.
Authors: Iwanij V , Chua N H , Siekevitz P
Published: May 2004

The chloroplast enzyme ribulose-1,5-bisphosphate (Ru-1,5-P2) carboxylase (EC 4.1 1.39) is made up ot two nonidentical subunits, one synthesized in the chloroplast and the other outside. Both of these subunits of the assembled enzyme are synthesized in a stepwise manner during the synchronous cell cycle of the green alga Chlamydomonas reinhardtii. The activity of this enzyme increases in the light and this increase is due to de novo protein synthesis as shown by the measurement of the amount of protein and by the pulse incorporation of radioactive arginine in the 18S enzyme peak in linear sucrose density gradients. During the dark phase of the cell cycle, there is little change in the enzymatic activity as well as in the amount of this enzyme. Pulse-labeling studies using radioactive arginine indicated that there is a slow but detectable rate of synthesis of the carboxylase and of its subunits in the dark. Ru-1,5-P2 carboxylase, prelabeled with radioactive arginine throughout the entire light period, shows a similarly slow rate of degradation in the following dark period. This slow turnover of the enzyme in the dark accounts for the steady levels of carboxylase protein and of enzymatic activity during this period. A wide variety of inhibitors of protein synthesis by 70S and 80S ribosomes abolished the incorporation of [3H]arginine into total Ru-1,5-P2 carboxylase during short-term incubation. These results suggest a tight-coordinated control of the biosynthesis of the small and large subunits of the enzyme. This stringent control is further substantiated by the finding that both subunits are synthesized in sychrony with each other, that the ratio of radioactivity of the small to the large subunit remains constant throughout the entire light-dark cycle, and that the rates of synthesis and of degradation of both subunits are similar to that of the assembled enzyme.
Modulation of uterine morphology and growth by estradiol-17beta and an estrogen antagonist.
Authors: Kang Y H , Anderson W A , DeSombre E R
Published: May 2004

The estrogen antagonist C1628 maintains sustained hypertrophy of the uterine epithelium and the synthesis of many proteins including peroxidase. C1628 is a progestogen, inducing secretion of the protein by surface epithelial and glandular cells. C1628 is a connective tissue mitogen, inducing DNA synthesis in fibroblasts and the endothelium. C1628 and estrogen share these properties mentioned above. Estrogen, however, induced moderate growth of the mucosa within a 24-h period and massive hyperplasia of the mucosa within a 24-h period thereafter. C1628 alone, or in combination with estradiol, does not have mitogenic effect on the mucosa, and in fact blocks the mitotic response normally induced by estrogen alone.
Estrogen and antagonist-induced changes in endometrial topography of immature and cycling rats.
Authors: Anderson W A , Kang Y H , DeSombre E R
Published: May 2004

The topographical changes of the luminal surface of the endometrium of immature and ovariectomized rats treated with estrogen, antagonists to estrogen, and progesterone. and during various stages of the estrous cycle and in pregnancy were examined by scanning electron microscopy. Massive increases in numbers and length of endometrial cell microvilli were observed at estrus, after injection of estradiol-17beta, diethylstilbestrol, estrogen plus progesterone. or the inhibitor C1628 to immature and ovariectomized rats. Withdrawal of the estrogen stimulus results in diminution of microvilli, producing a state identical to diestrus, during pregnancy, and after injection of progesterone, The estrogen antagonist appears to have both estrogenic and progestogenic properties, stimulating endometrial cell hypertrophy, secretion of protein, and production of numerous apical microvilli.
Formation of enlarged mitochondria in a liver cell line in response to a synthetic glucocorticoid
Authors: Berliner JA
Published: May 2004

For a number of years it has been recognized that glucocorticoids cause alterations in liver cell morphology (6, 9). Several investigators have shown that in liver in vivo mitochondria can be enlarged to many times their normal volume by treatment with cortisone (13, 15). There is a concomitant decrease in mitochondrial number, and the results of Kimberg and Loeb suggest that this is due to mitochondrial fusion (7). However, the exact mechanism whereby mitochondrial volume is altered and whether in fact cortisone is the direct causal agent are not known due to the complexity of studying these questions in a whole animal system. We have found that dexamethasone sodium phosphate (dex), a synthetic glucocorticoid, causes the formation of enlarged mitochondria in a liver cell line RLC-GAI, which grows in defined medium. In this paper we present our observations on the amount of enlargement that occurs after 5 days of treatment. The formation of enlarged mitochondria is reversible upon removal of the hormone from the medium, and we have attempted to determine whether "mitochondrial" or "nonmitochondrial" inhibitors are more effective in blocking the return of mitochondria to their normal size when the hormone is removed.
The use of flow microfluorimetry in the analysis of the phenotype expression of mouse histocompatibility antigens
Authors: Campbell GL , Goldstein LT , Knowles BB
Published: May 2004

