bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cross‐linkings between spectrin and band 3 in human erythrocyte membranes
Authors: Liu Shih‐Chun , Palek Jiri 
Published: May 2005

AbstractA 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

AbstractChemical 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

AbstractWe 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

AbstractWe 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

AbstractUsing 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

AbstractAddition 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

AbstractThe 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

AbstractSuperpacking 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

AbstractTubulin 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

AbstractComplexes 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

AbstractSimian 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

AbstractIn 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

Abstract12‐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

AbstractThe 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

AbstractWe 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.