bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycolipids as indicators of tumorigenesis
Authors: Morré D. J. , Kloppel T. M. , Merritt W. D. , Keenan T. W.
Published: May 2005

Hyperplastic liver nodules and hepatocellular carcinomas were induced in rats by oral administration of the carcinogen N‐2‐fluorenylacetamide. Neoplastic tissue was compared with control, fetal, neonatal, and precancerous liver tissues. The development of the tumors was slow, such that temporal changes in the biochemical and morphologic development of carcinogenesis could be identified. Ganglioside sialic acid levels were elevated in all but the most poorly differentiated tumors. Experiments to monitor individual enzymes suggested that the alterations in glycolipid composition were a direct effect of alterations in biosynthetic activities. The pattern during tumorigenesis was the inverse of that during normal development. Also, ganglioside patterns showed a progressive simplification from hyperplastic nodules to well‐differentiated hepatomas and through two grades of poorly differentiated hepatomas. An increase in the activity of the branchpoint enzyme of ganglioside biosynthesis preceded both a decrease in the branchpoint enzyme of the disialoganglioside pathway and a marked increase in the galactosyltransferase of GM1 formation. The results indicate that ganglioside deletions are the end result of a cascade of events in the tumorigenic transformation. The onset of ganglioside deletions but not of the cascade per se may correlate with the onset of malignancy.

Glycolipid levels are elevated early in certain surrounding tissues especially in the blood. In rats bearing transplantable hepatomas, serum levels of lipidbound sialic acid were elevated 2.5‐fold. Similar results were obtained with sera of mice bearing transplantable mammary carcinomas and of cancer patients. These findings provide new emphasis for gangliosides in both cancer detection and as regulatory signals for growth and multiplication of cells.
Lipopolysaccharide effects on sensitive and resistant variant chinese hamster ovary cell lines
Authors: Brunson Kenneth W. , Nicolson Garth L.
Published: May 2005

Chinese hamster ovary (CHO · K1 · PRO) cell growth was inhibited by addition of a gram‐negative bacterial lipopolysaccharide (LPS) to the cell culture medium. Growth inhibition began after three or four days of incubation, was dose‐dependent up to a maximum at an LPS concentration of 500 μg/ml and was accompanied by cell shape changes and enhanced cytoplasmic vacuolization. Formation of bizarre CHO · K1 · PRO cell shapes and vacuole formation were most pronounced after seven days of incubation with LPS and could be observed by light and electron microscopy. An LPS‐resistant cell population was obtained by intermittent in vitro exposure to high levels of LPS; these variant cells or clones derived from them failed to display growth inhibition in the presence of LPS. A clone from the LPS‐resistant variant population showed altered cell properties compared to the parental cell line which included changes in cell morphology, adhesion, and endocytosis. Parental cells were markedly density‐inhibited, whereas the variant clone exhibited considerable growth after confluency. The LPS‐resistant variant cells showed a more elongated morphology than the parental line. No significant differences were observed between rates of detachment of parental and variant cells when sparse cultures of either line were removed from tissue culture dishes by ethylenediaminetetracetate (EDTA). However, at confluency approximately 100% of the variant cells versus 35% of the parental cells were removed by EDTA in one hour. Measurements of 125I‐ferritin uptake by parental and variant cells showed approximately twenty‐fold and twofold increases, respectively, in uptake induced by LPS when compared to untreated control cultures.
Binding of ricin A chain to rat liver ribosomes: Relationship to ribosome inactivation
Authors: Hedblom Mary L. , Cawley Daniel B. , Boguslawski Sophie , Houston L. L.
Published: May 2005

Ricin A chain was radioactively labeled using reductive alkylation, lactoperoxidase catalyzed iodination, and reaction with iodoacetamide or N‐ethylmaleimide (NEM). The inhibition of cell‐free rat liver protein synthesis by the modified A chains and the ribosome binding characteristics of each of the labeled derivatives was examined. [3H] NEM was found to quantitatively react with the A chain sulfhydryl group normally involved in a disulfide bond with the B chain in intact ricin. Labeling the protein with [3H] NEM had no effect on the in vitro inhibition of protein synthesis by the A chain. [3H] NEM‐labeled A chain binds to rat liver ribosomes in a manner which is dependent on the concentrations of NaCl and Mg2+. At optimal Mg2+ concentration (5.5 mM), A chain binding to ribosomes is saturable and fully reversible either by dilution of the reaction mixture or by addition of unlabeled A chain. At 5.5 mM Mg2+, A chain was found to bind to a single site on rat liver ribosomes with a dissociation constant of 6.2 X 10−8 M. [3H] NEM‐labeled A chain did not bind to isolated 40S ribosomal subunits and bound to 60S ribosomal subunits with a 1 : 1 molar stoichiometry and a dissociation constant of 2.2 X 10−7 M. The relationship between ribosome binding and A chain inhibition of eucaryotic protein synthesis is discussed.
Transmembrane action of thrombin initiates chick cell division
Authors: Carney Darrell H. , Cunningham Dennis D.
Published: May 2005

