bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of ribosomal protein S1 of Escherichia coli to the 3' end of 16S rRNA.
Authors: Dahlberg A E , Dahlberg J E
Published: May 2006

Ribosomal protein S1 reversibly binds the 49-nucleotide fragment that is cleaved from the 3' end of 16S rRNA in ribosomes by colicin E3. The fragment has secondary structure in the form of a hairpin loop. At the base of the stem is a sequence (A-C-C-U-C-C) thought to be involved in the base pairing with complementary sequences in mRNA during the initiation of protein synthesis. The role of S1 may be to stabilize this region of the fragment in an open conformation to allow for base pairing to mRNA. This model is supported by the observation that S1 binds specifically to this region of the fragment. In addition, aurin tricarboxylic acid, an inhibitor of protein synthesis, reverses this effect by disrupting the S1-RNA complex. These results can explain why S1 is an essential component of the ribosome for translation of natural mRNA and why aurin tricarboxylic acid blocks initiation.
Interaction of elongation factor Tu with 2'(3')-O-aminoacyloligonucleotides derived from the 3' terminus of aminoacyl-tRNA.
Authors: Ringer D , Chládek S
Published: May 2006

The interaction between Escherichia coli elongation factor-Tu-GTP complex and chemically synthesized 2'(3')-O-aminoacyldinucleoside phosphates with the nucleotide sequence of the 3' terminus of aminoacyl-tRNA (AA-tRNA) has been studied. It was found that C-A-Phe, C-A-Pro, and C-A-Asp interact with EF-Tu-GTP, causing the release of GTP bound to the enzyme. The specificity of this interaction closely resembles that of AA-tRNA since C-A and C-A(Ac-Phe) as well as the corresponding tRNAs are inactive. The 3'-O-aminoacyl derivative C-2'-dA-Phe does not interact with EF-Tu-GTP, whereas the 2'-O-aminoacyl derivative C-3'-dA-Phe is almost as active as the 2'(3')-O-aminoacyl derivative, C-A-Phe. C-A-Phe also interacts with the EF-Tu-GDP complex in a manner similar to its interaction with EF-Tu-GTP. It is concluded that interaction of 2'(3')-O-aminoacyloligonucleotides possessing the sequence of the 3' terminus of AA-tRNA is analogous to the interaction of that terminus with EF-Tu and it is suggested that EF-Tu is specific for 2'-O-AA-tRNA.
Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin.
Authors: Tomita M , Marchesi V T
Published: May 2006

Glycophorin, the major sialoglycoprotein of the human erythrocyte membrane, is composed of 131 amino acids and an average of 16 oligosaccharide chains. Fifteen oligosaccharides are linked to threonine/serine residues via O-glycosidic bonds, and one more complex unit is attached to asparagine. The location of each of these oligosaccharides and the complete amino-acid sequence of this molecular have been determined by Edman degradation techniques. Glycophorin appears to be organized into three distinct "domains" on the basis of the locations of glycosylated amino acids and the clustering of residues of similar type. These include (i) a glycosylated segment composed of approximately 64 residues from the NH2-terminus, (ii) a "hydrophobic" segment of approximately 32 nonpolar residues, and (iii) a COOH-terminal segment, composed of approximately 35 residues, which has an unusual concentration of hydrophilic amino acids. This unique structure is consistent with the earlier suggestions that glycophorin is one of the major "intrinsic" membrane proteins which has a transmembrane orientation.
Amino acids are not all initially attached to the same position on transfer RNA molecules.
Authors: Fraser T H , Rich A
Published: May 2006

Escherichia coli tRNA has been modified by replacement of the 3'-terminal AMP with either 3'-amino-3'-deoxy AMP of 2'-amino-2'-deoxy AMP. These tRNA analogs have enabled us to determine the initial site of enzyme-catalyzed aminoacylation of different tRNAs by the formation of aminoacyl-tRNA molecules in which the amino acid is linked to the 3'-terminal ribose through a stable amide bond. The tRNA species specific for glutamic acid, glutamine, leucine, phenylalanine, tyrosine, and valine are all aminoacylated on the 2'-hydroxyl group. The tRNA species specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, and threonine are aminoacylated on the 3'-hydroxyl group. The amino acids arginine, isoleucine, methionine, proline, serine, and tryptophan form stable amide bonds with both amino tRNA analogs. This might suggest that the synthetases for these amino acids can acylate both the 2'- and 3'-hydroxyl groups, but it is more likely that these enzymes can acylate both hydroxyl and amino groups at either the 2' or 3'-position of the tRNA. These results clearly illustrate a fundamental heterogeneity which is apparent in the mechanism of action of aminoacyl-tRNA synthetases.
Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.
Authors: Reed L J , Pettit F H , Eley M H , Hamilton L , Collins J H , Oliver R M
Published: May 2006

