bluuebunny/crossref_metadata_2025 huggingface dataset:
Origin of replication of pBR345 plasmid DNA.
Authors: Bolivar F , Betlach M C , Heyneker H L , Shine J , Rodriguez R L , Boyer H W
Published: May 2006

A small (approximately 1100 base pairs) ColE1-type plasmid, pBR345, was constructed from plasmid pMB1 by a series of in vitro recombinant manipulations. Approximately 9% of the supercoiled pBR345 DNA obtained from cultures amplified with chloramphenicol appears to be replicative intermediates with replicating "eye" structures of uniform size. Results obtained from electron microscopy and biochemical analyses have enabled us to localize the origin of replication at the same position as that reported for ColE1. A sequence of 420 nucleotides surrounding this origin has been determined. A comparison between this sequence and the one determined for the origin of replication of ColE1 is presented.
An internal deletion mutant of a myosin heavy chain in Caenorhabditis elegans.
Authors: MacLeod A R , Waterston R H , Brenner S
Published: May 2006

Unc-54 I is the structural gene for a myosin heavy chain present in a major fraction of the total myosin of Caenorhabditis elegans. The allele e675, which possesses a normal amount of myosin but fails to assemble thick filaments, has been shown previously to contain a novel heavy chain of molecular weight 2 X 10(5), shorter by 10(4) than the wild-type (N2) unc-54 gene product. The structural alteration of the E675 heavy chain is an internal deletion of 10(4) molecular weight near the COOH terminus of the molecule. This has been determined by mapping the partial digestion products of heavy chain fragments labeled specifically at their NH2 termini.
Guanosinetriphosphatase activity of tubulin associated with microtubule assembly.
Authors: David-Pfeuty T , Erickson H P , Pantaloni D
Published: May 2006

Tubulin, purified by cycles of assembly followed by phosphocellulose chromatography, exhibits a characteristic GTPase activity that is polymerization dependent and can be attributed to the tubulin itself. This activity has been observed, in a standard reassembly buffer containing low Mg2+, under three conditions that induce microtubule assembly: in the presence of microtubule-associated proteins, in the presence of DEAE-dextran, or after addition of high Mg2+ and glycerol. The phosphocellulose-purified tubulin showed no GTPase activity under the following nonpolymerizing conditions: in buffer with low Mg2+ in the absence of microtubule-associated proteins or DEAE-dextran, in buffer with high Mg2+ and glycerol at tubulin concentrations below the critical concentration, or when microtubule assembly was inhibited by vinblastine. Colchicine, on the other hand, while blocking microtubule assembly, induced a significant GTPase activity in the phosphocellulose-purified tubulin. During the process of assembly, GTP appears to be hydrolyzed as a free tubulin dimer polymerizes into a microtubule. A constant GTPase activity when polymerization equilibrium is reached apparently reflects the cyclic polymerization-depolymerization of tubulin dimers at the ends of the microtubules.
Use of gene fusions to study outer membrane protein localization in Escherichia coli.
Authors: Silhavy T J , Shuman H A , Beckwith J , Schwartz M
Published: May 2006

Escherichia coli strains have been isolated that produce hybrid proteins comprised of an NH2-terminal sequence from the lamB gene product (an outer membrane protein) and a major portion of the COOH-terminal sequence of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23; a cytoplasmic protein). These proteins exhibit beta-galactosidase activity. One such strain, pop 3105, produces a hybrid protein containing very little of the lamB gene protein; the protein is found in the cytoplasm. The protein found in a second strain, pop 3186, contains much more of the lamB gene protein; a substantial fraction of the beta-galactosidase activity is found in the outer membrane, probably facing outward. These results indicate that information necessary to direct the lamB gene product to its outer membrane location is located within the lamB gene itself. The properties of such fusion strains open up the prospect of a precise genetic analysis of the genetic components involved in protein transport.
Thermosensitive mutation in Escherichia coli simultaneously causing defects in penicillin-binding protein-1Bs and in enzyme activity for peptidoglycan synthesis in vitro.
Authors: Tamaki S , Nakajima S , Matsuhashi M
Published: May 2006

A thermosensitive mutant of Escherichia coli K-12 was isolated in which the membrane fractions were deficient both in penicillin-binding protein-1Bs, the major components of protein 1 [Spratt, B.G. & Pardee, A.B. (1975) Nature 254, 516-517] and in activity for in vitro peptidoglycan synthesis. The mutant was also supersensitive to many kinds of beta-lactam antibiotics. All these phenotypic changes were found to be caused by a single mutation (mrc). Genetic mapping studies show that the mrc mutation was located at about 3.3 min on the E. coli chromosome linkage map [Bachmann, B.J., Low, K.B. & Taylor, A.L. (1976) Bacteriol. Rev. 40, 116-167]. Penicillin-binding protein-1Bs seemed to be identical to one of the essential enzymes involved in crosslinking of peptidoglycan and the target of cell-lytic action of penicillins. Possible functions of some other penicillin-binding proteins in compensating for lack of protein-1Bs were also proposed.
Alternative aspirins as antisickling agents: acetyl-3,5-dibromosalicylic acid.
Authors: Walder J A , Zaugg R H , Iwaoka R S , Watkin W G , Klotz I M
Published: May 2006