Thrombin immobilized on polystyrene beads initiates DNA synthesis and cell division in quiescent cultures of chick embryo (CE) cells in serum‐free medium. These thrombin beads also produce morphological changes in CE cells similar to those produced by soluble thrombin. The amount of acid‐precipitable material released from 125I‐thrombin‐beads into the culture medium was 60‐fold less than the amount of soluble thrombin required to produce an equivalent increase in cell number. Moreover, EM autoradiography of CE cells 10 h after 125I‐thrombin‐bead addition showed that there was no direct release and accumulation of radioactive material in the cytoplasm of these cells. These results demonstrate that thrombin action at the cell surface is sufficient to initiate proliferation of CE cells.
Resistance of the intact and reconstituted adipocyte hexose transport system to irreversible inhibition by sulfhydryl and amino reagents
Authors: Czech Michael P. , Pillion Dennis J. , Shanahan Michael F.
Published: May 2005

Sensitivity of the adipocyte D‐glucose transport system in intact plasma membranes or following solubilization and reconstitution into phospholipid vesicles to several protein‐modifying reagents was investigated. When intact plasma membranes were incubated with N‐ethylmaleimide (20 mM) or fluorodinitrobenzene (4 mM), D‐glucose transport activity was virtually abolished. However, washing the membranes free of unreacted reagents restored transport activity, indicating that covalent interaction with the membranes did not mediate the transport inhibition. Reaction of [3H] N‐ethylmaleimide with plasma membranes under similar conditions resulted in extensive labeling of all protein fractions resolved on dodecyl sulfate gels. Similarly, addition of N‐ethyl‐maleimide to cholate‐solubilized membrane protein had no effect on transport activity in artifical phospholipid vesicles reconstituted under conditions where the membrane protein was free of unreacted N‐ethylmaleimide. Transport activity in plasma membranes was also inhibited by both reduced and oxidized dithiothreitol or glutathione (15 mM) in a readily reversible manner, consistent with a noncovalent mode of inhibition. Thus, the insulin‐responsive adipocyte D‐glucose transport system differs from the red cell hexose transport system in its remarkable insensitivity to modulation by covalent blockade of sulfhydryal or amino groups by the reagents studied.
Photolabile and paramagnetic reagents for the investigation of transmembrane signaling events
Authors: Bramhall John , Ishida Brian , Wisnieski Bernadine
Published: May 2005

To investigate the dynamics of membrane processes that may be integral components of specific transmembrane signaling events we have synthesized several novel paramagnetic probes and their photoreactive counterparts. The structure of these probes was designed to (1) restrict “flipping” across the membrane bilayer; (2) contain paramagnetic or photoreactive moieties that could be placed at specific depths within the bilayer; (3) provide information about membrane structure as well as dynamics of protein movement; and (4) in the case of the photoreactive probes, be of high specific radioactivity.

The molecules described in this paper consist of amino acid, dipeptide, or carbohydrate groups attached to arylazide‐ or nitroxide‐bearing fatty acids. The synthesis and initial characterization of these membrane probes is described.
Redistribution of intramembrane particles of human erythrocytes induced by HVJ (Sendai virus): A prerequisite for the virus‐induced cell fusion
Authors: Asano Akira , Sekiguchi Kiyotoshi
Published: May 2005

Aggregation of intramembrane particles of human erythrocytes was found to be induced by HVJ (Sendai virus) under conditions which lead to cell fusion. Degree of polyerythrocyte formation was compared under a variety of conditions with extent of cluster formation observed with the same preparations. Both structural changes of the membranes, ie, fusion and clustering of the particles, behaved very similarly under widely different virus‐to‐cell ratios and over the time course of cell fusion. Furthermore, by inclusion of high concentrations of antispectrin antibodies within the ghosts, inhibition of clustering of intramembrane particles and hindrance of virus‐induced cell fusion were found to occur simultaneously. Antibodies by themselves did not induce aggregation of particles under isotonic conditions, whereas particle clustering could be induced under hypotonic conditions at antibody concentrations causing partial cross‐linking of spectrin molecules.

In conclusion, clustering of intramembrane particles seems to be required for virus‐induced fusion of human erythrocytes.
Intracellular compartmentation and transport of metabolites
Authors: Davis Rowland H. , Bowman Barry J. , Weiss Richard L.
Published: May 2005

The intracellular locations of enzymes and metabolites were determined for ornithine metabolism in Neurospora. Pulse label experiments were used to measure the rates of intracellular translocations and the sizes of compartmented pools of metabolites in the mitochondrial, cytosolic and vesicular compartments. The results indicate that rapid equilibration occurs between these pools during growth in minimal medium, although the vast majority of the ornithine is confined to the vesicular compartment. Arginine, the biosynthetic end‐product of ornithine metabolism, regulates ornithine utilization through a combination of feedback inhibition, repression, and control of intracellular translocations. The last phenomenon plays a decisive role indicating that the regulation of intercompartmental translocations may be a common mechanism in rapid adaptation responses in eukaryotic cells.
Increase in the affinity of the uridine phosphorylation system for ATP after serum or insulin activation of 3T3 fibroblasts
Authors: Goldenberg Gerald J. , Stein Wilfred D.
Published: May 2005