Quantitation of the expression of cell surface antigens has hitherto been limited to analysis by either cytotoxicity tests or radioimmune assays (5, 15). We report here the use of a new methodology to analyze and quantitate the expression of mouse histocompabililty antigens (H-2 locus) in hybrid clones and parental cell types. The binding of fluorescein-tagged antibody is measured on a cell-to-cell basis in large viable cell populations using flow microfluorimetric techniques. These techniques have been used to measure hapten and immunoglobulin binding to lymphocyte populations (8, 9, 14). However, this is the first report in which these techniques have been used to examine the expression of the H-2 locus. The advantage of this approach is twofold: first, a large and statistically significant sample population may be analyzed one cell at a time, thus revealing the fine detail of heterogeneity in the expression of the cell surface markers within a population. Second, as has been demonstrated for analysis of specific components of the immune system, this method does permit fluorescence-activated sorting of cell types according to their different surface populations (8, 9, 14).
On the connection between the transverse tubules and the plasma membrane in frog semitendinosus skeletal muscle
Authors: Zampighi G , Vergara J , Ramon F
Published: May 2004

The transverse tubular system (TTS) of skeletal muscle fibers represents the morphological basis for the inward spread of conduction of the electrical signal that triggers muscle contraction. A historical account of the main steps contributing to the elucidation of the structure and function of the TSS has been presented by Huxley (1971). While the localization of the TSS and its association with the sarcoplasmic reticulum (SR) is well documented; there is still a need further to develop our knowledge of the morphology of the connection between the TSS and the plasma membrane. It is generally believed that the TSS opens directly to the extracellular space and that there is continuity between its membrane and the sarcolemma. However, direct observation of such a connection has been clearly shown only for the myotome of fish (Franzini-Armstrong and Porter, 1964). In other muscle fibers, only indirect evidence of the connection has been provided by experiments showing penetration of extracellular tracers into the TSS. These extracellular markers were also observed inside another membrane-bounded compartment consisting of round profiles named "caveolae" (Yamada, 1955) or "pinocytotic vesicles" (Ashurst, 1969). The present study deals with the communication between the TTS, caveolae, and plasma membrane (Peachey, 1965); Ezerman and Ishikawa, 1967; Schiaffino and Margreth, 1968; and Rayns et al., 1968). A detailed study of the caveolae compartment was undertaken with ruthenium red as an electron-dense tracer. As a result of this study, we propose that in certain species the caveolae compartment represents the transitional region in the connection between the TSS and the sarcolemma.
Mitochondrial and cytoplasmic ribosomes and their activity in blood and culture form Trypanosoma brucei.
Authors: Hanas J , Linden G , Stuart K
Published: May 2004

Ribosomes of Trypanosoma brucei, a parasitic, flagellated protozoan (order Kinetoplastida), were identified on sucrose density gradients by their radioactively labeled nascent peptides. Ultraviolet absorption revealed only cytoplasmic ribosomes which served as internal sedimentation markers. Synthesis on cytoplasmic ribosomes was completely inhibited by cycloheximide. In the presence of this antibiotic, nascent peptides were associated with ribosomes of lower sedimentation coefficient than the cytoplasmic ribosomes. Chloramphenicol blocked synthesis on these ribosomes which are probably the mitochondrial ribosomes. These ribosomes differed from the cytoplasmic ribosomes in several ways. Their sedimentation coefficient was about 72S rather than 84S. The stability of the 72S ribosomes was less sensitive to pancreatic ribonuclease and low Mg-++ concentrations, dissociating below 0.1 mM Mg++. The 72S ribosomes were more sensitive to elevated KCl concentrations, dissociation above 0.25 M. Protein synthetic activity associated with the 72S class of ribosomes was found in trypanosomes grown in rats. Under these conditions no cytochromes or fully active Krebs cycle is present in these cells and respiration is insensitive to cyanide.
Liposomes containing chelating agents. Cellular penetration and a possible mechanism of metal removal.
Authors: Rahman Y E , Wright B J
Published: May 2004