The binding of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (flavoprotein) to dihydrolipoyl transacetylase, the core enzyme of the E. coli pyruvate dehydrogenase complex [EC 1.2.4.1:pyruvate:lipoate oxidoreductase (decaryboxylating and acceptor-acetylating)], has been studied using sedimentation equilibrium analysis and radioactive enzymes in conjunction with gel filtration chromatography. The results show that the transacetylase, which consists of 24 apparently identical polypeptide chains organized into a cube-like structure, has the potential to bind 24 pyruvate dehydrogenase dimers in the absence of flavoprotein and 24 flavoprotein dimers in the absence of pyruvate dehydrogenase. The results of reconstitution experiments, utilizing binding and activity measurements, indicate that the transacetylase can accommodate a total of only about 12 pyruvate dehydrogenase dimers and six flavoprotein dimers and that this stoichiometry, which is the same as that of the native pyruvate dehydrogenase complex, produces maximum activity. It appears that steric hindrance between the relatively bulky pyruvate dehydrogenase and flavoprotein molecules prevents the transacetylase from binding 24 molecules of each ligand. A structural model for the native and reconstituted pyruvate dehydrogenase complexes is proposed in which the 12 pyruvate dehydrogenase dimers are distributed symmetrically on the 12 edges of the transacetylase cube and the six flavoprotein dimers are distributed in the six faces of the cube.
Stabilization of electron spin resonance probes for photosynthesis studies.
Authors: Sun A S , Calvin M
Published: May 2006

The major obstacle to the study of functional/structural interrelationships of spinach chloroplasts by using spin labels has been the rapid loss of the electron paramagnetic resonance (EPR) signals upon illumination with visible light. The present study demonstrates that the addition of ferredoxin and NADP+ in the presence of N-tris(hydroxymethyl)methylglycine (Tricine) buffer at pH 7.1 or higher mitigates the rapid loss of Biradical X [N,N'-bis(1-oxyl - 2,2,5,5 - tetramethylpyrroline-3-carboxy)-1,2-diaminoethane] and Monradical A (2,2,5,5-tetramethyl-3-carbamidpyrroline-1-oxyl). However, the 5-line EPR spectrum characteristic of Biradical X in aqueous solution was changed to a dominantly 3-line spectrum within a few minutes after illumination in the presence of ferredoxin and NADP+. Analysis of the double integration of the first derivative EPR spectrum revealed no decrease in Biradical X concentration for more than 30 min of illumination. Our data suggest that Biradical X attaches to some soluble macromolecule(s) and that illumination of chloroplasts promotes such an attachment.
General model for osmotic and pressure-induced flow in plant roots
Authors: Fiscus Edwin L. , Kramer Paul J.
Published: May 2006

This paper presents a general model to describe coupled solute and water flow through plant roots when they are subjected to osmotic or hydrostatic pressure gradients, or both simultaneously. The model is based on well-established membrane transport equations derived from irreversible thermodynamic considerations. A variety of experimentally observed phenomena such as changes in root resistance with flow rate, apparent negative resistance effects, xylem sap dilution, and apparent non-osmotic water transport can be adequately explained with this model. The model also predicts that an exuding excised root system will be very insensitive to changes in the hydraulic conductivity coefficient. Previous work with a flat membrane of unit surface area and uniform properties is confirmed for a cylindrical coordinate system of nonuniform characteristics.
Growth-dependent alterations in oligomannosyl cores of glycopeptides.
Authors: Ceccarini C , Muramatsu T , Tsang J , Atkinson P H
Published: May 2006