The stimulation of uridine uptake, brought about by the addition of serum or insulin to quiescent 3T3 fibroblasts, is associated in the half‐saturation concentration of the uridine phosphorylating system for the substrate ATP, with relatively little change in the maximum uptake or in the affinity for uridine. In stimulated cells the Km towards ATP fell in the range 0.053–0.187 mM, while V max was 34 to 52 pmoles/106 cells/min. In quiescent cells these values were 2.89–4.22 mM and 74.5–126 pmoles/106 cells/min, respectively. No difference was found, however, between the Km's for ATP when phosphorylation of uridine was determined using cell‐free extracts prepared from either quiescent cells or from stimulated cells.
Steroid hormone receptor characterization of several histologic variants of a rat prostatic adenocarcinoma
Authors: Markland Francis S. , Chopp Richard T. , Cosgrove Malcolm D. , Howard Edwin B.
Published: May 2005

Several histologic variants of the transplantable R‐3327 prostatic adenocarcinoma carried in male Copenhagen rats have been characterized and the histologic types have been correlated with steroid hormone receptor content. One type is clearly an adenocarcinoma; this tumor is hormonally responsive and contains substantial amounts of both androgen and estrogen receptors. In contrast, another histologic type, a fibrosarcoma, is hormonally nonresponsive and does not contain either receptor. A third histologic variant is classified as a carcinosarcoma and contains histological elements of both adenocarcinoma and fibrosarcoma and is also hormonally responsive. This tumor contains lower receptor levels than the adenocarcinomas but more than the fibrosarcomas. The androgen receptor appears to be identical in the different histologic forms of the tumor; the sedimentation coefficient is 7.8S and the dissociation constant for methyltrienolone is 4 X 10−9 M. Similarly, the estrogen receptor from the different histologic forms of the tumor has a sedimentation coefficient of 8.3S and the dissociation constant for estradiol is 7 X 10−10 M. These findings clearly distinguish the cytosol binding macromolecules from plasma binding proteins, and classify them as steroid hormone receptors. Further, rat serum was devoid of androgen and estrogen binding in the 8S region. Normal prostate tissue from Copenhagen rats contained low levels of an androgen receptor, but no estrogen receptor. It is possible that during growth and/or passage of the R‐3327 tumor, the hormonally responsive adenocarcinoma cells do not survive and there is a gradual emergence of the nonresponsive fibrosarcoma. If, as we suspect, the receptors are found in the epithelial cells and not the stromal cells, there clearly should be considerable variation of receptor content in the different intermediary histologic forms of the tumor.
Requirement for guanosine triphosphate in the activation of adenylate cyclase by cholera toxin
Authors: Enomoto Keiichi , Gill D. Michael
Published: May 2005

The activation of adenylate cyclase in lysed pigeon erythrocytes requires, among several cofactors, a nucleotide which may be ATP, GTP, or many other triphosphates. However, after removal of endogenous nucleotides by gel filtration or by adsorption onto charcoal the requirement can be met only by GTP, or an analog of GTP. The GTP is required during the activation of the cyclase by toxin even if GTP is also included during the subsequent adenylate cyclase assay, conducted without toxin. In the presence of GTP it is possible to assay for the cytosolic protein that is also required for the action of cholera toxin. By gel filtration, its apparent molecular weight is 15,000–20,000.
Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells
Authors: Maher P. , Molday R. S.
Published: May 2005

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either 125I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 107 binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy‐dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein‐tagged Con A and rhodamine‐tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.
Cross‐linkings between spectrin and band 3 in human erythrocyte membranes
Authors: Liu Shih‐Chun , Palek Jiri
Published: May 2005

A specific structural association between spectrin component 1 and band 3 in human erythrocyte membrane has been demonstrated by covalent cross‐linkings, specific labeling, and the technique of two‐dimensional gel electrophoresis. A complex of 330,000 daltons, representing 1 + 3, was produced in mildly oxidized membranes at physiologic pH and isotonic conditions but not at hypotonic conditions (< 10 mM KCl or NaCl). The yield of this complex decreased dramatically as the monovalent cation concentration decreased from 90 mM to 30 mM. The presence of Mg++ or Ca++ (2 mM) at low ionic strength promoted 1 + 3 cross‐linking in an amount similar to that produced at isotonic conditions. The specific segment of band 3 involved in the cross‐linking was also investigated by means of chymotrypsin digestion of band 3 in the intact red cells. The results showed the cross‐links between spectrin component 1 and the 55,000‐dalton fragment of band 3 at physiologic pH and isotonic conditions. This is consistent with the idea that band 3 is anchored on or contacted with the submembrane meshwork at the cytoplasmic membrane surface.
Structure, function, and antigenicity of cholera toxin
Authors: Markel Daiel E. , Hejtmancik Kelly E. , Peterson Johnny W. , Kurosky Alexander
Published: May 2005