Electron microscope studies were done on mouse liver, from 5 min to 8 wk after an intravenous injection of liposomes containing ethylenediaminetetraacetic acid (EDTA). Livers of mice receiving an injection of liposomes containing KCL instead of EDTA or an injection of a solution of EDTA were also examined. Liposomes were shown to be phagocytized by hepatocytes as well as by Kupffer cells within minutes after the injection. Initially, there was a close contact between the liposomal membrane and the cellular membrane, followed by an invagination of the latter and the formation of a distinct vesicle surrounding a single liposome or a cluster of several liposomes. No fusion between the liposomal membrane and the cell membrane was observed. Between 15 min and 6 h after liposome injection, the Kupffer cells were found to have an increased number of lysosomes and autophagic vacuoles. Within the latter, morphologically intact liposomes or remnants of liposomes could be seen. At 12 h after injection, a striking increase in macrophages was observed in the liver sinusoids of EDTA-liposome-injected mice, but not in those of KCl-liposome-injected mice. Within the macrophages, remnants of liposomes occasionally could be observed. However, the origin and the physiological role of these cells are unknown. In the hepatocytes, morphological changes were first observed 24 h after injection; there were large numbers of autophagic vacuoles, and some cells showed extensive areas of focal cytoplasmic degeneration. The morphology of the liver cells returned to normal about 7 days after injection. No morphological changes were observed in livers of mice receiving EDTA solution without liposomes. A possible mechanism by which the liposome-encapsulated chelating agents can successfully remove intracellular toxic metals is discussed. The use of liposomes as carriers seems to be a useful tool for intracellular delivery of chelating agents or drugs in general.
Intramembrane organization of specialized contacts in the outer plexiform layer of the retina. A freeze-fracture study in monkeys and rabbits.
Authors: Raviola E , Gilula N B
Published: May 2004

Freeze-fracture analysis of the neural connections in the outer plexiform layer of the retina of primates (Macaca mulatta and Macaca arctoides) demonstrates a remarkable diversity in the internal structure of the synaptic membranes. In the invaginating synapses of cone pedicles, the plasma membrane of the photoreceptor ending contains an aggregate of A-face particles, a hexagonal array of synaptic vesicle sites, and rows of coated vesicle sites, which are deployed in sequence from apex to base of the synaptic ridge. The horizontal cell dendrites lack vesicle sites and have two aggregates of intramembrane A-face particles, one at the interface with the apex of the synaptic ridge, the other opposite the tip of the invaginating midget bipolar dendrite. Furthermore, the horizontal cell dendrites are interconnected by a novel type of specialized junction, characterized by: (a) enlarged intercellular cleft, bisected by a dense plate and traversed by uniformly spaced crossbars; (b) symmetrical arrays of B-face particles arranged in parallel rows within the junctional membranes; and (c) a layer of dense material on the cytoplasmic surface of the membranes. The plasmalemma of the invaginating midget bipolar dendrite is unspecialized. At the contact region between the basal surface of cone pedicles and the dendrites of the flat midget and diffuse cone bipolar cells, the pedicle membrane has moderately clustered A-face particles, but no vesicle sites, whereas the adjoining membrane of the bipolar dendrites contains an aggregate of B-face particles. The invaginating synapse of rod spherules differs from that of cone pedicles, because the membrane of the axonal endings of the horizontal cells only has an A-face particle aggregate opposite the apex of the synaptic ridge. Specialized junctions between horizontal cell processes, characterized by symmetrical arrays of intramembrane B-face particles, are also present in the neuropil underlying the photoreceptor endings. Small gap junctions connect the processes of the horizontal cells; other gap junctions probably connect the bipolar cell dendrites which make contact with each cone pedicle. Most of the junctional specializations typical of the primate outer plexiform layer are also found in the rabbit retina. The fact that specialized contacts between different types of neurons interacting in the outer plexiform layer have specific arrangements of intramembrane particles strongly suggests that the internal structure of the synaptic membranes is intimately correlated with synaptic function.
Release of growth hormone from ox pituitary slices after pronase treatment
Authors: Schofield JG , Orci L
Published: May 2004