Acetyl-3,5-dibromosalicylic acid (dibromoaspirin) is shown to be a potent acylating agent of intracellular hemoglobin in vitro. Transfer of the actyl group of dibromoaspirin to amino groups of hemoglobins A and S seems to occur predominantly at just two or three sites on these proteins. This acetylation produces moderate increases in the oxygen affinities of normal and sickle erythrocytes. Furthermore, treatment of intracellular hemoglobin S with dibromoaspirin directly inhibits erythrocyte sickling. This antisickling effect is paralleled by an increase in the minimum gelling concentration of deoxy hemoglobin S extracted from sickle erythrocytes that had been exposed to low concentrations of dibromoaspirin. These observations suggest that dibromoaspirin might be an effective antisickling agent in vivo.
Random walks and generalized master equations with internal degrees of freedom
Authors: Landman Uzi , Montroll Elliott W. , Shlesinger Michael F.
Published: May 2006

We present an extension of the continuous-time random walk formalism to include internal states and to establish the connection to generalized master equations with internal states. The theory allows us to calculate physical observables from which we can extract the characteristic parameters of the internal states of the system under study.
Robust nonlinear data smoothers: Definitions and recommendations
Authors: Velleman Paul F.
Published: May 2006

Nonlinear data smoothers provide a practical method of finding smooth traces for data confounded with possibly long-tailed or occasionally “spikey” noise. While they are natural tools for analyzing time-series data, they can be applied to any data set for which a sequencing order can be established. Their resistance to the effects of unsupported extreme observations and their ability to respond rapidly to well-supported patterns make them valuable as tools for finding patterns not constrained to specific parametric form and as versatile data-cleaning algorithms. This paper defines some robust nonlinear smoothers that have performed well in Monte-Carlo trials and makes brief recommendations based upon that study.
Membrane-associated assembly of M13 phage in extracts of virus-infected Escherichia coli.
Authors: Wickner W , Killick T
Published: May 2006

Assembly of coliphage M13 is known to occur as the viral DNA crosses the cytoplasmic membrane, shedding its virus-coded DNA unwinding protein and acquiring from the membrane approximately 2400 copies of the major coat protein. Conditions are described in which extracts of M13-infected E. coli and membranes prepared from such extracts will support virus assembly at a rate equivalent to that of intact cells. Extracts prepared from cells infected with temperature-sensitive M13 mutants in genes 1, 3, 4, or 5 are temperature-sensitive in this cell-free assembly reaction. Phage assembly in vitro requires magnesium and as yet an unidentified heat-stable cofactor of low molecular weight. The rate of virus assembly is approximately linear with respect to extract concentration over a 10(4)-fold range, consistent with the observation that the entire M13 assembly activity copurifies with the cell membrane fraction.
Molecular description of dioxygen bonding in hemoglobin.
Authors: Olafson B D , Goddard W A
Published: May 2006

From ab initio quality calculations on model systems, we conclude that in unliganded Fe-porphyrin the FE lies in the plane for both the high-spin (q) and intermediate-spin (t) states. Thus, the high-spin d6 Fe is not too big to fit into the porphyrin plane (as often suggested). We find the q state lower for a porphyrin hole radius greater than 1.94 A and the t state lower for smaller sizes. For the five-coordinate complex including an axial nitrogenous ligand [a model for myoglobin (Mb) and hemoglobin (Hb)], we find the ground state to be q with the Fe 0.3 A out of the plane (recent x-ray data on deoxy Mb suggests about 0.4 A). The origin of this out-of-plane displacement is the nonbonded repulsions between the axial ligand and porphyrin nitrogen orbitals. Pushing the Fe of the five-coordinate complex into the plane does not lead to a stable low-spin state (as usually suggested), the q and t states being the low-lying states. Bonding the O2 to form the six-coordinate complex stabilizes the t form of the Mb model, leading to a singlet state of MbO2 with Fe in the plane. (It has often been suggested that the Fe of MbO2 and HbO2 is low-spin Fe2+; however, we find this not to be the case.) The bonding in the MbO2 model confirms the ozone model of the bonding, leading to a structure consistent with the Pauling model (our calculated FeOO bond angle is 119 degrees). The total charge transfer to the O2 is 0.10 electron, in disagreement with the Weiss model. Molecular orbital calculations (Hartree-Fock) incorrectly lead to septet ground state (S = 3) for the MbO2 model. The implications for the cooperative O2 binding in hemoglobin and protein modifications of the energetics of the active site are considered. Use of our calculated force constants for displacement of Fe perpendicular to the heme plane suggests that the movement of the Fe upon a change in the quaternary structure from the T to the R form is only about 0.04 A toward the heme plane.
Cellular uptake and nuclear binding of insulin in human cultured lymphocytes: evidence for potential intracellular sites of insulin action.
Authors: Goldfine I D , Smith G J , Wong K Y , Jones A L
Published: May 2006

Human cultured lymphocytes (IM-9) were used to demonstrate that insulin can enter the intact cell and bind to the nucleus. When these lymphocytes were incubated with 125I-labeled insulin, specific cellular uptake reached a maximum within 2 min and remained at a plateau for 90 min or longer. Partially purified nuclei from such cells contained approximately 15-20% of the total cellular radioactivity. Nuclei freed of all other cellular fractions (by washing the partially purified nucleic with Triton X-100) bound approximately 7% of the total cellular radioactivity. In contrast to the rapid uptake of labeled insulin into the intact cell, specific binding to the nucleus was half-maximal after 5 min of incubation and maximal after 90 min. Both the cellular uptake and subsequent nuclear binding of labeled insulin were progressively inhibited by increasing concentrations of unlabeled hormone. Independent evidence for the nuclear binding of insulin was obtained by preparing autoradiographs of lymphocytes incubated for various times with labeled insulin. Such preparations strongly suggest that insulin binds to the plasma membrane, enters the cytosol, and then binds to the nucleus.
Differential utilization of leucyl-tRNAs by Escherichia coli.
Authors: Holmes W M , Goldman E , Miner T A , Hatfield G W
Published: May 2006