Mannose-labeled glycopeptides from the surface of growing and nongrowing human diploid cells (KL-2) were separated into neutral glycopeptides and acidic glycopeptides by paper electrophoresis. Growth-dependent alterations occurred in oligomannosyl cores in the neutral glycopeptides; namely, the neutral glycopeptides from the surface of growing cells were more resistant to endo-beta-N-acetylglucosaminidase D but were more susceptible to alpha-mannosidase (EC 3.2.1.24; alpha-D-mannose mannohydrolase) than those derived from the surface of nongrowing cells. Another growth-dependent change was found when the endoglycosidase-resistant material from acidic glycopeptides was compared by paper electrophoresis at pH 1.9. The material from the surface of nongrowing cells contained a component that was absent or greatly reduced in growing cells.
On the alleged high sensitivity of mouse Ehrlich-Lettre ascites tumor cells to diphtheria toxin.
Authors: Pappenheimer A M , Randall V
Published: May 2006

It was recently reported by Iglewski and Rittenberg in THESE PROCEEDINGS (71, 2707-2710, 1974) that low doses of purified diphtheria toxin inhibit protein synthesis in mouse Ehrlich-Lettre ascites carcinoma cells cultured in vitro. These observations could not be confirmed by us nor could the authors' further claim that toxin can cause regression of well-established ascites tumors in preimmunized mice be confirmed. Although temporary regression of such tumors can be demonstrated in unimmunized mice following intraperitoneal injection of diphtheria toxin, the amounts of toxin required are high and approach the lethal dose. About the same amount of CRM45, a tox gene product serologically related to diphtheria toxin but only 1/10,000th as toxic for guinea pigs, will also cause temporary regression in tumor-bearing mice.
An inequality among determinants
Authors: Seiler E. , Simon B.
Published: May 2006

We prove that for any trace class operators, [Formula: see text]
Chemical and physical properties of fractionated chromatin.
Authors: Berkowitz E M , Doty P
Published: May 2006

Sonicated chicken reticulocyte chromatin was fractionated into transcriptionally active and transcriptionally repressed components. The active fraction is 8% of the whole chromatin but contains 70% of the newly synthesized chromosomal RNA. This RNA has five times as many hemoglobin RNA sequences as does the RNA in the repressed fraction. The amount of the active fraction in the chromatins of several tissues correlates with their synthetic activity. The molecular weight of the DNA of the repressed fraction is approximately twice that of the active fraction. Moreover, the configuration of repressed chromatin is much more compact, consistent with a much larger sedimentation constant. The transcriptionally active fraction displays a 6 degrees lower melting profile and is highly susceptible to DNase I relative to the repressed fraction. The active fraction contains twice as much non-histone protein and 15% less histone than the repressed fraction and is lacking the lysine-rich and much of the arginine-rich histones.
A model for compartmentation of de novo and salvage thymidine nucleotide pools in mammalian cells.
Authors: Kuebbing D , Werner R
Published: May 2006

When [3H]thymidine is added to HeLa cells grown in thymidine-free medium it is incorporated into DNA almost immediately at full specific activity, blocking any further incorporation of de novo synthesized thymidine nucleotides. Apparently, de novo nucleotide pools [Baumunk, C.N. & Friedman, D.L. (1971) Cancer Res. 31, 1930--35] and salvage nucleotide pools are compartmentalized in HeLa cells. After one generation in the presence of thymidine, the cells begin utilizing nucleotides from both pools to about equal extent.
Bacterial luciferase requires one reduced flavin for light emission.
Authors: Becvar J E , Hastings J W
Published: May 2006

Recent reports revive a hypothesis that the bacterial bioluminescence reaction involves two reduced flavin mononucleotide molecules per enzyme turnover. A two-flavin mechanism requires that the two flavins bind simultaneously or sequentially to the same or different sites on luciferase during a catalytic cycle. Measurements using equilibrium techniques show that the luciferase dimer has only a single reduced flavin binding site. Quantum yield results demonstrate that bioluminescence requires only one reduced flavin per luciferase, ruling out mechanisms involving either two reduced flavins or one reduced flavin plus one oxidized flavin per catalytic cycle.
Reversible effects of chaotropic agents on the proton permeability of Escherichia coli membrane vesicles.
Authors: Patel L , Schuldiner S , Kaback H R
Published: May 2006

