bluuebunny/crossref_metadata_2025 huggingface dataset:
Trypanosoma brucei brucei: inhibition of glycosylation of the major variable surface coat glycoprotein by tunicamycin.
Authors: Strickler J E , Patton C L
Published: May 2006

Trypanosoma brucei brucei incorporates D-[3H]mannose into protein in vitro in a medium we describe here. The label appears entirely in glycoproteins with approximately 90% in the major variable surface coat glycoprotein (VSCG). Incorporation is linear for 60 min and usually continues for an additional 30 min although at a decreased rate. In the same medium incorporation of L-[14C]serine is linear for 90 min. Incorporation of [3H]mannose is completely inhibited by tunicamycin at concentrations above 100 ng/ml, indicating that the label is being added as part of an N-linked oligosaccharide. This is reflected by a 5% decrease in the apparent molecular weight of VSCG on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Cycloheximide inhibits incorporation of both mannose and serine, although the rates and extent of inhibition differ. Based on the effects of tunicamycin or cycloheximide on incorporation of either precursor, we suggest that N-linked glycosylation occurs subsequent to synthesis of the VSCG polypeptide.
Studies on the mechanism of capacitation: albumin-mediated changes in plasma membrane lipids during in vitro incubation of rat sperm cells.
Authors: Davis B K , Byrne R , Bedigian K
Published: May 2006

Plasma membrane isolated from rat sperm cells after incubation in vitro had a significantly lower cholesterol/phospholipid mole ratio when the medium contained serum albumin. Transfer of albumin-bound phospholipids to the membrane can largely account for this effect. The result is broadly consistent with a previously proposed model for albumin-induced destabilization of sperm membrane (capacitation) and its reversal by seminal plasma membrane vesicles. Albumin also decreased sialic acid and, more specifically, ganglioside levels, presumably by promoting release of sperm neuraminidase. Cholesteryl ester comprised up to 0.5 mol/mol of cholesterol in these plasma membrane preparations.
Taxol stabilizes microtubules in mouse fibroblast cells.
Authors: Schiff P B , Horwitz S B
Published: May 2006

Taxol, a potent inhibitor of human HeLa and mouse fibroblast cell replication, blocked cells in the G2 and M phase of the cell cycle and stabilized cytoplasmic microtubules. The cytoplasmic microtubules of taxol-treated cells were visualized by transmission electron microscopy and indirect immunofluorescence microscopy. More than 90% of the cells treated with 10 micro M taxol for 22 hr at 37 degrees C displayed bundles of microtubules that appeared to radiate from a common site (or sites), in addition to their cytoplasmic microtubules. Untreated cells that were kept in the cold (4 degrees C) for 16 hr lost their microtubules, whereas cells that were pretreated with taxol for 22 hr at 37 degrees C continued to display their microtubules and bundles of microtubules in the cold. Taxol inhibited the migration behavior of fibroblast cells, but these cells did not lose their ability to produce mobile surface projections such as lamellipodia and filopodia.
Discrete stages of human intrathymic differentiation: Analysis of normal thymocytes and leukemic lymphoblasts of T-cell lineage
Authors: Reinherz Ellis L. , Kung Patrick C. , Goldstein Gideon , Levey Raphael H. , Schlossman Stuart F.
Published: May 2006

A series of monoclonal antibodies was used to define three discrete stages of human intrathymic T-cell differentiation. The earliest stage was confined to <10% of thymocytes, which were·reactive with both OKT9 and OKT10. Subsequently, approximately 70% of human thymocytes acquired a thymocyte-restricted antigen, OKT6, lost OKT9 antigen, and expressed reactivity with OKT4 and OKT5. These last two monoclonal antibodies were previously shown to define inducer (helper) and cytotoxic/suppressor populations, respectively, in peripheral blood. The OKT4
+
, OKT5
+
, OKT6
+
“common” thymocyte population represents the majority of thymocytes and accounts for more than 70% of thymocytes. With further maturation, thymocytes lose OKT6 reactivity, segregate into OKT4
+
and OKT5
+
subsets, and acquire reactivity with OKT3 (and OKT1). This latter stage corresponds to the more functionally mature subset. The possible relationship of acute lymphoblastic leukemia of T-cell lineage to these proposed stages of intrathymic differentiation was determined. Analysis of 25 tumor populations showed that 21 could be related to one or another differentiative stage. The majority (15/21) were derived from an early thymocyte or prothymocyte subpopulation, 5/25 were derived from a common thymocyte subpopulation, and 1/25 was derived from a mature (OKT3
+
) subpopulation. These data suggest that is it now possible to define stages of T-cell differentiation that can be related to T-cell malignancies in humans.
Sodium and potassium salt stimulation of taste receptor cells: An allosteric model
Authors: Mooser Gregory
Published: May 2006