The utilization of the isoaccepting species of leucyl-tRNA in protein synthesis has been examined in E. coli. Two minor leucyl-tRNA species, isoacceptors tRNA3Leu and tRNA4Leu, are the predominant species found bound to ribosomes during exponential growth in minimal medium. In rich medium, an increased proportion of tRNA1Leu is found bound to ribosomes. One species, tRNA5Leu, is always absent from ribosomes. In a mutant strain in which normal tRNA3Leu and tRNA5Leu are reduced or absent, tRNA1Leu is a major ribosome-bound species in minimal medium. Protein synthesis in vitro with RNA from E. coli further suggests that tRNA5Leu is rarely used for total protein synthesis; however, this species is active with RNA from MS2 phage. We propose that tRNA1Leu can substitute for tRNA3Leu under rapid growth conditions, and that tRNA5Leu is used minimally in total protein synthesis.
Inhibition of histidyl-tRNA-adenosine triphosphate phosphoribosyltransferase complex formation by histidine and by guanosine tetraphosphate.
Authors: Kleeman J E , Parsons S M
Published: May 2006

Formation of the complex between the first enzyme of histidine biosynthesis from Salmonella typhimurium, ATP phosphoribosyltransferase [1-(5'-phosphoribosyl)-ATP: pyrophosphate phosphoribosyltransferase; EC 2.4.2.17], and histidyl-tRNA is shown to be inhibited by L-histidine and by guanosine-5'-diphosphate-3'-diphosphate in the presence of histidine. Higher histodine levels make guanosine tetraphosphate a more effective inhibitor. Relatively high concentrations of guanosine-5'-triphosphate also inhibit complex formation, but this inhibition is not enhanced by histidine. The possible implications of these observations with respect to the gene regulatory activity of this enzyme are discussed.
Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA.
Authors: Bacchetti S , Graham F L
Published: May 2006

Transformation of human cells from a thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75)-negative to a thymidine kinase-positive phenotype has been achieved by using purified DNA from herpes simplex virus type 2. The specific activity of the DNA was in the range 0.5 to 2.0 transformants per microng and the efficiency of gene transfer was up to 1 transformant per 10(5) recipient cells. Several transformed lines able to grow continuously in medium selective for thymidine kinase-positive cells have been established. All of these lines express a thymidine kinase activity of viral origin but they differ from each other in the stability of enzyme expression. Subclones derived from a given transformed line inherited the degree of stability of the parental line.
Direct observations of atomic diffusion by scanning transmission electron microscopy
Authors: Isaacson M. , Kopf D. , Utlaut M. , Parker N. W. , Crewe A. V.
Published: May 2006

The feasibility of using a high-resolution scanning transmission electron microscope to study the diffusion of heavy atoms on thin film substrates of low atomic number has been investigated. We have shown that it is possible to visualize the diffusion of individual uranium atoms adsorbed to thin carbon film substrates and that the observed motion of the atoms does not appear to be induced by the incident electron beam.
Synthetic peptides with the biological activities and specificity of human C3a anaphylatoxin.
Authors: Hugli T E , Erickson B W
Published: May 2006

Two peptides identical to the COOH-terminal sequence of human C3a anaphylatoxin and two analogs were synthesized by the solid-phase method and tested for biological activity. The synthetic COOH-terminal octapeptide, C3a-(70-77) or Ala-Ser-His-Leu-Gly-Leu-Ala-Arg, caused contraction of guinea pig ileum and uterus, release of vasoactive amines from rat mast cells, and increased vascular permeability in guinea pig and human skin. On a molar basis, the synthetic octapeptide possessed 1-2% of the biological activities of C3a and specifically desensitized smooth muscle to stimulation by C3a. Like natural C3a, the synthetic C3a=(70-77) was inactivated by digestion with carboxypeptidase B [peptidyl-L-lysine(-L-arginine) hydrolase, EC 3.4.12.3], which removed the essential COOH-terminal arginine. A synthetic nonapeptide [C3a-(70-77)-Gly], containing a glycyl instead of an arginyl COOH terminus, was approximately 1% as active as the octapeptide when assayed with smooth muscle. The COOH-terminal 13-residue peptide of C3a, C3a-(65-77), was equal in activity to C3a=(70-77); similarly, C3a-(65-77)-Gly expressed the same activity as C3a-(70-77)Gly. It is concluded that both the biological specificity and the activity of human C3a anaphylatoxin are determined by eight or fewer residues located at the COOH terminus of the natural protein. However, expression of full activity requires additional groups and the secondary conformational integrity of the C3a molecule.
Role of DNA gyrase in phiX replicative-form replication in vitro.
Authors: Marians K J , Ikeda J E , Schlagman S , Hurwitz J
Published: May 2006