Chemical modification of intact cholera toxin or its B subunit by either partial nitration or reduction and alkylation did not result in significant loss of biological activity as determined by measurement of cyclic AMP in Chinese hamster overy cells. Complete nitration or succinylation in the presence of guanidine hydrochloride resulted in complete loss of biological activity and significantly affected the immunoreactivity of the toxin and B subunit. Compositional analyses of both the isolated α and γ chains of the toxin were typical of globular proteins and did not reveal significant hydrophobicity. Analysis of antigenic relationships by radioimmunoassay indicated a partial crossreactivity between the α chain and the B subunit of cholera toxin. Since previous structural studies of the β chain of cholera toxin indicated chemical similarity with the glycoprotein hormones [Kurosky et al. Science 195:299 (1977)], radioimmunoassay procedures were employed to investigate for possible crossreactivity. No evidence of crossreactivity between cholera toxin subunits and subunits of ovine luteinizing hormone was found.
Independence of the lethal actions of glucocorticoids on lymphoid cells from possible hormone effects on calcium uptake
Authors: Nicholson Mary L. , Young Donald A.
Published: May 2005

We have examined the possibility that hormone‐induced increases in calcium uptake might initiate the lethal actions of glucocorticoids in two types of lymphoid cells. Hormone‐induced increases in nuclear fragility are used as the measure of hormone action, since in both rat thymus cells and in mouse P1 798 lymphosarcoma cells increased nuclear fragility (the inability of nuclei to survive lysis of the cells by hypotonic shock) precedes other indices of cellular deterioration by several hours.

In the case of the tumor cells, those from corticosteroid‐sensitive lines are less able to withstand incubation in vitro than resistant cells. Such differences in cell survival are predicted both by earlier changes in nuclear fragility and also by differences in calcium uptake. However, there is no detectable early glucocorticoid effect on calcium uptake that precedes or coincides with the substantial hormone‐induced increases in nuclear fragility that develop in the sensitive cells by 2 h.

In rat thymus cells the absence of calcium in the medium does prevent some of the increase in nuclear fragility and cell disintegration that occurs spontaneously during incubation in vitro. Nevertheless, when cells are exposed to hormones the glucocorticoid effect on nuclear fragility develops in the absence of calcium and is similar in magnitude to that seen in the presence of calcium.

We conclude that calcium seems to enhance the spontaneous deterioration of lymphoid cells, and there is a large increase in calcium uptake that occurs as cells deteriorate. It nevertheless seems unlikely that hormone‐induced changes in calcium uptake initiate the lethal actions of glucocorticoids. The data also support a proposal made earlier [2] that resistance to glucocorticoids in tumor cells may develop by the selection of cells with hardier membranes.
Predictive value of the analogy between hormone‐sensitive adenylate cyclase and light‐sensitive photoreceptor cyclic GMP Phosphodiesterase: A specific role for a light‐sensitive GTPase as a component in the activation sequence
Authors: Shinozawa Takao , Sen Indira , Wheeler George , Bitensky Mark
Published: May 2005

We report experiments which involve a light sensitive GTPase in the light dependent activation of retinal rod 3′5′‐cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE). The data suggest that the light activated GTPase is intermediate between rhodopsin and PDE in the light‐dependent activation sequence. We list the many striking similarities between hormone sensitive adenylate cyclase and light activated PDE in order to emphasize that the findings presented herein may have predictive value for ongoing studies of the hormone sensitive adenylate cyclase specifically regarding the role of the hormone activated GTPase in the activation sequence.
Analysis of transmembrane dynamics of cholera toxin using photoreactive probes
Authors: Wisnieski B. J. , Shiflett M. A. , Mekalanos J. , Bramhall J. S.
Published: May 2005

Using sodium dodecyl sulfate‐‐polyacrylamide gel electrophoresis and autoradiography, we have shown that 125I‐labeled cholera toxin binds to Newcastle disease virus. Pretreatment of Newcastle disease virus with “cold” cholera toxin (at 37°C for 30 minutes) inhibits the binding of 125I‐labeled toxin in a subsequent incubation (at 37°C for 30 minutes). These results suggest that cholera toxin binds to Newcastle disease virus in a specific manner. The precise receptor for toxin is unknown in Newcastle disease virus but it is presumed to be the ganglioside GM1. We have previously shown that the photoreactive probe 12‐(4‐azido‐2‐nitrophenoxy)stearoylgucosamine[1‐14C] labels the membrane proteins of Newcastle disease virus. Since the reactive group of the probe, ie, N3, resides within the membrane bilayer, studies were initiated to determine which, if any, of the subunits of cholera toxin cross the membrane of Newcastle disease virus and become radioactively labeled upon photoactivation of the probe at 360 nm. After a 15‐minute incubation of cholera toxin with Newcastle disease virus containing the photoreactive probe, irradiation effected the 14C‐labeling of the active A1 subunit of cholera toxin. Irradiation of cholera toxin in solution with an equivalent amount of probe but without virus resulted in no labeling of toxin subunits.
Erythrocyte spectrin alteration induced by low‐density lipoprotein
Authors: Hui David Y. , Harmony Judith A. K.
Published: May 2005