Stimulation of taste receptors with sodium chloride, sodium acetate, sodium propionate, and the respective potassium salts gave concentration-response profiles, measured electrophysiologically, which are remarkably consistent with a two-state allosteric mechanism. The allosteric constant or equilibrium constant for the transition between the active and inactive receptor states is low, resulting in a condition in which small differences in ion affinities for the two states are sufficient to significantly alter the equilibrium. Receptor activators, such as sodium ion, displaced the equilibrium toward the active receptor state by virtue of a higher affinity for that state, whereas receptor inhibitors, such as acetate and propionate ions, displaced the equilibrium in the opposite direction as a result of a higher affinity for the inactive state. The low allosteric constant increased about 10-fold after treatment with the protein modification reagent dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide, resulting in a marked reduction in the response to sodium chloride and sodium propionate without a significant change in ion affinities. In order to fully resolve the potassium response characteristics, it was necessary to consider both a potassium activation site and a potassium inhibition site. Analysis of the response from sodium chloride/potassium chloride mixtures showed that sodium ion is competitive with potassium binding at the activation site but not the inhibition site. With potassium propionate as the stimulus, the effect of both a receptor activator and a receptor inhibitor was quantitatively consistent with depression of the response below a water baseline level at low stimulus concentrations. Estimation of active and inactive state dissociation constants for each anion and cation permitted accurate prediction of the response magnitude for a range of cation ratios in sodium chloride/potassium chloride mixtures and anion ratios in sodium chloride/sodium propionate mixtures. The association of salty taste with receptor activators and bitter taste with receptor inhibitors may be relevant to the generation of these taste qualities.
Somatostatin-like immunoreactivity in the retina.
Authors: Yamada T , Marshak D , Basinger S , Walsh J , Morley J , Stell W
Published: May 2006

A substance with somatostatin-like immunoreactivity (SLI) was found in extracts of goldfish, frog, and cow retina. Dilutions of retinal SLI parallel the standard curve for radioimmunoassay obtained with synthetic somatostatin. Chromatography of goldfish retinal extract on Sephadex G-50 revealed two peaks of SLI, one that coeluted with synthetic somatostatin and one that eluted as a larger molecule. Incubation in 8 M urea did not alter the chromatographic pattern of the extract. SLI was present in extracts of frog optic nerve and tectum in concentrations higher than those found in the retina. In goldfish retina, SLI was localized by immunofluorescence to four types of processes in the inner plexiform layer; their origins could be traced to three classes of SLI-containing cell bodies in the proximal row of the inner nuclear layer and one class in the ganglion cell layer. Localization of SLI to cells of the retina and characterizations of the molecular forms of retinal SLI suggest that the retina is a promising model system for studies on the potential neurotransmitter function of somatostatin.
Chloride-activated passive potassium transport in human erythrocytes.
Authors: Dunham P B , Stewart G W , Ellory J C
Published: May 2006

Passive K+ transport in human erythrocytes (defined as ouabain-insensitive transport) was inhibited 70% by replacement of Cl- by several permeant monovalent anions. The Vmax of Cl--dependent K+ influx was 1.14 mmol . liter-1, hr-1; its apparent Km for K+ was 4.7 mM. There was a much smaller component of Na+ influx dependent on Cl- (Vmax, 0.23 mmol . liter-1 . hr-1). Furosemide and other inhibitors of Cl- transport inhibited passive K+ transport to the same extent as replacement of Cl-, but 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, a specific inhibitor of anion exchange in erythrocytes, was ineffective. The Cl--dependent K+ transport, which may be K+/Cl- cotransport, could reflect a mechanism for regulating cell volume.
Population oscillations and energy reserves in planktonic cladocera and their consequences to competition
Authors: Goulden Clyde E. , Hornig Linda L.
Published: May 2006

Time lags in an individual's response to decreased food densities are responsible for oscillations in laboratory populations of
Daphnia galeata mendotae
. Visible energy reserves of triacylglycerols accumulate in the body of animals at low population densities when food is abundant and are later metabolized at high population densities when food is scarce to temporarily sustain activity and reproduction. After these energy reserves are metabolized many individuals, primarily juveniles, starve and die. The length of the time lags is a function of the amount of energy reserve accumulated in the individual. Because this sustained activity and reproduction further decreases food concentrations to very low levels, individuals of a second smaller-body-sized species,
Bosmina longirostris
, that do not have sufficient energy reserves quickly starve and die. Thus the accumulation of energy reserves in individuals underlies the time lags important in causing population oscillations and has consequences to interspecific competition.
X-ray-structure of a cytidylyl-3',5'-adenosine-proflavine complex: a self-paired parallel-chain double helical dimer with an intercalated acridine dye.
Authors: Westhof E , Sundaralingam M
Published: May 2006

The non-self-complementary dinucleoside monophosphate cytidylyl-3',5'-adenosine (CpA) forms a base-paired parallel-chain dimer with an intercalated proflavine. The dimer complex possesses a right-handed helical twist. The dimer helix has an irregular girth with a neutral adenine-adenine (A-A) pair, hydrogen-bonded through the N6 and N7 sites (C1'...C1' separation of 10.97 A), and a triply hydrogen-bonded protonated cytosine-cytosine (C-C) pair with a proton shared between the base N3 sites (Cl'...Cl' separation of 9.59 A). The torsion angles of the sugar-phosphate backbone are within their most preferred ranges and the sugar puckering sequence (5' leads to 3') is C3'-endo, C2'-endo. There is also a second proflavine molecule sandwiched between CpA dimers on the 21-axis. Both proflavines are necessarily disordered, being on dyad axis, and this suggests possible insights into the dynamics of intercalation of planar drugs. This structure shows that intercalation of planar drugs in nucleic acids may not be restricted to antiparallel complementary Watson-Crick pairing regions and provides additional mechanisms for acridine mutagenesis.
Cell wall appositions and plant disease resistance: Acoustic microscopy of papillae that block fungal ingress
Authors: Israel H. W. , Wilson R. G. , Aist J. R. , Kunoh H.
Published: May 2006