Preparations containing DNA gyrase activity Gellert, M., Mizuchi, K., O'Dea, M.H. & Nash, H.A. (1976) Proc. Natl. Acad. Sci. USA 73, 3872-3876] have been extensively purified from Escherichia coli. Such fractions, in the presence of ATP and Mg2+, catalyze supertwisting of relaxed circular double-stranded DNA replicative forms of a number of DNAs that results in the formation of superhelical replicative forms. Relaxed phiX174 replicative form (phiX RFIV) is not attacked by the A protein endonuclease coded for by the phiX DNA genome. After exposure to preparations of DNA gyrase, the relaxed phiX174 replicative form is converted to phiX RFI which can then be attacked by the phiX gene A protein and participate in replication of duplex phiX DNA.
Dynamic structure of whole cells probed by nuclear Overhauser enhanced nitrogen-15 nuclear magnetic resonance spectroscopy.
Authors: Lapidot A , Irving C S
Published: May 2006

The proton-decoupled 15N Fourier transform nuclear magnetic resonance (NMR) spectra of 15N-enriched Escherichia coli, Bacillus licheniformis, baker's yeast, and Friend leukemic cells were obtained. The 15N NMR spectra of whole cells displayed 15N resonances originating from (i) protein backbones with lysine, arginine, and histidine side chains, (ii) ribonucleic acids, (iii) peptidoglycan, and (iv) phospholipids. Several additional amino and amide resonances were observed but not identified. In bacteria and yeast, the cell wall was found to be the site of a relatively mobile group of molecules, whose resonances dominate the proton-decoupled 15N NMR spectra of whole cells. 15N NMR chemical shifts and nuclear Overhauser effects have provided information on the in vivo structure of cell wall peptidoglycan. In Staphylococcus aureus the pentaglycine cross-bridge of cell wall peptidoglycan was found to have a random coil conformation. In B. licheniformis considerable segmental motional freedom was detected in teichuronic acid and peptidoglycan polysaccharide chains in the wall of the intact cell.
Structure of the gramicidin A channel: Discrimination between the π
L,D
and the β helix by electrical measurements with lipid bilayer membranes
Authors: Bamberg E. , Apell H. J. , Alpes H.
Published: May 2006

Measurements with different chemically modified gramicidins in lipid bilayer membranes were used to discriminate between the dimeric π
L,D
helix proposed by Urry and the dimeric parallel or antiparallel helices proposed by Veatch and Blout. Evidence for the π
L,D
helix was obtained on the basis of the different actions of a negatively charged
O
-pyromellitylgramicidin and a negatively charged
N
-pyromellityldesformylgramicidin on lipid bilayer membranes.
O
-Pyromellitylgramicidin forms ionic channels in lipid membranes when it is applied to both sides of the membrane. In contrast to unmodified gramicidin,
O
-pyromellitylgramicidin is inactive when it is applied only to one side of the membrane.
N
-Pyromellityldesformylgramicidin does not form ionic channels in lipid bilayer membranes whether it is applied to one or both sides of the membrane. These results support the view that the gramicidin channel is formed by two π
L,D
helices. Dimer formation by head-to-head association of two π
L,D
helices needs six intermolecular hydrogen bonds, which are located at the formyl end of the molecule and which occur deep within the lipid membrane. In the head-to-head associated π
L,D
helix the absence of the formyl group leads to an inactivation of the peptide, whereas in a parallel or antiparallel double-stranded helix the absence of the formyl group should have only minor effects.
Excited state lifetimes in cytochromes measured from Raman scattering data: evidence for iron-porphyrin interactions.
Authors: Friedman J M , Rousseau D L , Adar F
Published: May 2006

Resonance Raman scattering excitation profile data have been obtained on ferrocytochromes c and b5 in the alpha absorption band region. We observe in cytochrome c that the shape of the excitation profile agrees with the absorption band shape, while in cytochrome b5 it does not. In addition, we observe in cytochrome b5 a linewidth substantially larger than that in cytochrome c. From our data we conclude that the excited state lifetime in cytochrome c is longer than that in cytochrome b5 and that in cytochrome b5 the relaxation of the pi-pi* excited state configuration of the porphyrin ring is different in the x direction than in the y direction. Possible origins of these effects due to coupling to the d-d transitions of the iron atom are discussed.
Classification of the irreducible representations of semisimple Lie groups
Authors: Vogan David A.
Published: May 2006

We obtain a classification of the irreducible (nonunitary) representations of a connected semisimple Lie group
G
, in terms of their restriction to a maximal compact subgroup
K
of
G
. (A classification in terms of analytic properties of the representations has been given by R. P. Langlands [(1973), mimeographed notes, Institute for Advanced Study, Princeton, NJ] for linear groups.) We first define a norm on the representations of
K
: if μ ∈
K̂
, ‖μ‖ is a nonnegative real number. Then if π ∈
Ĝ
, μ is called a
lowest K-type of
π if ‖μ‖ is minimal among the
K
-types occurring in π. We announce a parameterization of the set of representations containing μ as a lowest
K
-type by the orbits of a finite group acting in a complex vector space (the dual of the vector part of a certain Cartan subgroup of
G
), and the result that μ necessarily occurs with multiplicity one in such representations.
Analytic torsion and Reidemeister torsion
Authors: Cheeger Jeff
Published: May 2006