Addition of human plasma low‐density lipoproteins (LDL) to intact human erythrocytes induces the erythrocytes to undergo morphologic transition from biconcave disks to echinocytes and spherocytes. The transformation is time‐dependent. Two hours are required before echinocytes are detected by scanning electron microscopy. After two hours, LDL also decrease the phosphate content of spectrin by 40% relative to the control, suggesting that these lipoproteins modulate cell shape by influencing phosphorylationdephosphorylation of a membrane‐associated cytoskeletal protein. LDL do not induce depletion of intracellular adenosine triphosphate (ATP), nor do they inhibit cyclic adenosine monophosphate‐independent protein kinases which phosphorylate spectrin. LDL stimulate membrane‐bound phosphatases by a factor of two, thereby reducing the amount of phosphate covalently bound to membrane proteins. The observed effects are specific for LDL. High‐density lipoproteins (HDL) do not stimulate dephosphorylation of spectrin or alter erythrocyte morphology. However, HDL protect the erythrocytes against LDL‐induced alterations. These data suggest that the circulating lipoproteins have a role in maintaining erythrocyte morphology by regulating the extent of phosphorylation of spectrin.
Relationship between steps in 8‐anilino‐1‐naphthalene sulfonate (ANS) fluorescence and changes in the energized membrane state and in intracellular and extracellular adenosine 5′‐triphosphate (ATP) levels following bacteriophage T5 infection of Escherichia coli
Authors: Braun V. , Oldmixon E.
Published: May 2005

The addition of bacteriophage T5 to anaerobic, fermenting cells of Escherichia coli B or K‐12 in the presence of 8‐anilino‐1‐naphthalene sulfonate (ANS), N‐phenylnaphthyl‐1‐amine (NPN), or dansyl ethylamine causes the fluorescence of these probes to rise in two steps, the first occurring immediately upon addition, the second delayed by 6 min. The conditions necessary for observing this phenomenon are defined (cell density, probe concentration, substrate, absence of an electron acceptor, multiplicity of infection, growth, and harvesting conditions).

The magnitudes of the first and second steps in fluorescence are dependent upon the multiplicity of infection; the timing of the steps is not. The first step correlates with a breakdown in the potassium or rubidium permeability barrier of the cell, and it occurs either aerobically or anaerobically, with fermentable or nonfermentable substrates. The second step occurs only with cells that are without an available electron acceptor, are fermenting, and which have a functional membrane‐bound, Ca2+‐Mg2+‐dependent adenosine triphosphatase (ATPase). The results are consistent with disturbance of energization of the cell membrane by the membrane‐bound ATPase at the time of the second step in fluorescence. No change in the intracellular level of adenosine 5′‐triphosphate (ATP) was seen, whereas the extracellular level increased sharply, starting 3–6 min after phage addition. The quantity of ATP found in the medium by 30 min after infection amounted to about four times the amount present inside the cells at the time of infection. The quantity and rate of efflux of ATP was similar under aerobic and anaerobic conditions.
Superstructures of wet inactive chromatin and the chromosome surface
Authors: Basu Samarendra
Published: May 2005

Superpacking of chromatin and the surface features of metaphase chromosomes have been studied by SiO replication of wet, unstained, and unfixed specimens in an exceedingly thin (≤ 1 nm) aqueous layer, keeping them wet. Hydrophilic Formvar substrates allow controlled thinning of the aqueous layer covering the wet specimens. Whole mounts of chromatin and chromosomes were prepared by applying a microsurface spreading method to swollen nuclei and mitotic cells at metaphase.

The highest level of nucleosome folding of the inactive chromatin in chicken erythrocytes and rat liver nuclei is basically a second‐order superhelical organization (width 150–200 nm, pitch distance 50–150 nm) of the elementary nucleosome filament. In unfavorable environments (as determined by ionic agents, fixative, and dehydrating agents) this superstructure collapses into chains of superbeads and beads. Formalin (10%) apparently attacks at discrete sites of chromatin, which are then separated into superbeads. The latter consist of 4–6 nucleosomes and seemingly correspond to successive turns of an original solenoidal coil (width 30–35 nm), which forms the superhelical organization. When this organization is unfolded, eg, in 1–2 mM EDTA, DNAse‐sensitive filaments (diameter 1.7 nm) are seen to be wrapped around the nucleosomes.

The wet chromosomes in each metaphase spread are held to each other by smooth microtubular fibers, 20–30 nm in diameter. Before they enter into a chromsome, these fibers branch into 9–13 protofilaments, each 5 nm wide. The chromosome surface contains a dense distribution of subunits about 10–25 nm in diameter. This size distribution corresponds to that of nucleosomes and their superbeads. Distinct from this beaded chromosome surface are several smooth, 23–30‐nm‐diameter fibers, which are longitudinal at the centromere and seem to continue into the chromatid structure. The surface replicas of dried chromosomes do not show these features, which are revealed only in wet chromosomes.
Tubulin rings: Curved filaments with limited flexibility and two modes of association
Authors: Voter William A. , Erickson Harold P.
Published: May 2005