Plant cells react to localized stress by forming wall appositions outside their protoplasts on the inner surface of their cellulose walls. For many years it has been inferred that appositions elicited by encroaching fungi, termed “papillae,” may subsequently also deter them and thus represent a disease-resistance mechanism. Recently, it has been shown that preformed, oversized papillae, experimentally produced in coleoptile cells of compatible barley,
Hordeum vulgare
, can completely prevent direct entry of
Erysiphe graminis
f. sp.
hordei
that ordinarily penetrates and causes disease. To discover how these papillae may function, acoustic microscopy was used to contrast their
in vivo
elastic properties with those of ineffective normal papillae and contiguous cell wall. Raster and line scans showed intense acoustic activity at sites of preformed papillae; scans in selected focal planes identified this activity with the papillae, not with subtending cell wall. Minimal acoustic activity was found in normal papillae. It is suggested that some wall appositions could serve in disease resistance as viscoelastic barriers to mechanical forces exerted by the special penetration structures of advancing pathogenic fungi.
Localization of ecdysterone on polytene chromosomes of Drosophila melanogaster.
Authors: Gronemeyer H , Pongs O
Published: May 2006

Ecdysterone has been crosslinked in situ to polytene chromosomes of salivary glands of Drosophila melanogaster by photoactivation. The crosslinked hormone has been localized on the chromosomes by indirect immunofluorescence microscopy. At different developmental stages the hormone was detected at different chromosomal loci. These chromosomal sites correspond to ecdysterone-inducible puff sites. Thus, the hormone binds directly to chromosomal loci, whose transcription depends on the presence of the hormone. Ecdysterone was also crosslinked to one puff site that regresses during larval development. This indicates that (i) hormone binding to polytene chromosomes activates transcription at specific loci, and (ii) hormone-dependent regression of intermolt puff 68C is mediated by direct binding of ecdysterone. Ecdysterone was not detected on puff sites that are independent of hormone action or in chromosomal interbands. After heat shock, no ecdysterone became crosslinked to polytene chromosomes.
Radioimmunoassay for the vitamin K-dependent protein of bone and its discovery in plasma.
Authors: Price P A , Nishimoto S K
Published: May 2006

The vitamin K-dependent protein of bone has been detected in human plasma by radioimmunoassay at 4.5 ng per ml. The plasma protein has the same apparent molecular weight as the pure bone Gla protein (BGP) and other studies indicate the plasma protein is probably the intact bone protein. BGP also has been detected in bovine serum by radioimmunoassay. The bovine serum levels of BGP decrease with developmental age from 200 ng per ml in fetal calves to 26 ng per ml in adult cows. The implications of the discovery of BGP in plasma to the function of this unique protein are discussed. This assay employs rabbit antibody directed against calf BGP and has a sensitivity of 0.1 ng. The antibody crossreacts with purified human BGP but not with BGP from rat or rabbit bone. Studies with peptides of known structure derived from enzymatic digests of BGP indicate that the rabbit antibody recognizes the COOH-terminal region of the 49-residue calf bone protein.
2,3-
trans
-3,4-
trans
-3,4-Dihydroxy-L-proline: An amino acid in toxic peptides of
Amanita virosa
mushrooms
Authors: Buku A. , Faulstich H. , Wieland T. , Dabrowski J.
Published: May 2006

Among the four possible stereoisomers of 3,4-dihydroxy-L-proline,2,3-
trans
-3,4-
trans
-3,4-dihydroxy-L-proline (IV) had not been found in nature previously. It has now been detected as a component of virotoxins, toxic peptides of
Amanita virosa
mushrooms. Because periodate failed to effect an oxidative glycol splitting reaction, the two hydroxyl groups in positions 3 and 4 were expected to be in a
trans
configuration. Furthermore, the formation of a 4-lactone on treatment with acids pointed to the carboxyl group and the hydroxyl group at position 4 being in a
cis
configuration. These results are in agreement with structure IV only. Final proof for structure IV was given by NMR spectroscopy and direct comparison with the 2,3-
cis
-3,4-
trans
-3,4-dihydroxy-L-proline isomer.
Asymmetry of interoceanic fresh-water and heat fluxes
Authors: Stommel Henry
Published: May 2006

According to recent hydrological and meteorological studies, the meridional flux of fresh water and heat is remarkably different from ocean to ocean. These fluxes have been found to be consistent with the temperature and salinity distribution in the northern hemisphere. However, an attempt to map these fluxes on the temperature-salinity plane of a southern latitude leads to such large amplitudes of water-mass volume flux that it seems that there may be something wrong about them.
Targeted deletions of sequences from closed circular DNA.
Authors: Green C , Tibbetts C
Published: May 2006