Proteolytic enzymes have been used both to modify properties of the cell membrane and to dissociate cells from many tissues including pituitary (4, 5, 12). Exposure of secretory tissues to pronase can alter their secretory response. Thus incubation of pancreatic islets of Langerhans in the presence of low concentrations of pronase increased the subsequent release of insulin in the presence of stimulatory and nonstimulatory glucose concentrations (7). The purpose of the present investigation was to determine whether low concentrations of pronase have the same stimulatory effect on the release of a pituitary hormone, growth hormone. Such an effect on hormone release could be of some importance in view of the development of dissociated cell systems as models for the study of the control of hormone release (4, 5).
Tannic acid-stained microtubules with 12, 13, and 15 protofilaments
Authors: Burton PR , Hinkley RE , Pierson GB
Published: May 2004

Subunit structure in the walls of sectioned microtubules was first noted by Ledbetter and Porter (6), who clearly showed that certain microtubules of plant meristematic cells have 13 wall protofilaments when seen in cross section. Earlier, protofilaments of microtubular elements had been described in negatively stained material, although exact counts of their number were difficult to obtain. In microtubular elements of axonemes, some success has been achieved in visualizing protofilaments in conventionally fixed and sectioned material (8, 10); much less success has been achieved in identifying and counting protofilaments of singlet cytoplasmic microtubules. By using glutaraldehyde-tannic acid fixation, as described by Misuhira and Futaesaku (7), Tilney et al. (12) studied microtubules from a number of sources and found that all have 13 protofilaments comprising their walls. These authors note that "...the number of subunits and their arrangement as protofilaments appear universal...". Preliminary studies of ventral nerve cord of crayfish fixed in glutaraldehyde-tannic acid indicated that axonal microtubules in this material possess only 12 protofilaments (4). On the basis of this observation, tannic acid preparations of several other neuronal and non-neuronal systems were examined. Protofilaments in microtubules from these several cell types are clearly demonstrated, and counts have been made which show that some kinds of microtubules have more or fewer protofilaments than the usual 13 and that at least one kind of microtubule has an even rather than an odd number.
Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules
Authors: Dentler WL , Granett S , Rosenbaum JL
Published: May 2004

Microtubules isolated from brain extracts by in vitro assembly (1, 19, 23) are composed principally of two tubulins and two high molecular weight proteins (microtubule-associated proteins [MAPS] 1 and 2) (2,5,7,20). Recently, it was demonstrated that in vitro-assembled brain microtubules (neurotubules) are coated with filaments (5, 7) which are similar to the filaments attached to neurotubules in situ (4, 15, 21, 24, 25), and it was suggested that the filaments are composed of the higher molecular weight MAPs (5, 7, 12). In this study, microtubules were assembled in the presence and absence of the MAPs, and thin sections of the microtubules were examined by electron microscopy. The results show that the filaments only occur on microtubules assembled in the presence of the MAPs and it is therefore concluded that the filaments are composed of the high molecular weight MAP's.
Membrane proteins synthesized by rabbit reticulocytes.
Authors: Lodish H F , Small B
Published: May 2004

Intact rabbit reticulocyte cells synthesize two predominant species of polypeptides which are components of the cell plasma membrane. Previous work (Lodish, H. F. 1973. Proc. Natl. Acad. Sci. U. S. A. 70:1526-1530.) showed that these proteins were synthesized by polyribosomes not attached to membranes. We show here that both polypeptides are confined to the cytoplasmic surface of the cell membrane. These studies utilized iodination of whole cells and of membranes with lactoperoxidase, and digestion of whole cells and membranes with chymotrypsin, One of these proteins is synthesized as a precursor, and about 20-40 amino acids are removed after it is incorporated into the membrane, We discuss the probable sites of synthesis of these and other classes of membrane proteins.
The basal bodies of Chlamydomonas reinhardtii. Formation from probasal bodies, isolation, and partial characterization.
Authors: Gould R R
Published: May 2004