We announce a proof of the conjecture of Ray and Singer that for a compact Riemannian manifold the analytic torsion and Reidemeister torsion are equal. The proof involves studying the heat equation for certain manifolds M, equipped with metrics

g
u

, 0 <
u
< 1 which degenerate in a prescribed way at the boundary δM, as
u
→ 0,1.
Biotinylinsulins as potential tools for receptor studies
Authors: Hofmann Klaus , Finn Frances M. , Friesen Heinz-Jürgen , Diaconescu Cornelia , Zahn Helmut
Published: May 2006

The preparation of affinity columns that contain insulin attached to Sepharose in a targeted manner by way of biotin-avidin noncovalent bonds is described. Insulin was acylated selectively at the amino terminus of the B chain with the
N
-hydroxysuccinimido ester of biotin to form
N

α,B
1

-biotinylinsulin. The ability of this modified insulin to stimulate rat epididymal adipocytes was (mean ± SD) 94 ± 9.6% (
P
, 0.05) that of the control insulin.
N

α,B
1

-Biotinylinsulin displaced 4-hydroxyazobenzene-2′-carboxylic acid from avidin, demonstrating affinity for this protein. The formation of the
N

α,B
1

-biotinylinsulin-avidin complex was visualized by cellulose acetate electrophoresis at pH 4.
N

α,B
1

-Biotinylinsulin combined with avidin attached to Sepharose to form affinity columns in which the hormone was attached to the support by strong noncovalent bonds. The determination of the loading of avidin-Sepharose columns with biotinylinsulin was greatly facilitated by the attached biotin which provided a marker whose concentration could be assessed accurately by titration with avidin. Biotinylinsulin attached to avidin-Sepharose beads retained the ability to stimulate rat epididymal adipocytes. The activity of several samples of these beads was about 15% that of free biotinylinsulin, based on the amount of biotinylinsulin anchored to the support. The advantages of biotinylated hormones for the targeted attachment of hormones to solid supports are discussed.
Concept of internal structural controls for evaluation of inactive synthetic peptide analogs: synthesis of [Orn13,14]apamin and its guanidination to an apamin derivative with full neurotoxic activity.
Authors: Cosand W L , Merrifield R B
Published: May 2006

The importance of arginine residues 13 and 14 in the bee venom neurotoxin, apamin, was teste by the synthesis of replacement analogs. [13,14-di-Ndelta-trifluoroacetylornithine]Apamin was synthesized by the solid phase method on a benzhydrylamine resin. It was deprotected to [13,14-diornithine]apamin, which was then guanidinated to produce the 4-homoarginine-13,14-diarginine analog, [Har4]apamin. Neither the trifluoroacetylornithine analog nor the ornithine analog produced any detectable symptoms when injected intravenously into mice. However, the synthetic [Har4]apamin exhibited the full neurotoxic activity of native apamin and of [Har4]apamin derived from the natural toxin. This provided an internal structural control for the correctness of the primary structure of the inactive synthetic analogs and strengthened the conclusion that one, or both, of the arginine residues plays an important role in the action of apamin.
Food, drug, and cosmetic dyes: biological effects related to lipid solubility.
Authors: Levitan H
Published: May 2006

Food, drug, and cosmetic dyes of the xanthane type (analogs of fluorescein) were applied to isolated molluscan ganglia and changes in the electrophysiological properties of identified neurons were monitored. The synthetic coloring agents increased the resting membrane potential and conductance of the neurons in a dose-dependent manner by increasing the potassium permeability of the membrane relative to that of other ions. The relative activity of these anionic dyes was highly correlated with their lipid solubility. The structure-activity study of the effects of the dyes on molluscan neurophysiology provides a basis for estimating the toxicity and brain uptake of the dyes in vertebrates, and predicting their effects on metabolism and blood clotting.
Epigenetic variation of cultured somatic cells: evidence for gradual changes in the requirement for factors promoting cell division.
Authors: Meins F , Binns A
Published: May 2006

Cells of higher plant species in culture sometimes lose their requirement for an exogenous supply of a cell division factor that, thereafter, they are able to produce. This heritable change, known as cytokinin habituation, appears to be an epigenetic one rather than a classical mutation because it is directed, potentially reversible, leaves the cell totipotent, and involves the expression of a latent differentiated function. By using cloned cell lines derived from pith parenchyma of tobacco, we have obtained evidence that the habituation process is gradual rather than all-or-none and leads to progressively more autotrophic tissues. Cells in culture show reversible shifts among a range of habituated states but remain totipotent and can be induced to regain their requirement for a cell division factor. Thus, it appears that habituation involves epigenetic changes in a quantitative cellular phenotype. Our findings support the hypothesis that tumor progression in crown gall, a neoplastic disease of higher plants, can be accounted for by heritable changes in the pattern of gene expression.
Defective organization of actin in cultured skin fibroblasts from patients with inherited adenocarcinoma
Authors: Kopelovich Levy , Conlon Susan , Pollack Robert
Published: May 2006

In the cytoplasm of well-spread cultured normal fibroblasts, actin is organized into a network of cables that run the length of the cell just inside the adherent cell membrane. A diffuse matrix replaces the cables in fibroblasts that have become tumorigenic as a result of oncogenic transformation. We have found a similar disruption in actin organization in cultured skin fibroblasts (passage 6-10) obtained by biopsy from patients with the inherited colonic cancer, adenomatosis of the colon and rectum (ACR). Because ACR is inherited as an autosomal dominant trait, about half the children of ACR patients will develop colon cancer, but they typically remain asymptomatic until at least the second decade of life. Actin distribution within cultured cells from children of ACR patients was identical either to that seen in cultured cells from normal persons or to that seen in cultured cells from ACR patients. The two different patterns were independent of age, sex, drug treatment, or infections of the donors. Apparently, this class of colonic carcinoma is accompanied by a systemic aberration in the organization of fibroblast cytoplasm, and this aberration can be detected by immunofluorescent localization of actin within cultured skin fibroblasts, prior to manifestation of any colonic symptoms.
A protonmotive force drives bacterial flagella.
Authors: Manson M D , Tedesco P , Berg H C , Harold F M , Van der Drift C
Published: May 2006