Closed circular DNA interacts with complementary sequences of single-stranded DNA to form displacement loop (D loop) structures in vitro. The site of D-loop formation can be directed by using single-stranded DNA derived from a selected restriction fragment. Circular DNA containing a D loop can then be linearized by cleavage with endonuclease S1. This cleavage appears to remove a limited number of nucleotides from each strand of the circular DNA substrate. Incubation with polynucleotide ligase followed by propagation in vivo leads to circular DNA molecules that bear small, single deletions in the region of the single-stranded DNA sequence chosen for the formation of the D loops. We have utilized these manipulations of DNA to construct tetracycline-sensitive deletion mutants of plasmid pBR322. The level of mutagenesis obtained by the procedure is sufficiently high that selective growth and screening procedures are not necessary for the isolation or identification of mutants. The frequency, variety, and small size of the deletions obtained within the selected target regions present considerable advantage for genetic and biochemical analysis. The method is quite general in rationale and should be immediately applicable to phage and viruses having infectious circular DNA genomes or recombinant DNA species propagated in circular plasmid vectors.
Functional domain structure of fibronectin.
Authors: Sekiguchi K , Hakomori S
Published: May 2006

Structural domains of fibronectin (FN) and their ability to associate with cell surface components have been systematically investigated. Plasma FN was cleaved into three structural domains (Mr 150,000-140,000, 40,000, and 32,000) by sequential digestion with trypsin and thermolysin. A single digestion with thermolysin alone generated Mr 150,000-140,000, 40,000, and smaller fragments. With the inclusion of thermolysin, but not with other proteases, one can, with a high yield dissect FN simultaneously into three clearly distinctive functional domains. Of three major fragments only the Mr 40,000 fragment bound to a gelatin column; this fragment contained essentially all of the carbohydrates present in the original FN. In contrast, heparin-binding sites were localized on both the Mr 150,000-140,000 and 32,000 fragments but not on the Mr 40,000 fragment. Only the Mr 150,000-140,000 fragments and intact FN promoted cell spreading, whereas the Mr 40,000 and 32,000 fragments could induce cell attachment but failed to promote cell spreading. These results indicate that FN is composed of (at least) three structural domains that are functionally distinct from each other.
Evidence for participation of transglutaminase in receptor-mediated endocytosis.
Authors: Levitzki A , Willingham M , Pastan I
Published: May 2006

We report evidence that the enzyme transglutaminase (glutaminyl-peptide gamma-glutamyltransferase; R-glutaminyl-peptide:amine gamma-glutamyltransferase, EC2.3.2.13) participates in receptor-mediated endocytosis. Clustering and internalization of rhodamine-labeled alpha 2-macroglobulin (R alpha 2 M) in normal rat kidney (NRK) cells is inhibited by a wide spectrum of compounds that inhibit transglutaminases, including that from NRK cells. The pattern of clustering inhibition resembles the pattern of transglutaminase inhibition as follows: (i) The most potent transglutaminase inhibitors are dansylcadaverine and the transglutaminase-directed affinity label N-benzyloxy-carbonyl-5-diazo-4-oxonorvaline p-nitrophenyl ester; these were also the most potent inhibitors of clustering and internalization of R alpha 2M. (ii) The inhibition of clustering of R alpha 2M occurs in the same concentration range as that required for transglutaminase inhibition. (iii) Linear primary amines are more effective blockers than the iso-chain primary amines. (iv) The transglutaminase affinity label N-benzyloxycarbonyl-5-diazo-4-oxonorvaline p-nitrophenyl ester irreversibly inhibits a significant fraction of the NRK transglutaminase and the clustering and internalization of R alpha 2M. A closely related compound, N-trifluoroacetyl-6-diazo-5-oxonorleucine ethyl ester, does not significantly inhibit transglutaminase or clustering and internalization. (v) Clustering and internalization is inhibited 10-fold more effectively by the heptapeptide Ac-Gly2-LLeu-LLys-Gly3 than by the heptapeptides Ac-Gly2-LLeu-DLys-Gly3 or AcGly3-DLys-DLeu-Gly2. This is the pattern of stereospecificity for the inhibition of purified transglutaminases.
Proportion of fetal hemoglobin synthesis decreases during erythroid cell maturation.
Authors: Chui D H , Wong S C , Enkin M W , Patterson M , Ives R A
Published: May 2006

Peripheral blood mononuclear cells from pregnant and postpartum women were cultured in vitro with erythropoietin. Burst-forming unit (BFU-E)-derived erythroid colonies composed of immature erythroblasts with low hemoglobin contents were observed by day 8 of culture. By day 12 of culture, numerous BFU-E-derived erythroid colonies with high hemoglobin contents were present. The gamma/(gamma + beta) globin synthetic ratio was approximately 12% in the early cultures and 6% in the late cultures, indicating that the proportion of fetal hemoglobin synthesis decreases during erythroid cell maturation. These studies also reveal that the capacity fof fetal hemoglobin production by peripheral blood BFU-E in vitro is not altered during pregnancy.
Superoxide dismutase: correlation with life-span and specific metabolic rate in primate species.
Authors: Tolmasoff J M , Ono T , Cutler R G
Published: May 2006

Much evidence now suggests that superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) may be a major intracellular protective enzyme against oxygen toxicity by catalyzing the removal of the superoxide radical. We examined the possible role this enzyme may have in determining the life-span of primate species. Superoxide dismutase specific activity levels were measured in cytoplasmic fractions of liver, brain, and heart of 2 rodent and 12 primate species. These species had maximum life-span potentials ranging from 3.5 to 95 years. Liver, brain, and heart had similar specific activity levels for a given species, but the levels for different species varied over 2-fold, with man having the highest level. No general correlation was found in the levels with life-span. However, the ratio of superoxide dismutase specific activity to specific metabolic rate of the tissue or of the whole adult organism was found to increase with increasing maximum lifespan potential for all the species. This correlation suggests that longer-lived species have a higher degree of protection against by-products of oxygen metabolism.
Localization of loci for hypoxanthine phosphoribosyltransferase and glucose-6-phosphate dehydrogenase and biochemical evidence of nonrandom X chromosome expression from studies of a human X-autosome translocation.
Authors: Pai G S , Sprenkle J A , Do T T , Mareni C E , Migeon B R
Published: May 2006