Tubulin rings have been previously identified as composed of linear polymers of tubulin subunits, equivalent to a protofilament in the microtubule wall but in a curved rather than a straight conformation. We have examined and measured a number of different ring structures obtained under different conditions. The preferred curvature is indicated by a single ring of 380 Å outside diameter. Radially double rings consist of two coplanar rings of 460 Å and 350 Å outside diameter, held together by a pattern of eight identical contacts between the 40 Å subunits in the inner and outer rings. In some circumstances a larger ring, 570 Å diameter, can be added to the outside, or a smaller ring, 240 Å diameter, may be added to the inside of the radially double ring, in both cases repeating the pattern of eight radial contacts. The distortion of the filament from its relaxed 380 Å diameter curvature apparently can be made without disrupting the longitudinal bond between subunits in the filament, but must be stabilized by the energy of the radial contacts. All of these rings (single and radially double and triple) are observed to associate axially to form pairs or in some cases larger stacks. The radially double rings or an axially associated pair of these (quadruple ring) may also associate to form crystals. These are thin plates, up to 100 μm in extent and several μm thick which have been of limited use so far in diffraction studies because of irregularities in the packing of adjacent rings.
Preliminary molecular replacement results for a crystalline gene 5 protein–deoxyoligonucleotide complex
Authors: Fitzgerald Paula M. D. , McPherson Alexander , Jurnak Frances A. , Wang Andrew H. J. , Kolpak Frank , Rich Alexander , Molineux Ian
Published: May 2005

Complexes of the gene 5 protein from bacteriophage fd with a variety of oligodeoxynucleotides, ranging in length from two to eight and comprised of several different sequences, have been formed and crystallized for X‐ray diffraction analysis. The crystallographic parameters of four different unit cells, all of which are based on hexagonal packing arrangements, indicate that the fundamental unit of the complex is composed of six gene 5 protein dimers. We believe this aggregate has 622 point group symmetry and is a ring formed by end‐to‐end closure of a linear array of six dimers. From our results we have proposed a double‐helix model for the gene 5 protein–DNA complex in which the protein forms a spindle or core around which the DNA is spooled. Currently 5.0‐Å X‐ray diffraction data from one of the crystalline complexes is being analyzed by molecular replacement techniques to obtain a direct image of the protein–nucleic acid complex.
Flow cytometry analysis of early DNA content changes in human and monkey cells following infection with simian virus 40
Authors: Lehman John M. , Klein Iris B. , Cram L. Scott
Published: May 2005

Simian virus 40 (SV40) is capable of inducing cellular DNA synthesis in permissive and nonpermissive cells. Utilizing flow cytometry, we analyzed the DNA content changes in two diploid human cell strains and two monkey cell lines. The osteogenesis imperfects (OI) human skin fibroblasts were induced into DNA synthesis, and within one to two cell generations, a polyploid cell population was produced. With WI‐38 phase II cells, a similar pattern of increased cycling of cells into DNA synthesis was observed; however, the majority (∼60%) of the cells were blocked in the G2 + M phase of the cell cycle. At later time intervals, an increase in the G1 population was demonstrated. The two monkey cell lines responded to SV40 virus with an accumulation of cells in the G2 + M phase of the cell cycle. Thus, two diploid human cell strains exhibited different cell cycle kinetics early after infection with SV40 virus. The one strain (WI‐38) behaved similarly to the two monkey cell lines studied. The other strain (OI) responded similarly to nonpermissive (transformin) cells infected with SV40 virus.
Endogenous cyclic AMP does not modulate transport of hexoses, nucleosides, or nucleobases in chinese hamster ovary cells
Authors: Wohlhueter Robert M. , Plagemann Peter G. W. , Sheppard J. R.
Published: May 2005

In a previous study we have demonstrated that neither extracellular nor intracellular cyclic adenosine monophosphate (AMP) levels directly affect the uptake of nucleosides, nucleobases, or hexoses by various types of cultured mammalian cells. Uptake of these nutrients into cells, however, involves two processes operating in tandem: facilitated transport across the membrane and intracellular phosphorylation; and uptake rates generally reflect the rates of substrate phosphorylation rather than of transport. In the present study we have examined the question of whether substrate transport per se is regulated by intracellular cyclic AMP. Initially various cell lines, grown both in suspension and monolayer culture, were screened for their cyclic AMP response to prostaglandin E1, isoproterenol, and inhibitors of cyclic AMP phosphodiesterase. Prostaglandin E1 treatment of Chinese hamster ovary cells was selected as the systems giving the largest and most consistent (50‐fold to 100‐fold) elevation of cyclic AMP. Rapid kinetic techniques were used to measure the transport of 3‐O‐methylglucose, thymidine, adenosine, hypoxanthine, and adenine in wild‐type cells and in mutant sublines incapable of phosphorylating these substrates. In no case was an increse in intracellular cyclic AMP accompanied by a singinficant change in the rate of transport of these substrates, although prostaglandin E1 slightly inhibited the transport of various substrates.
Synergistic stimulation of early events and DNA synthesis by phorbol esters, polypeptide growth factors, and retinoids in cultured fibroblasts
Authors: Dicker Phillip , Rozengurt Enrique
Published: May 2005