Streptococcus strain V4051 is motile in the presence of glucose. The cells move steadily along smooth paths (run), jump about briefly with little net displacement (twiddle), and then run in new directions. They stop swimming when deprived of glucose. These cells become motile when an electrical potential or a pH gradient is imposed across the membrane. Starved cells suspended in a potassium-free medium respond to the addition of valinomycin by a brief period of vigorous twiddling. They also twiddle, although less vigorously, when the external pH is lowered. Valinomycin-induced twiddling occurs in the absence of external alkali or alkaline earth cations and without significant net synthesis of ATP. When a chemoattractant is added to cells swimming in the presence of glucose, twiddles are transiently suppressed, and the cells run for a time. Similarly, when starved cells are suspended in a potassium-free medium containing both valinomycin and an attractant, many cells initially run rather than twiddle. We conclude that the flagella are driven by a protonmotive force.
Reinnervation of original synaptic sites on muscle fiber basement membrane after disruption of the muscle cells.
Authors: Marshall L M , Sanes J R , McMahan U J
Published: May 2006

Regenerating axons form new synapses precisely at sites of original synapses in denervated skeletal muscle. To determine what role the muscle cell plays in this phenomenon, we studied reinnervation of frog muscle at intervals after crushing the nerve and damaging the muscle fibers. Damaged muscle fibers degenerate and are phagocytized, but their basement membrane persists and acts as a scaffold for regenerating muscle cells. Specializations of the basement membrane serve to mark original synaptic sites after nerve and muscle have degenerated. Regenerating axons enter the region of damage and form functional synapses with regenerating myofibers. The new nerve terminals are found almost exclusively at the original synaptic sites, demonstrating that the integrity of the original postsynaptic cell is not necessary for topographically precise reinnervation of denervated muscle.
Modulation of acetylcholine receptor by antibody against the receptor
Authors: Heinemann S. , Bevan S. , Kullberg R. , Lindstrom J. , Rice J.
Published: May 2006

Antibody against acetylcholine receptor induces an increase in the rate of degradation of acetylcholine receptors on a mouse cell line (BC
3
H-1) and cultured rat skeletal muscle. The increased rate of degradation results in a lowered density of acetylcholine receptors on muscle membrane and a lowered sensitivity to iontophoretically applied acetylcholine. The modulation of acetylcholine receptor is energy, temperature, and time dependent and may be related to antigenic modulation found in other systems. Acetylcholine noise analysis demonstrates that antibody against acetylcholine receptor reduces the channel mean conductance and mean open time slightly.

It is concluded that antibody binds to the acetylcholine receptor, impairs its function, and induces receptor degradation. This results in a lowered density of acetylcholine receptor and a lowered sensitivity to acetylcholine. Patients with myasthenia gravis have antibodies to their acetylcholine receptor in their serum. Antigenic modulation of receptor in the muscle of patients with myasthenia gravis could contribute to the observed decrease in amplitudes of miniature endplate potentials and in muscle acetylcholine sensitivity, and the symptoms of muscular weakness.
Spliced segments at the 5′ terminus of adenovirus 2 late mRNA
Authors: Berget Susan M. , Moore Claire , Sharp Phillip A.
Published: May 2006

An mRNA fraction coding for hexon polypeptide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA⋅DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonuclease cleavage fragments of viral DNA were visualized in the electron microscope, branched forms were observed in which 160 nucleotides of RNA from the 5′ terminus were not hydrogen bonded to the single-stranded DNA. DNA sequences complementary to the RNA sequences in each 5′ tail were found by electron microscopy to be located at 17, 20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of viral RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA.
Adenosine 3′:5′-cyclic monophosphate- and guanosine 3′:5′-cyclic monophosphate-dependent protein kinases: Possible homologous proteins
Authors: Lincoln Thomas M. , Corbin Jackie D.
Published: May 2006

The properties of purified mammalian adenosine 3′:5′-cyclic monophosphate (cAMP)- and guanosine 3′:5′-cyclic monophosphate (cGMP)-dependent protein kinases were compared. Several physical characteristics of the two enzymes were similar, including size, shape, affinity for cyclic nucleotide binding, and
K
m
for ATP. In addition, the amino acid composition of the two proteins indicated a close composition homology (70-90%). Both cyclic nucleotide-dependent protein kinases catalyzed phosphorylation of rat liver pyruvate kinase (EC 2.7.1.40) and fructose 1,6-diphosphatase (EC 3.1.3.11), rabbit skeletal muscle glycogen synthase (EC 2.4.1.11) and phosphorylase
b
kinase (EC 2.7.1.38), and calf thymus histone H
2
b. The phosphorylation of several synthetic peptides and of trypsin-sensitive and trypsin-insensitive sites in glycogen synthase suggested similar recognition sites on the protein substrates for the two kinases. The cAMP-dependent protein kinase was the better catalyst with each protein or peptides substrate. The results suggest that the two enzymes evolved from a common ancestral protein.
In vitro translation yields a possible Rous sarcoma virus src gene product.
Authors: Beemon K , Hunter T
Published: May 2006