We report a unique and complex karyotypic rearrangement involving chromosomes X, 3, 7, and 21. Blood cells and fibroblasts from the proband do not express the maternal allele for glucose-6-phosphate dehydrogenase (G6PD), providing biochemical evidence for nonrandom expression of X-linked genes in balanced X-autosome translocations. The break point on the X chromosome, at the junction of Xq27-Xq28, separates the loci for hypoxanthine phosphoribosyltransferase (HPRT) and G6PD. Studies of mouse-human hybrids derived from the proband's cells indicate that G6PD, at q28, is clearly distal to all other X loci now assigned. From these and previous studies, we can localize HPRT to that segment between Xq26 and Xq27. The studies also provide further evidence for the stability of the inactive X phenotype in hybrid cells.
Isolation and localization of DNA segments from specific human chromosomes
Authors: Gusella James F. , Keys Cheryl , Varsanyi-Breiner Aviva , Kao Fa-Ten , Jones Carol , Puck Theodore T. , Housman David
Published: May 2006

Recombinant DNA techniques have been combined with somatic cell genetic methods to identify, isolate, and amplify fragments of human DNA localized at specific regions of human chromosome 11 selected as a model system. A library of genomic DNA segments has been constructed, in λ Charon 4A bacteriophage, from the DNA of a somatic cell hybrid carrying a portion of human chromosome 11 on a Chinese hamster ovary cell background. Using a nucleic acid hybridization technique that distinguishes human and Chinese hamster interspersed, repetitive DNA, we have been able to distinguish recombinant phages carrying DNA segments of human origin from recombinant phages carrying DNA segments of Chinese hamster origin. We have isolated 50 human DNA segments thus far and have characterized 5 in detail. For each DNA segment characterized, a subsegment that carries no repetitive human DNA sequences has been identified. These segments have been used as hybridization probes in experiments that localize the DNA fragment on the chromosome. In each case an unequivocal chromosomal localization has been obtained with reference to a panel of hybrid cell clones each of which carries a deletion of a portion of the short arm of chromosome 11. At least one DNA segment has been identified which maps to each of the four regions on the short arm defined by the panel of hybrid cell clones used. The approaches described here appear to be general. They can be extended to produce a fine structure map of human chromosome 11 and other human chromosomes. This approach promises implications for human genetics generally, for the human genetic diseases, and possibly for understanding of gene regulation in normal and abnormal differentiation.
Structure and organization of transposable of transposable mating type cassettes in Saccharomyces yeasts.
Authors: Strathern J N , Spatola E , McGill C , Hicks J B
Published: May 2006

Cell type in Saccharomyces yeasts is regulated by two transposable blocks of DNA, the a and alpha cassettes. There are three loci where either cassette can exist. At the HML and HMR loci the cassettes are not expressed. The cassette at the MAT locus is expressed and controls the cell type. Changes of cell type involve transposition-substitution of cassettes from HML or HMR into MAT. We recently reported the molecular cloning of the alpha cassette at the HML locus, HML alpha, and showed that it contained sequences homologous to HMR and MAT. Using HML alpha as a hybridization probe, we have isolated HMLa, HMR alpha, HMRa, MAT alpha, and MATa. Heteroduplex analysis and restriction endonuclease mapping studies indicate that the a and alpha cassettes differ by a substitution corresponding to about 750 base pairs in alpha and about 600 base pairs in a. The HML, HMR, and MAT loci have regions of homology flanking the position of the a versus alpha substitution. We have used specific chromosome rearrangements fusing MAT and HML and MAT with HMR to orient the cloned sequences on the genetic map and have found that all three genes have the same left-to-right polarity on the chromosome.
Persistence of increased platelet cyclic AMP induced by prostaglandin E1 after removal of the hormone.
Authors: Sinha A K , Colman R W
Published: May 2006

Prostaglandin E1 (PGE1) covalently linked to omega-NH2-hexyl-agarose (PGE1-hexyl-agarose) stimulates the activity of human platelet adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] 3-fold over control level at 60 sec when stirred at 1200 rpm at 37 degrees C. The time course exhibited a lag phase of 20 sec, a rapid increase to a maximum plateau between 60 and 80 sec, and a more gradual decrease to basal level at about 120 sec. During this entire period of incubation PGE1-hexyl-agarose could be easily separated; therefore it bound only transiently to the platelet. No prostaglandin(s) were found to be released into plasma. The stimulation of adenylate cyclase activity by the insolubilized hormone was dependent on the rate of collision as influenced by the speed of stirring. Removal of the insoluble PGE1 before the end of the lag period (10 sec) prevented the increase of adenylate cyclase. In contrast, separation of PGE1-hexyl-agarose from platelets by filtration after the lag period (at 30 or 40 sec) allowed the stimulation of the enzymatic activity to continue to completion in the absence of the hormone. The results suggest that PGE1 initiates molecular events leading to an increase of adenylate cyclase that do not require its continued presence for maintenance of the stimulated state.
Reconstitution of purified acetylcholine receptors with functional ion channels in planar lipid bilayers.
Authors: Nelson N , Anholt R , Lindstrom J , Montal M
Published: May 2006