Extraction of E. coli ML 308-225 membrane vesicles with chaotropic agents causes the vesicles to become specifically permeable to protons. As a result, the vesicles no longer generate a membrane potential, interior negative, and they do not catalyze respiration-dependent lactose or proline transport. Treatment of the extracted vesicles with various carbodiimides decreases the permeability of the vesicle membrane to protons, causing them to regain their ability to generate a membrane potential. By this means, active transport is completely reactivated. Exposure of the vesicles to carbodiimides prior to extraction with chaotropic agents makes transport activity impervious to the effects of the chaotropes.
Conformational change and cooperativity in actin filaments free of tropomyosin.
Authors: Loscalzo J , Reed G H , Weber A
Published: May 2006

The decrease in amplitude of the electron spin resonance spectrum of the cysteine-bound spin-label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinoxyl, brought about by the magnetic interaction with tightly bound manganous ion, was used as a probe of conformational change in actin on binding myosin. The magnitude of this "spin--spin" interaction first decreased then increased on increasing saturation of the actin filament with heavy meromyosin subfragment-1. That the "spin--spin" interaction occurred between spins of adjacent monomers was demonstrated by the observation that the change in magnitude of the "spin--spin" interaction was maintained on binding of heavy meromyosin subfragment-1 to copolymers in which actin monomers containing both manganous ion and spin label were diluted 7-fold with native actin monomers. These data provide evidence for a conformational change in actin on interacting with heavy meromyosin subfragment-1. Further, the fact that not only the magnitude but also the sense of the change in the "spin--spin" interaction is a function of increasing saturation with heavy meromyosin subfragment-1 indicates that the monomers of the actin filament are capable of cooperative interaction in the absence of tropomyosin.
In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.
Authors: Cameron J R , Panasenko S M , Lehman I R , Davis R W
Published: May 2006

DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells.
Central role for magnesium in coordinate control of metabolism and growth in animal cells.
Authors: Rubin H
Published: May 2006

The rate of DNA synthesis in cultures of chicken embryo fibroblasts is reduced by deprivation of serum, high population density, and other "physiological" effectors, through a reduction in the number of cells in the S-period of the cell cycle. The same effect can be produced by drastically reducing the concentration of Mg++ added to the medium. This effect is erratic, however, and better control of [Mg++] can be achieved with phosphorylated compounds which preferentially bind Mg++. Both ATP and ADP, at concentrations in the medium less than or equal to [Mg++], stimulate DNA synthesis in cultures, and at greater concentrations inhibit DNA synthesis by affe-ting the proportion of cells in the S-period. Sodium pyrophosphate, which strongly complexes Mg++, causes little stimulation of DNA synthesis at low concentrations, but causes a striking decrease at concentrations exceeding [Mg++] of the medium. The inhibition can be fully reversed by adding an excess of Mg++, and the kinetics of increase in DNA synthesis resemble those which follow the restoration of serum to serum-deprived cultures. Limitation of [Mg++] by pyrophosphate also reduces the rates of RNA and protein synthesis, 2-deoxy-D-glucose uptake, and lactic acid production to an extent comparable to the reduction caused by the removal of serum from the medium. A model for the coordinate control of metabolism, differentiated function, and growth through the activity of divalent cations is described. The compartmentalization of Mg++ within the cell serves as the key element in this coordinate control by regulating those metabolic pathways in which the rate-limiting steps are transphosphorylation reactions.
Inhibition of tubulin assembly by RNA and other polyanions: evidence for a required protein.
Authors: Bryan J B , Nagle B W , Doenges K H
Published: May 2006