In vitro translation of Rous sarcoma virus (RSV) virion RNA in the messenger-dependent reticulocyte lysate system yielded polypeptides that were not synthesized by translation of RNA from a transformation-defective deletion mutant of RSV. These RSV-specific products migrated on sodium dodecyl sulfate/polyacrylamide gels as two doublets of approximately 25,000 and 17,000 daltons. Synthesis of these proteins was not sensitive to inhibition by m7GTP; however, synthesis of the 76,000-dalton precursor of the internal structural proteins was sensitive to inhibition by m7GTP. Tryptic peptide maps showed the 25,000- and 17,000-dalton proteins to be related to one another but to be distinct from the 76,000-dalton protein. The 25,000-dalton protein was translated only from a polyadenylylated RNA of approximately 2500 nucleotides, whereas the 76,000-dalton protein was translated from 38S RNA, corresponding to the entire viral genome. A 180,000-dalton protein was also synthesized from 38S RSV virion RNA. From the absence of the 25,000- and 17,000-dalton proteins in the translation products of transformation-defective RSV RNA and the size of their RNA templates, we conclude that these proteins may be derived from coding sequences within the RSV src gene.
Identification of prelarge and presmall basic proteins in mouse myelin and their structural relationship to large and small basic proteins
Authors: Barbarese E. , Braun P. E. , Carson J. H.
Published: May 2006

A new technique is described to identify antigenically related proteins by radioimmunoassay after sodium dodecyl sulfate/polyacrylamide gel fractionation. When adult mouse myelin was examined by this technique, four proteins that are antigenically related to the small myelin basic protein were identified. They were designated: prelarge (molecular weight 21,500), large (18,500), presmall (17,000), and small (14,000). The four proteins were isolated by elution from polyacrylamide gels, and each protein migrated as a single band when analyzed by either sodium dodecyl sulfate or acidic polyacrylamide gel electrophoresis. Serial dilutions of the purified proteins were measured by radioimmunoassay. Both the slope of the inhibition curve and the level of maximal inhibition for each protein were the same as for the small myelin basic protein, indicating that each of the four proteins contains all of the antigenic sites present in the small basic protein. Structural relationships among the four proteins were examined by using two-dimensional analysis of tryptic digests. The results showed that: large was similar in amino acid sequence to the major myelin basic protein from other species; small was identical in sequence to large, except for an internal deletion of approximately 40 amino acid residues: prelarge contained the sequence of large plus an additional sequence of 25-35 amino acid residues; and presmall contained the sequence of small plus the same additional sequence as in prelarge. The four proteins were also treated with 2-(2-nitrophenylsulfenyl)-3-methyl-3′-bromoindolienine (BNPS-skatole) which cleaves proteins specifically at tryptophan residues. Analysis of the cleavage products indicated that the additional amino acid sequence in both prelarge and presmall extends from the amino terminus of the molecule. Several implications of these results are discussed.
Flagellar mutants of
Chlamydomonas
: Studies of radial spoke-defective strains by dikaryon and revertant analysis
Authors: Luck David , Piperno Gianni , Ramanis Zenta , Huang B.
Published: May 2006

The motility mutant of
Chlamydomonas reinhardtii pf14
lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a two-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its
35
S-labeled flagellar proteins. Six of these same proteins are missing in
pf1
, which lacks spoke-heads. To determine whether any of the missing proteins represent the mutant gene product two experimental approaches have been applied. The first makes use of the fact that gametes of either mutant strain when fused with wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with
35
S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The second technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of
pf14
leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For
pf1
only two radioactive proteins are restored; proteins 4, 6, 9, and 10 were not radioactive. Analysis of revertants of
pf1
gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of
pf14
, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product.
Expression in
Escherichia coli
K-12 of the structural gene for catabolic dehydroquinase of
Neurospora crassa
Authors: Vapnek Daniel , Hautala Judith A. , Jacobson James W. , Giles Norman H. , Kushner Sidney R.
Published: May 2006

The inducible quinic acid catabolic pathway of
Neurospora crassa
is controlled by four genes, the
qa
cluster which includes structural genes
qa-2, qa-3, qa-4
for three enzymes and a regulatory gene,
qa-1
. In this paper we report the molecular cloning of at least the
qa-2
gene which encodes the catabolic dehydroquinase (5-dehydroquinate hydro-lyase, EC 4.2.1.10). Endo·R·
Hin
dIII restriction endonuclease fragments of
N. crassa
DNA from a

qa-1
c

(constitutive) mutant and of
Escherichia coli
plasmid pBR322 DNA were ligated
in vitro
and used to transform an
aroD6
(5-dehydroquinate hydrolyase deficient) strain of
E. coli
K12. The recombinant plasmid (pVK55) isolated from one AroD
+
transformant (SK1518) contained, in addition to pBR322, two
N. crassa Hin
dIII fragments with molecular weights of 2.3 × 10
6
and 1.9 × 10
6
. Derivatives of SK1518 cured of plasmid DNA were phenotypically Amp
s
and AroD
-
. These cured strains, retransformed with pVK55, were phenotypically Amp
R
and AroD
+
. Strains transformed with pVK55 possessed 5-dehydroquinate hydrolyase activity but no activity was present in any AroD
-
strain. The enzyme extracted from strains containing the recombinant plasmid was identical to
N. crassa
catabolic dehydroquinase by the criteria of heat stability, ammonium sulfate fractionation, immunological crossreactivity, molecular weight, and purification characteristics. This identity demonstrates that the