Acetylcholine receptor, solubilized and purified from Torpedo californica electric organ under conditions that preserve the activity of its ion channel, was reconstituted into vesicles of soybean lipid by the cholate-dialysis technique. The reconstituted vesicles were then spread into monolayers at an air-water interface and planar bilayers were subsequently formed by apposition of two monolayers. Addition of carbamoylcholine caused an increase in membrane conductance that was transient and relaxed spontaneously to the base level (i.e., became desensitized). The response to carbamoylcholine was dose dependent and competitively inhibited by curare. Fluctuations of membrane conductance corresponding to the opening and closing of receptor channels were observed. Fluctuation analysis indicated a single-channel conductance of 16 +/- 3 pS (in 0.1 M NaCl) with a mean channel open time estimated to be 35 +/- 5 ms. Thus, purified acetylcholine receptor reconstituted into lipid bilayers exhibited the pharmacological specificity, activation, and desensitization properties expected of this receptor in native membranes.
Novel features in the genetic code and codon reading patterns in
Neurospora crassa
mitochondria based on sequences of six mitochondrial tRNAs
Authors: Heckman Joyce E. , Sarnoff Joshua , Alzner-DeWeerd Birgit , Yin Samuel , RajBhandary Uttam L.
Published: May 2006

We report the sequences of
Neurospora crassa
mitochondrial alanine, leucine
1
, leucine
2
, threonine, tryptophan, and valine tRNAs. On the basis of the anticodon sequences of these tRNAs and of a glutamine tRNA, whose sequence analysis is nearly complete, we infer the following: (
i
) The
N. crassa
mitochondrial tRNA species for alanine, leucine
2
, threonine, and valine, amino acids that belong to four-codon families (GCN, CUN, ACN, and GUN, respectively; N = U, C, A, or G) all contain an unmodified U in the first position of the anticodon. In contrast, tRNA species for glutamine, leucine
1
, and tryptophan, amino acids that use codons ending in purines (CA
G
A
, UU
G
A
, and UG
G
A
, respectively) contain a modified U derivative in the same position. These findings and the fact that we have not detected any other isoacceptor tRNAs for these amino acids suggest that
N. crassa
mitochondrial tRNAs containing U in the first position of the anticodon are capable of reading all four codons of a four-codon family whereas those containing a modified U are restricted to reading codons ending in A or G. Such an expanded codon-reading ability of certain mitochondrial tRNAs will explain how the mitochondrial protein-synthesizing system operates with a much lower number of tRNA species than do systems present in prokaryotes or in eukaryotic cytoplasm. (
ii
) The anticodon sequence of the
N. crassa
mitochondrial tryptophan tRNA is U
*
CA and not CCA or CmCA as is the case with tryptophan tRNAs from prokaryotes or from eukaryotic cytoplasm. Because a tRNA with U
*
CA in the anti-codon would be expected to read the codon UGA, as well as the normal tryptophan codon UGG, this suggests that in
N. crassa
mitochondria, as in yeast and in human mitochondria, UGA is a codon for tryptophan and not a signal for chain termination. (
iii
) The anticodon sequences of the two leucine tRNAs indicate that
N. crassa
mitochondria use both families of leucine codons (UU
A
G
and CUN; N = U, C, A, or G) for leucine, in contrast to yeast mitochondria [Li, M. & Tzagoloff, A. (1979)
Cell
18, 47-53] in which the CUA leucine codon and possibly the entire CUN family of leucine codons may be translated as threonine.
Specificity of oligodeoxynucleotide binding of mouse uterine cytosol estradiol receptors.
Authors: Kumar S A , Beach T A , Dickerman H W
Published: May 2006

The relative capacities of oligodeoxynucleotides, covalently linked to cellulose, to bind estradiol receptor complexes (E2R) of mouse uterine cytosol have been shown to follow the order oligo(dG) > oligo(dT) greater than or equal to oligo(dC) > oligo(dA). The E2R . oligo(dT)-cellulose-binding reaction is more sensitive to Cibacron blue F3GA than is E2R . oligo(dG)-cellulose or oligo(dC)-cellulose binding. Preformed E2R . oligo(dT)- or oligo(dC)-cellulose complexes are dissociated more readily by lower concentrations of KCl or Cibacron blue F3GA than is the E2R . oligo(dG)-cellulose complex. Preincubation of E2R at 37 degrees C results in a rapid loss of binding ability towards oligo(dT)- and oligo(dC)-cellulose, while up to 90% of the binding ability to oligo(dG)-cellulose is retained. On the basis of the differential sensitivities of E2R to temperature and the inhibition by Cibacron blue F3GA of the binding reaction, it is suggested that the polynucleotide-binding domain consists of two types of subsites, one of which has a higher affinity for oligo(dG) residues and the other of which recognizes oligo(dT), oligo(dC), and, to a lesser extent, oligo(dA).
The cytoskeleton of digitonin-treated rat hepatocytes.
Authors: Fiskum G , Craig S W , Decker G L , Lehninger A L
Published: May 2006

Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes.
Continuous, clonal, insulin- and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor.
Authors: Gazdar A F , Chick W L , Oie H K , Sims H L , King D L , Weir G C , Lauris V
Published: May 2006