The assembly and composition of basal bodies was investigated in the single-celled, biflagellate green alga, Chlamydomonas reinhardtii, using the cell wall-less strain, cw15. In the presence of EDTA, both flagellar axonemes remained attached to their basal bodies while the entire basal body-axoneme complex was separated from the cell body, without cell lysis, by treatment with polyethylene glycol-400. The axonemes were then removed from the basal bodies in the absence of EDTA, leaving intact basal body pairs, free from particulate contamination from other regions of the cell. The isolated organelles produced several bands on sodium dodecyl sulfate-urea polyacrylamide gels, including two tubilin bands which co-electrophoresed with flagellar tubulin. The formation of probasal bodies was observed by electron microscopy of whole mount preparations. Synchronous cells were lysed, centrifuged onto carbon-coated grids, and either negatively stained or shadowed with platinum. The two probasal bodies of each cell appeared shortly after mitosis as thin "annuli," not visible in thin sections, each consisting of nine rudimentary triplet microtubules. Each annulus remained attached to one of the mature basal bodies by several filaments about 60 in diameter, and persisted throughout interphase until just before the next cell division. It then elongated into a mature organelle. The results revive the possibility of the nucleated assembly of basal bodies.
Structural and transcriptional features of the mouse spermatid genome.
Authors: Kierszenbaum A L , Tres L L
Published: May 2004

A whole-mount electron microscope technique has allowed direct visualization of the transcription process in mouse spermatids. Thes observations have been supported by light and electron microscope autoradiographic techniques that employ [3H]uridine and [3H]arginine in attempts to clarify mechanisms of RNA synthesis and their relationship to nuclear histone changes throughout spermiogenesis. Early spermatid genomes are dispersed almost completely, whereas in later spermiogenic steps the posterior or flagellar nuclear region is readily dispersed and the anterior or subacrosomal nuclear region remains compact. Display of genome segments permits identification of regions where transcription complexes, presumably heterogeneous nuclear RNA species, are seen related to chromatin. These complexes appear as ribonucleoprotein chains, some of them of considerable length, decreasing progressively in number in late spermiogenic steps. This decrease coincides with diminishing rates of [3H]uridine incorporation. Two distinct patterns of chromatin have been identified: a beaded chromatin type associated with transcription complexes encounterd in early spermatids; and a smooth chromatin type not involved in transcriptive activity observed in advanced spermiogenic genomes. Protein particles staining densely with phosphotungstic acid become apparent in nuclei of spermatids after [3H]arginine incorporation becomes significant. There is no structural or autoradiographic evidence for the presence of nucleoli during spermiogenesis. From these data and from previous experimental findings, we conclude that: (a) spermatogonia, spermatocytes and Sertoli cells are transcriptionally expressed into heterogeneous nuclear RNA and preribosomal RNA species whereas transcription in spermatids is predominantly heterogeneous nuclear RNA; and (b) the modification of the chromatin patterns in late spermiogenic steps indicates a stabilized genome that restricts transcriptive functions.
Response of myogenic and fibrogenic cells to cytochalasin B and to colcemid. I. Light microscope observations.
Authors: Croop J , Holtzer H
Published: May 2004

Cytochalasin B (CB) induces a biphasic retraction is some cell types. The rapid response that peaks in 30 min leads to the "dendritic" condition. Replicating myogenic and fibrogenic cells, as well as postmitotic myoblasts and myotubes, participate in this reaction. This is followed by a slower phase that requires 40 h for stabilization and leads to the fully "absorized" state. Only replicating myogenic and fibrogenic cells participate in this reaction. Postmitotic myoblasts and myotubes do not arborize but round up and float off into the medium. Pretreatment with Colcemid does not block the rapid response to CB, but does block arborization. CB-arborized cells exposed to Colcemid while in the presence of CB develop sufficient tension to pull themselves apart. If CB depolymerizes actin-like filaments, and if such filaments constitute the only contractile system in the cell, then it is difficult to visualize how cells in CB develop such tension. Colcemid induces twisting, birefringent bands in interphase- and metaphase-arrested myogenic and fibrogenic cells, and in postmitotic myotubes. Such bands are more evident when CB-arborized cells are removed from CB and allowed to relax in Colcemid. These birefringent bands assemble in the prescence of cycloheximide, and may constitute 20% of the volume of the cell.
The role of extracellular materials in cell movement. I. Inhibition of mucopolysaccharide synthesis does not stop ruffling membrane activity or cell movement.
Authors: Spooner B S , Conrad G W
Published: May 2004