12‐O‐Tetradecanoyl‐phorbol‐13‐acetate (TPA), in the absence of serum, acts synergistically with a range of polypeptide growth factors to stimulate DNA synthesis in quiescent Swiss 3T3 cells. These growth factors include epidermal growth factor (EGF), insulin, and the peptide produced by BHK cells transformed by SV‐40 virus (fibroblast‐derived growth factor, FDGF). Retinoids also show mitogenic synergism with TPA or polypeptide growth factors. The spectrum of mitogenic synergisms displayed by TPA are similar to those of vasopressin, a pituitary peptide. However, TPA and vasopressin do not synergistically interact to stimulate DNA synthesis in quiescent 3T3 cells. This suggests that TPA and vasopressin act via an identical biochemical pathway. Several lines of evidence suggest rapid postreceptor convergence of the mitogenic mechanisms of action of the hormone and the tumor promotor. Thus, vasopressin and TPA both inhibit EGF binding to cellular receptors. Furthermore, TPA and vasopressin induce a similar array of early events in quiescent cells ‐ most strikingly, identical stimulation of Rb+ influx. Stimulation of ion flux is suggested as the possible convergence point of the pathway by which TPA and vasopressin act as mitogens.
NMR of fd coat protein
Authors: Cross T. A. , Opella S. J.
Published: May 2005

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the 13C and 1H nuclei of the coat protein. Both the 13C and 1H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance 13C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The 1H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a 1H spectra gives the pKas for the tyrosines.
Pyrimidine biosynthesis in normal and transformed cells
Authors: Uziel Mayo , Selkirk James
Published: May 2005

We have developed procedures for sensitive measurement of specific radioactivities of pyrimidine nucleosides excreted from cells in culture. The changes in the observed values reflect dilution of the added isotope through de novo biosynthesis of nonradioactive pyrimidine nucleosides or by shifting and equilibration of other nucleotide pools into the free uridine pool. It is thus possible to monitor uridine biosynthesis occurring in intact cells without destroying or disrupting the cell population. On comparing a series of normal and transformed lines, we have observed several growth‐dependent patterns of change in specific activity and levels of uridine excretion and the temporal appearance of these changes.

Hamster embyro fibroblasts slows pyrimidine biosynthesis at mid‐growth while the hamster cell line V79 continues to dilute the pyrimidine pool at about 7% of the rate observed during exponential growth at confluence. Both cells exhibit Urd excretion beginning at one‐half maximal growth.

Passageable normal rat liver cells (IARC‐20) also show a cessation of pyrimidine biosynthesis with a prior increase in uridine excretion. Two chemically transformed lines IARC‐28 and IARC‐19 derived from IARC‐20 show different patterns. IARC‐19 begins uridine excretion in early log growth and the specific activity continues to decrease at about 2% of the rate observed during exponential growth at confluence. The IARC‐28 cells also begin excretion in early log growth but pyrimidine biosynthesis stops at about midlog. This method may prove to be an additional aid in recognizing and differentiating transformed cells in culture that do not exhibit the transformed phenotype.
Isolation and characterization of a variant of B16‐mouse melanoma resistant to MSH growth inhibition
Authors: Niles Richard M. , Logue Mary P.
Published: May 2005

A variant of B‐16 F1 mouse melanoma was selected for its ability to survive and replicate in the presence of melanocyte‐stimulating hormone (MSH). Although the variant (MR‐4) was completely resistant to growth inhibition by MSH, cyclic AMP was still able to block cell replication. Tyrosinase activity in MR‐4 cells was considerably lower than in B‐16 F1 cells. MSH induced a twofold to three‐fold increase in tyrosinase activity in both cell types, but the absolute activity in MR‐4 remained significantly less than in the parental cells. MR‐4 cells were also found to have a markedly depressed cyclic AMP‐dependent protein kinase activity relative to B‐16 F1 cells. The protein kinase from both cell types was stimulated by cyclic AMP, but the level of MR‐4 kinase activity at maximal cyclic AMP concentrations remained considerably lower than B‐16 F1 kinase activity under the same conditions. In both cell types adenylate cyclase activity was markedly stimulated by MSH. When equal numbers of viable F1 and MR‐4 cells were injected subcutaneously into C57/B1 mice, the MR‐4 cells formed tumors earlier and killed the host sooner than the parental F1 cells. We conclude that the biochemical alteration which allows MR‐4 cells to replicate in the presence of MSH is a low level of tyrosinase activity, which in turn may be the result of low cyclic AMP‐dependent protein kinase activity.
Mechanisms of thrombin‐stimulated cell division
Authors: Cunningham Dennis D. , Glenn Kevin C. , Baker Joffre B. , Simmer Robert L. , Low David A.
Published: May 2005

Addition of highly purified thrombin t o cultures of several kinds of nondividing fibroblasts brings about cell division. This stimulation occurs in serum‐free medium, permitting studies on its mechanism under chemically defined conditions. Previous studies have shown that action of thrombin a t the cell surface is sufficient to cause cell division and that the proteolytic activity of thrombin is required for its mitogenic effect. These results prompted experiments which showed that there is a cell surface receptor for thrombin and that thrombin must hind to its receptor and cleave it to stimulate cell division. Some of the thrombin that hinds to its receptors becomes attached to them by a linkage that appears to be covalent. However, it is presently unknown whether this direct thrombin receptor complex plays a role in the stimulation.