Continuous cell lines that secrete both insulin and somatostatin were established by two cooperating groups of investiagtors from a serially transplantable, radiation-induced, rat islet cell tumor. The cell lines, named RIN-r and RIN-m, were initiated from tumors maintained in inbred rats or in athymic nude mice, respectively. The cultured cells are epithelioid, free of fibroblast contamination, and can be cloned. They have a hypodiploid chromosome number, are tumorigenic, and posses amine-handling properties, including high levels of L-dopa decarboxylase and formaldehyde-induced fluorescence. Preliminary analysis of clones revealed a spectrum of insulin secretion from undetectable to relatively high. These clonal cell lines provide important systems to study the biology of insulin and somatostatin.
Experimental animal model for mucopolysaccharidosis: suramin-induced glycosaminoglycan and sphingolipid accumulation in the rat.
Authors: Constantopoulos G , Rees S , Cragg B G , Barranger J A , Brady R O
Published: May 2006

Intracerebral injection of the trypanocidal drug suramin in rats caused the formation of membranous neuronal and neuroglial inclusions. Here we show that intravenous administration suramin, 500 mg/kg, to 2-month-old rats causes a 5- to 8-fold increase of glycosaminoglycan concentration in the liver within 10 days and a 6-fold increase in urinary glycosaminoglycan excertion. The excess glycosaminoglycans consist of heparan sulfate and dermatan sulfate. Intracerebral injection of 250 micrograms of suramin results in a small increase of glycosaminoglycan and larger increase of ganglioside GM2, GM3, and GD3 concentrations in the treated region of the brain. The activities of the lysosomal enzymes iduronate sulfatase, beta-glucuronidase, and hyaluronidase in the liver of the suramin-treated mature rats were consistently decreased, whereas those of alpha-L-iduronidase, heparan N-sulfatase, arylsulfatase B, and others were considerably increased. The activity of iduronate sulfatase was completely inhibited in vitro by suramin at concentrations of 50 microM or higher. The activity of beta-glucuronidase was also strongly inhibited by low concentrations of suramin, but this inhibition was partially decreased at higher concentrations of the drug. The inhibition of both enzymes by suramin was noncompetitive. The suramin-treated rat may be a useful experimental animal model of mucopolysaccharidosis.
Field-induced trapping as a probe of dimensionality in molecular crystals
Authors: Scher H. , Alexander S. , Montroll E. W.
Published: May 2006

The combined effect of an electric field and reduced dimensionality on the hopping motion of a charge carrier in an anisotropic molecular crystal is determined. New, exact expressions for
S
(
t
), the number of distinct sites occupied by a carrier in time
t
, are presented for two-dimensional and three-dimensional anisotropic lattices. The general results are used to model the unusual phenomenon of field-induced charge carrier trapping reported to occur in a quasi-one-dimensional molecular crystal. An analytic expression for the carrier lifetime τ, as a function of the anisotropy in the molecular hopping rates and electric field, is developed and is shown to be in excellent agreement with experimental results. A major conclusion of the theory is that
E
c
, the electric field characterizing the
E
dependence of τ, can be a more direct probe of the intrinsic anisotropy than is the observed mobility ratio.
The pitch of chromatin DNA is reflected in its nucleotide sequence.
Authors: Trifonov E N , Sussman J L
Published: May 2006

A correlation analysis of chromatin DNA nucleotide sequences reveals the clear tendency of some of the dinucleotides to be repeated along the sequences with periods of 3 and about 10.5 bases. This latter period, which is equal within experimental error to recent estimates of the pitch of the DNA double helix [Wang, J. (1979) Proc. Natl. Acad. Sci. USA 76, 200-203; Trifonov, E. & Bettecken, T. (1979) Biochemistry 18, 454-456] is interpreted as a reflection of the deformational anisotropy of the DNA molecule that facilitates its smooth folding in chromatin.
Posttranscriptional regulation of an erythromycin resistance protein specified by plasmic pE194.
Authors: Shivakumar A G , Hahn J , Grandi G , Kozlov Y , Dubnau D
Published: May 2006

Induction of the synthesis of a plasmid-encoded polypeptide (E3) by erythromycin is known to be required for the inducible expression of resistance to the macrolide-lincosamide-streptogramin B group of antibiotics in Bacillus subtilis strains carrying the plasmid pE194. This resistance is mediated by a specific N6-dimethylation of adenine in the 23S rRNA of the large ribosomal subunit. We show in this report that E3 induction is regulated posttranscriptionally in the sense that it can occur when RNA synthesis is blocked and that induction is accompanied by an increase in the functional half-life of E3 mRNA but not of the mRNA species that code for the remaining four known pE194 polypeptides. The induction of E3 is subject to feedback regulation and involves the ribosome. Modification of the erythromycin binding site on the ribosome by methylation or by mutation interferes with induction.
Can the binding of Ca2+ to two regulatory sites on troponin C determine the steep pCa/tension relationship of skeletal muscle?
Authors: Brandt P W , Cox R N , Kawai M
Published: May 2006