Nonneural cell extracts contain a heat-stable, nondialyzable activity that will inhibit the spontaneous assembly in vitro of partially purified brain tubulin. The sensitivity of this inhibitory activity to ribonucleases but not to a variety of other hydrolytic enzymes indicates that the inhibitor is an RNA. This conclusion is supported by the observation that purified RNAs from sea urchins, chinese hamster ovary cells, and brain all inhibit spontaneous microtubule assembly in vitro. The synthetic polynucleotides [poly(A), (C), (G), and (U)] are also inhibitory. This inhibition, however, appears to be nonspecific since the RNA base composition is unimportant and a variety of other nonnucleic acid polyanions also function as inhibitors. The treatment of assembly competent tubulin preparations with an insoluble RNA in the form of poly(A) covalently linked to agarose beads produces a "stripped" tubulin which will not assemble microtubules unless a heat-stable, trypsin-sensitive fraction eluted with increased ionic strength is mixed with the "stripped" tubulin. Similar results can be obtained with other cation exchangers, including phosphocellulose and carboxymethylcellulose. The heat-stable protein sequestered by poly(A)-agarose appears to be identical to the "tau" factor recently described by Kirschner and coworkers. Reconstitution experiments indicate that there is a stoichiometric requirement for these factors. These results suggest that spontaneous assembly of microtubules in nonneural cell extracts is blocked because the endogenous factors are complexed with RNA. This idea is supported by the observation that the ratio of tubulin to RNA is low in cultured cell extracts but very high in neural tissue extracts.
Brain tryptophan hydroxylase: purification of, production of antibodies to, and cellular and ultrastructural localization in serotonergic neurons of rat midbrain.
Authors: Joh T H , Shikimi T , Pickel V M , Reis D J
Published: May 2006

Tryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropteridine:oxygen oxidoreductase (5-hydroxylating)], the enzyme catalyzing the rate-limiting step in the biosynthesis of serotonin, was purified 79-fold from the region of the raphe nucleus of rat midbrain by sequential column chromatography and disc-gel electrophoresis. In electrophoresis three bands were distinguished, A, B, and C, which, when separated and submitted individually to electrophoresis, reproduced the same three bands. Bands A and C were enzymatically active and inhibited by para-chlorohenylalanine. Antibodies produced to each of the three bands crossreacted by immuno double diffusion and electrophoresis with each other and homogenates of raphe nuclei; they completely inhibited enzyme activity only of tryptophan hydroxylase. Tryptophan hydroxylase was localized by light and electron immunohistochemistry to serotonin neutrons of the raphe. Ultrastructurally, in cell bodies, the enzyme was distributed in cytoplasm and in association with endoplasmic reticulum and Golgi apparatus. In dendrites and axons, it was associated with microtubules. Tryptophan hydroxylase in brain is only neuronal and cytoplasmic, exists in multiple forms, and is associated with microtubules, suggesting it may be transported from sites of synthesis in cell body into axons.
Complementation of H-2-linked Ir genes in the mouse.
Authors: Dorf M E , Benacerraf B
Published: May 2006

The immune response to the random linear terpolymer of L-glutamic acid, L-lysine, and L-phenylalanine (GLphi) is under dominant H-2-linked Ir gene control in the mouse. Matings between two nonresponder strains produced responder F1 hybrids, demonstrating complementation of the nonresponder alleles. This observation, coupled with the fact that several intra H-2 recombinant strains derived by recombination between two nonresponder parental haplotypes are also GLphi responders, indicated at least two dominant loci are concerned with responsiveness to this terpolymer. The complementary genes were termed alpha (tentatively localized in a new subregion of the H-2 complex, I-F) and beta, which maps in the I-A subregion. Generally, both the alpha(+) and beta(+) alleles are required for responsiveness. However, in the (C57BL/6J X SJL)F1 hybrid we noted complementation between two parental nonresponder strains, each of which carried beta genes derived from different H-2 haplotypes, yet lacked functional alpha genes. The possible cell levels at which these genes may function in the regulation of the immune response are discussed.
A normal level of adenosine deaminase activity in the red cell lysates of carriers and patients with severe combined immunodeficiency disease.
Authors: Trotta P P , Smithwick E M , Balis M E
Published: May 2006