The involvement of mucopolysaccharide synthesis in cell locomotion was investigated by determining the effects of inhibition of synthesis on ruffling membrane activity and cell movement by embryonic heart fibroblasts. Mucopolysaccharide synthesis was inhibited directly by treatment with a glutamine analog, 6-diazo-5-OXO-L-norleucine (DON), and indirectly with cycloheximide. DON treatment reduced synthesis to 20% of control values, and cycloheximide reduced synthesis to less than 10% of control values, as measured by incorporation of [35S]sulfate into mucopolysaccharides. Nevertheless, ruffling membrane activity and cell locomotion continued under both conditions. Cytochalasin B did not inhibit mucopolysaccharide synthesis, although it did stop ruffling and locomotion. These results suggest that if mucopolysaccharides are required for cell movement, they must have long half-lives or represent only a minute fraction of the normal synthetic load.
Nucleolar necklaces in chick embryo fibroblast cells. I. Formation of necklaces by dichlororibobenzimidazole and other adenosine analogues that decrease RNA synthesis and degrade preribosomes.
Authors: Granick D
Published: May 2004

A number of chemicals, mostly adenosine analogues, cause the nucleolus of the chick embryo fibroblast to lose material and unravel over a period of several hours into beaded strands termed nucleolar necklaces (NN). The results of analyses of the fibroblasts, treated with the NN-forming chemical dichlororibobenzimidazole (DRB), suggests that the following biochemical alterations occur: DRB almost completely prevents the increase in both messenger RNA (mRNA) and heterogeneous nuclear RNA. It interferes with ribosome synthesis by decreasing the rate of 45S ribosomal RNA (rRNA) accumulation by 50%, slowing the rate of 18S rRNA appearance by 50%, and causing an extensive degradation (80%) of the 32S and 28S rRNA-containing preribisomes. Most of this preribosome degration probably occurs at or before the 32S rRNA preribosome stage. The degradation of these preribosomes appears to be due to the formation of defective 45S rRNA preribosomes rather than to a direct DRB interference with preribosome processing enzyme action. DRB inhibits total cellular RNA synthesis in less than 15 min, suggesting a direct interference with RNA synthesis. DRB also inhibits the uptake of nucleosides into the cell. DRB in the concentrations used does not appear to directly interfere with the translation of mRNA (i.e., protein synthesis). Other NN-forming adenoside analogues and high concentrations of adenosine (2 mM) cause biochemical alterations similar to those produced by DRB. To explain the preribosome degradation, we propose the hypothesis that DRB inhibits the synthesis of mRNA; as a consequence, some of the preribosomal proteins that normally coat the 32S rRNA portion of the 45S precursor RNA become limiting, and this defective portion is then subject to degradation by nucleases.
Isolation and separation of highly enriched fractions of viable mouse gastric parietal cells by velocity sedimentation.
Authors: Romrell L J , Coppe M R , Munro D R , Ito S
Published: May 2004

Methods of tissue dissociation and cell separation have been modified to obtain highly enriched fractions of mouse gastric parietal cells. Suspension of gastric mucosal cells are prepared by pronase digestion of the glandular portion of the stomach from adult mice. By utilizing the velocity sedimentation technique to separate cells of different sizes it is possible to recovery parietal cells, which are larger than the other cell types, in fractions with purity of 75-95%. The homogeneity of cell fractions has been assessed by light and electron microscopy. The ability of the isolated cells to exclude the dye trypan blue, to incorporate labeled substrate, to consume oxygen, and to retain their structural integrity indicates that they are viable and still capable of functional activity.
Localization and characterization of carbohydrates in adrenal medullary cells.
Authors: Cantin M , Benchimol S
Published: May 2004

The localization and characterization of carbohydrates in adrenal medullary cells were studied by histochemical and cytochemical methods. Adrenaline (A)-and noradrenaline (N)-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. A small amount of glycogen in the form of single beta-particles as well as lysosomes were, however, visualized by this technique. The entire core of the A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded medullae were stained with phosphotungstic acid (PTA) at low pH (0.3). The N granules, in contrast, were mostly unreactive. In the A cells, PTA stained a large part of the Golgi complex, whereas in the N cells the Golgi complex was mostly unstained. In both cell types, the cell coat, lysosomes, and multivesticular bodies reacted to PTA. The periodic acid-Schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation, and greatly diminished by sulfation. In ultrathin sections of GMA- or Araldite-embedded medullae incubated with colloidal iron according to various techniques, the cell coat and lysosomes of both cell types were stained, unlike all the other cytoplasmic organelles. These results indicate that A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins which are probably not acidic.
Direct association of messenger RNA with microsomal membranes in human diploid fibroblasts.
Authors: Lande M A , Adesnik M , Sumida M , Tashiro Y , Sabatini D D
Published: May 2004