The relationship between tension and Ca2+ concentration in single skinned muscle fibers has been determined with a high density of experimental points and the data have been fitted by a least squares method to the Hill equation. We find that the mean Hill coefficient for the slope of the tension/Ca2+ relationship is between 5 and 6, and the pKd is about 5.9. Because there are four Ca2+ binding sites on troponin C, and only two of these regulate hydrolysis of MgATP, we conclude that the regulation of tension by Ca2+ binding is greatly modified by other factors. One important factor is the time required for a cross-bridge to complete a cycle once initiated, relative to the time Ca2+ remains bound to troponin C. The pCa/tension relationship will shift to higher pCa values as the ratio of cross-bridge cycle time to the Ca2+ bound time increases. For example, the pCa/tension curve may progressively shift to the left with increase in tension because strain in the myofilament lattice progressively increases the cycle time. This left shift will produce a pCa/tension relationship that is steeper than the actual Ca2+ binding curve. The anticipated shift of the pCa/tension curve with cycle time also bears on interpretations of earlier experiments on the "active state" and on the effects of Ca2+ on the maximal velocity of shortening.
Bioenergetic aspects and polymer length distribution in steady-state head-to-tail polymerization of actin or microtubules.
Authors: Hill T L
Published: May 2006

Wegner's theory of steady-state head-to-tail polymerization of actin (or microtubules) is extended somewhat in order to show the explicit role of the ATP (or GTP) free energy of hydrolysis (X) in the steady-state kinetics. The monomer flux and the ATP flux can both be expressed in terms of X and rate constants of the model. Both fluxes approach zero as X leads to 0 (by variation of the concentrations of ATP, ADP, and Pi); this limit corresponds to ATP equilibrium. The dependence of rate constants on these concentrations is examined. Free energy levels of the monomer kinetic cycle and the rate of free energy dissipation are discussed. The steady-state polymer length distribution is derived for a special case.
Synthesis of new proteins associated with the induction of interferon in human fibroblast cells.
Authors: Raj N B , Pitha P M
Published: May 2006

The relative amounts of translatable cellular mRNAs and newly synthesized cellular proteins were examined in poly(I) x poly(C)-induced human fibroblast cells early during induction. At this time interferon and interferon mRNA synthesis are maximal and cells have not acquired their antiviral thesis are maximal and cells have not acquired their antiviral state. Translation of the mRNA from poly(I) x poly(C)-induced cells in a wheat germ cell-free system led to the synthesis of a [35S]methionine-labeled 22,000-dalton protein that is precipitated by antiserum to highly purified human fibroblast interferon. The synthesis of this protein was detected only with the mRNA preparations that, when translated in Xenopus oocytes, coded for the synthesis of biologically active human interferon. Two-dimensional gel analysis of the [35S]methionine-labeled polypeptides translated from the total mRNA of the induced and uninduced cells revealed the presence of 23 new proteins that were translated from mRNAs of the induced cells but not from the mRNAs of the controls. These polypeptides ranged from 15,000 to 70,000 daltons. Thirteen of these proteins were detected in induced cells labeled with [35S]methionine. It is concluded that, in human fibroblasts, poly(I) x poly(C) induces, in addition to interferon, the synthesis of a variety of "interferon-associated" proteins.
Scanning calorimetric evidence for a third phase transition in phosphatidylcholine bilayers.
Authors: Chen S C , Sturtevant J M , Gaffney B J
Published: May 2006

A third phase transition, centered at about 18 degrees C, was observed by differential scanning calorimetry in a multilamellar suspension of dipalmitoyl phosphatidylcholine that had been held at 0 degrees C for several days. This transition is less cooperative than the other two transitions which are well known for this system, and it is accompanied by an enthalpy increase about three times as large as that of the so-called pretransition at 35 degrees C and about half that of the main gel to liquid crystal transition at 41 degrees C. The reversal of this transition on cooling is extremely slow.
Reconstitution of the hepatic asialoglycoprotein receptor with phospholipid vesicles.
Authors: Klausner R D , Bridges K , Tsunoo H , Blumenthal R , Weinstein J N , Ashwell G
Published: May 2006

A solubilized detergent-free preparation of the hepatic binding protein specific for asialoglycoproteins associates spontaneously with small unilamellar lipid vesicles. This process is independent of the phase transition of the lipid and effectively restores the specific binding activity of the receptor protein. The insensitivity of the resulting lipid-protein complex to ionic strength provides evidence for a hydrophobic interaction. There is a perturbation of the lipid phase transition concomitant with addition of the protein. Circular dichroism studies indicate that the protein undergoes a conformational change on association with lipid. Binding of specific ligand produces further physical changes in the receptor as indicated by alterations in the tryptophan fluorescence quenching pattern.
Adenovirus DNA replication in vitro: characterization of a protein covalently linked to nascent DNA strands.
Authors: Challberg M D , Desiderio S V , Kelly T J
Published: May 2006

The 5' terminus of each nascent daughter strand of adenovirus DNA replicated in vitro is covalently linked to a protein with an apparent Mr of 80,000. This protein may represent a precursor to the 55,000-dalton protein known to be linked to the 5' ends of mature adenovirus DNA strands. Partial proteolysis experiments indicate that the 80,000-dalton and 55,000-dalton proteins are structurally related. Furthermore, both proteins are attached to DNA by the same linkage: a phosphodiester bond between the beta-OH of a serine residue in the protein and the 5'-OH of the terminal deoxycytidine residue of the DNA. The role of the 80,000-dalton protein in adenovirus DNA replication is not yet clear, although one reasonable possibility is that it serves as the primer for daughter strand synthesis.