N. crassa qa-2
+

gene is carried by the recombinant plasmid and is apparently transcribed and translated with complete fidelity. Furthermore, subunit assembly of the
N. crassa
polypeptides also occurs in
E. coli
, because the catabolic dehydroquinase is a multimer composed of approximately 20 identical subunits.
Enzyme replacement therapy in Gaucher's disease: large-scale purification of glucocerebrosidase suitable for human administration.
Authors: Furbish F S , Blair H E , Shiloach J , Pentchev P G , Brady R O
Published: May 2006

Enzyme replacement therapy for the alleviation of Gaucher's disease has been impeded because of the difficulty in preparing large amounts of glucocerebrosidase, the enzyme that is deficient in patients with this disorder. A large-scale procedure for the purification of human placental glucocerebrosidase has been developed. The method uses cholate extraction, ammonium sulfate fractionation, acid precipitation, butanol extraction, and hydrophobic chromatography; the final enzyme preparation has a specific activity of more than 10(6) units/mg of protein with an overall recovery of 30%. In addition, the contamination of enzyme preparations, intended for human infusion, prepared by isolation procedures involving concanavalin A columns has been studied and is reported here.
A general treatment of relaxation phenomena
Authors: Lin S. H. , Eyring H.
Published: May 2006

The singular perturbation method has been applied to solve the quantum mechanical Liouville equation for the relaxation phenomenon of the system in thermal contact with a heat bath. The master equation derived gives the proper expressions for both diagonal and off-diagonal elements of the density matrix and is capable of describing the time-dependent behavior of the system in the time range comparable with the reciprocal of the damping constants and the time range
t
→ ∞ compared with the reciprocal of the damping constants.
Division of BALB/c mouse 3T3 and simian virus 40-transformed 3T3 cells in cellular aggregates
Authors: Carrino D. , Gershman H.
Published: May 2006

BALB/c mouse 3T3 cells and 3T3 cells transformed by simian virus 40 (SV40) were cultured as aggregates in agitated liquid medium. When maintained with daily medium changes, 3T3 cells incorporated [
3
H]thymidine into acid-insoluble material at a rate ¼ that of 3T3 cells in logarithmic-phase flat cultures but 16 times that of the same cells in stationary-phase flat cultures. Similarly, SV40-transformed 3T3 cells in aggregates incorporated [
3
H]thymidine at ⅓ the rate of SV40-3T3 cells in logarithimic-phase flat culture and 14 times that of these cells in stationary-phase flat culture. Autoradiographs of aggregates of 3T3 and SV40-3T3 cells incubated for 2 hr in the presence of [
3
H]uridine and [
3
H]thymidine indicated that penetration of the nucleosides into aggregates during this period was limited to the outer four to six cell layers. Aggregates were also incubated in the presence of radiolabeled thymidine and uridine continuously for 4 days. Under these conditions, where nucleoside penetration was not limiting, 100% of the SV40-3T3 cells and 56% of the 3T3 cells incorporated [
3
H]thymidine into acid-insoluble material.

The rates of cell division, cell loss, and net cell accumulation in aggregates of 3T3 and SV40-3T3 cells were measured by techniques not influenced by possible alterations in transport, pool size, or penetration. SV40-3T3 cells divided with a doubling time for the total population of 26.0 hr. The total cell number increased more slowly (doubling time of 48.3 hr) because of cell loss, which occurred with a half-time (time for 50% of the cells to be lost) of 53.3 hr. 3T3 cells in aggregates began to divide only after 3 days, then did so with a doubling time for the total population of 76.4 hr. Total cell number decreased (half-time of 26.3 hr) because this rate of cell division was exceeded by the rate of cell loss, which was constant with a half-time of 22.9 hr. The results suggest that 3T3 and SV40-3T3 cells display growth properties in aggregates consistent with their previously reported behavior in conventional flat culture: SV40-transformed 3T3 cells can proliferate under conditions of high cell density to a much greater extent than 3T3 cells can. Both cell lines, however, display an increased capacity to divide in aggregates relative to confluent flat culture, despite conditions of high cell density and absence of anchorage to an artificial solid substrate.
Insulin-like growth factor: a model for tertiary structure accounting for immunoreactivity and receptor binding.
Authors: Blundell T L , Bedarkar S , Rinderknecht E , Humbel R E
Published: May 2006

A model for the three-dimensional structure of insulin-like growth factor (IGF) is proposed based on the close sequence homology of IGF with insulin, the tertiary structure of which is known. The IGF molecule is postulated to have an insulin-like main chain conformation for residues equivalent to B6--B27 and A1--A21 and a hydrophobic core nearly identical to that of insulin. A short connecting peptide of twelve residues and an extension at the COOH-terminus are easily accommodated on the molecular surface. The surface involved in dimer formation in insulin is largely conserved, but the zinc-binding histidine and many residues involving hexamerization are very different from those of insulin and it is unlikely that IGF forms zinc hexamers. The model provides a ready explanation for the inability of IGF to bind antibodies to insulin and for its ability to bind insulin receptors with low affinity.