Messenger RNA (mRNA) of membrane-bound polysomes in a membrane fraction of WI-38 cells remains associated with the microsomal membranes even after ribosomes and their nascent polypeptide chains are removed by using puromycin in a high salt buffer or by disassembling the ribosomes in a medium of high ionic strength lacking magnesium. mRNA either was specifically labeled in the presence of actinomycin D, or it was recognized by virtue of its affinity for oligo-dT. Poly A segments in bound mRNAs have an electrophoretic mobility in acrylamide gels which is characteristic of cytoplasmic mRNAs and corresponds to 150-200 adenyl residues. Extensive RNase treatment did not lead to release of the poly A segments of membrane-associated mRNA molecules either from an intact membrane fraction or from a membrane fraction previously stripped of ribosomes. On the other hand, RNase treatment led to the release and digestion of the nonpoly A segments of the mRNA molecules, indicating that the site of attachment of mRNA to the ER membranes is located near or at the 3' end of the molecule which contains the poly A. A direct association of mRNAs and endoplasmic reticulum membranes is considered in a modelto explain the assembly of bound polysomes and protein synthesis in a membrane-associated apparatus.
Comparision of the structure and function of polysomal and helical ribosomes from Entamoeba invadens.
Authors: Kusamrarn T , Vinijchaikul K , Bailey G B
Published: May 2004

Some structural and functional properties of ribosomes from polysomes and from helix aggregates of Entamoeba invadens have been compared by sucrose gradient analysis and assays of in vitro protein synthesis. Actively growing trophozoites, lacking helices, presented normal polysome profiles in sucrose gradients. The single large ribosomal helix aggregate (chromoatoid body) of cysts diappeared as the cells were disrupted. Gradient profiles of cyst extracts contained predominantly large and small ribosome subunit peaks and no evidence of remaining helix fragments of mRNA-bound polysomes. Sequential profiles of trophozoites incubated with NaF or cycloheximide (which both stimulate ribosome aggregation, but at different rates) showed that polysome breakdown occurred before aggregates appeared and, again, that helices broke down to subunits in vitro. Radioactive ribosomes synthesized during vegetative growth were collected into helices during encystation. Subunits of these ribosomes cosedimented with comparable particles isolated from trophozoites. Ribosomes from both trophozoites and cysts were active in cell-free protein synthesis, although activity in cyst extracts required the addition of trophozoite-soluble fraction. It was concluded that ribosomes from polysomes and helices in E. invadens were probably identical and that the ability to form helices was an intrinsic property of mature mRNA-free ribosomes of this organism.
Pressure-induced depolymerization of spindle microtubules. I. Changes in birefringence and spindle length.
Authors: Salmon E D
Published: May 2004

Changes in birefringence retardation (BR) and length of Chaetopterus meiotic metaphase-arrested spindles produced by increased hydrostatic pressure were observed with polarized-light microscopy using a newly developed optical pressure chamber. Increased pressure produced rapid, reversible decreases in spindle BR and length. Pressures of 3,500 psi or higher at 22 degrees C caused complete disappearance of spindle BR within 3 min. Up to 6,000 psi, the rates of both BR decay and spindle shortening increased progressively with increasing pressure. At 6,000 psi or above, the BR decreased rapidly but there was no evidence of spindle shortening. The general observations are consistent with results of earlier classical experiments on effects of pressure on mitosis, and with experiments that used colchicine or low temperature as microtubule-depolymerizing agents. The kinetics of spindle depolymerization and repolymerization showed two phases: an initial phase of rapid decreases or increase in half-spindle microtubule BR; and a second phase of nearly constant BR during which most of the spindle shortening or growth occurs. BR is assumed to be directly related to the number of microtubules in a spindle cross section. It is hypothesized that microtubules in the spindle have different stabilities depending on the attachment of nonattachment of their ends. This hypothesis is used to explain the two phases of spindle depolymerization and repolymerization as well as several other observations.