These results raise a number of question that should be explored in future studies. They also provide a foundation on which to build hypotheses about tentative molecular mechanisms that might be involved in the stimulation. Knowledge that thrombin must cleave its receptor to bring about cell division suggests two alternative mechanisms for stimulation by proteolysis. In one the receptor is a negative effector which prevents cell division when it is intact, but not after it has been cleaved. Alternatively, a fragment of the receptor could be a positive effector. In this mechanism, proteolysis by thrombin would produce a specific receptor fragment which brings about cell proliferation. If every protease which cleaves the receptor also stimulates cell division, the receptor is probably a negative effector. In contrast, if certain proteases cleave the receptor but do not stimulate the cells, a fragment of the receptor is likely a positive effector. With negative regulation by the receptor, the controlling events would occur before proteolysis of it, and it might be possible to find putative regulatory molecules by identification of nearest neighbors of the receptor. This should be possible by using bifunctional crosslinking reagents. If a fragment of the thrombin receptor turns out to be a positive effector, it should be possible to identify and study fragments by analyzing the metabolic fate of the receptor. Techniques are now available for this kind of analysis and it should also be possible to determine whether receptor fragments remain in the membrane or whether they are translocated to specific sites within the cell. A critical question to be asked is which of these events and interactions involving the thrombin receptor are necessary for stimulation of cell division. It now appears that the best way to answer this question is to examine these events in a large number of cloned cell populations that are responsive or unresponsive to the mitogenic action of thrombin. If a thrombin‐mediated event occurs in all responsive clones but is altered or absent in sonie unresponsive clones, it is probably necessary for stimulation of cell division.
Specific interaction of cytochalasins with muscle and platelet actin filaments in vitro
Authors: Howard Thomas H. , Lin Shin
Published: May 2005

The cytochalasins (CE, CD, CB and H2CB) inhibit numerous cellular processes which require the interaction of actin with other structural and contractile proteins. In this report we describe the effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin. The cytochalasins decreased the viscosity of F‐actin solutions. The effect of H2CB, CB and CD on F‐actin viscosity was maximal at concentrations of 20–50μM and did not increase with time. In contrast, CE caused a progressive decrease in the viscosity of F‐actin solutions which was dependent upon the concentration of CE and the duration of incubation of the CE‐actin mixture. After two hours of incubation of drug‐actin mixtures, the relative effectiveness of the cytochalasins in reducing the viscosity of F‐actin was CE > CD > CB = H2CB. The effects of CD and CE were paralleled by morphologic changes in negatively stained actin filaments. The effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin were the same whether the drugs were added before or after the polymerization of the protein.

These studies show that the interaction of the cytochalasins with actin is highly specific. Because the relative potencies of these drugs for affecting motile processes and the relative affinities of the drugs for binding sites within a variety of cells are CE > CD > CB = H2CB, the effects of cytochalasins on actin described here may contribute to some of the biological effects of the drugs on motile processes.
Polypeptide subunits of dynein 1 from sea urchin sperm flagella
Authors: Bell Christopher W. , Fronk Earl , Gibbons I. R.
Published: May 2005

A high‐resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to show the presence, in both whole sperm and isolated flagellar axonemes, of eight polypeptides migrating in the 300,000–350,000 molecular weight range characteristic of the heavy chains of dynein ATPase. Previously, only five such chains have been discernible. Extraction of isolated axonemes for 10 min at 4°C with a solution containing 0.6 M NaCl, pH 7, releases a mixture of particles that separate, in sucrose density gradient centrifugation, into a major peak, dynein 1 ATPase, sedimenting at 21 S and a minor peak at 12–14S. The polypeptide compositions of these two peaks are different. The dynein 1 peak, which contains most of the protein on the gradient, contains approximately equal quantities of two closely migrating heavy chains, with a small amount of a third, more slowly migrating chain; no other heavy chains appear in this peak. Two groups of smaller polypeptides (three intermediate chains, within the apparent molecular weight range 76,000–122,000 and four newly discovered light chains, within the apparent molecular weight range 14,000–24,000) cosediment with the 21 S peak. The heavy chain composition of the 12–14S peak is more complex, all eight heavy chains occurring in approximately the same ratios as occur in intact axonemes.
Fibrous projections from the core of a bacteriophage T7 procapsid
Authors: Serwer Philip
Published: May 2005

A cylindrical core previously demonstrated in a bacteriophage T7 procapsid (capsid I) has been further examined by electron microscopy. Fibrous extensions of the core have been observed; these fibers appear to connect the core to the capsid I envelope. After infection of a nonpermissive host with bacteriophage T7 amber mutant in any gene coding for a core protein, the resulting lysates contained more noncapsid assemblies of capsid envelope protien than did wild‐type lysates; these assemblies had a mass two to at least 500 times greater than the mass of capsid I. This suggests that the internal core and fibers assist the assembly of subunits in the envelope of capsid I.