The red cell lysates of two children with severe combined immunodeficiency disease (SCID) exhibited a virtually total absence of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) when standard volumes were assayed. Under these conditions the parents exhibited depressed specific activity except for one mother, whose lysate showed a normal value for activity. Upon storage of the lysate at 4 degrees, a significant amount of activity appeared in one of the SCID children, and the activity of the heterozygous carriers was stimulated. With the use of a sensitive spectrophotometric assay based on conversion of inosine to uric acid, it was shown that the specific enzymatic activity in each of the SCID patients increased progressively as the volume of lysate assayed was lowered. With the smallest amount of lysate this specific activity was in the normal range. Similarly, the specific activity of each of the parents' lysates increased to the level of normal (or, in one case, about twice normal) as smaller volumes were assayed. The activity in the SCID patient was inhibitable by 2-fluoroadenosine and N6-methyladenosine, known competitive inhibitors of human red cell adenosine deaminase. The lysate from the SCID patient was also shown to inhibit adenosine deaminase partially purified from a normal individual. The results are interpreted in terms of a genetically programmed production of an adenosine deaminase inhibitor in at least one variant of the severe combined immunodeficiency disease.
Oxidation of ferrocytochrome c by mitochondrial cytochrome c oxidase.
Authors: Errede B , Haight G P , Kamen M D
Published: May 2006

Attempts to rationalize the kinetics of cytochrome c oxidation catalyzed by solubilized mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) have been based on assumptions of productive complex formation (Michaelis-Menten approach). However, the range of substrate concentrations used has not, in general, been sufficient to establish a general rate equation. Data adequate to derive such a rate expression are presented, as well as a method for estimation of constants which appear in the rate law deduced and reported herein. It is shown that either of two types of mechanisms, one assuming productive complex formation, as opposed to the other postulating dead-end complex formation, accurately predict the rate equation as deduced from experiment.
Synapse formation between clonal neuroblastoma X glioma hybrid cells and striated muscle cells.
Authors: Nelson P , Christian C , Nirenberg M
Published: May 2006

Clonal neuroblastoma X glioma hybrid cells were shown to form synapses with cultured, striated muscle cells. The properties of the synapses between hybrid and muscle cells were similar to those of the normal, neuromuscular synapse at an early stage of development. The number of synapses formed and the efficiency of transmission across synapses were found to be regulated, apparently independently, by components in the culture medium. Under appropriate conditions synapses were found with 20% of the hybrid-muscle cell pairs examined; thus, the hybrid cells form synapses with relatively high frequency.
Affinity of myosin S-1 for F-actin, measured by time-resolved fluorescence anisotropy.
Authors: Highsmith S , Mendelson R A , Morales M F
Published: May 2006

The association constant for myosin subfragment-1 (S-1) and actin was measured, using a new application of fluorescence depolarization which capitalizes on the fact that S-1 has high rotational mobility while F-actin does not. Uncoupling of the time dependences of the anisotropy decay and the association/dissociation phenomena allowed the experimentally determined anisotropy decay curve to be fitted by a sum of two terms weighted by the mole fractions of the free and bound S-1. At 4 degrees C, ionic strength 0.16 M, and pH 7.0, the association constant Ka is (1.73 +/- 0.35) X 10(6) M-1 at infinite dilution. This makes the -deltaG degrees of binding of F-actin to S-1 similar to the -deltaG degrees of binding of ATP to S-1, and the possible physiological relevance of the similarity to muscle contraction is discussed.
Translation of nonpolyadenylylated messenger RNA of sea urchin embryos.
Authors: Fromson D , Verma D P
Published: May 2006

A large proportion of newly synthesized polyribosomal RNA of sea urchin blastulae is not polyadenylylated. The size distributions of the polyadenylylated and nonpolyadenylylated RNA are indistinguishable (mean size of 26 S). Upon translation of sea urchin polyribosomal RNA containing poly(A) and that without poly(A) in a wheat embryo cell-free protein-synthesizing system, where polypeptide synthesis is dependent on added messenger, both classes of RNA support peptide synthesis to the same extent. A preliminary analysis of the proteins synthesized in response to added mRNA (polyadenylylated and nonpolyadenylylated) indicates that these classes of mRNA molecules may code for different populations of proteins.
Novel F prime factors able to replicate in Escherichia coli Hfr strains.
Authors: Hiraga S
Published: May 2006

A novel type of F' plasmid that can replicate extrachromosomally in cells of Hfr strains has been isolated. Genetic analysis of these plasmids indicates that all carry the dnaA-bglA region of the E. coli chromosome as well as ilv+ used as a selective marker. It is suggested that a specific site, designated poh+ (permissive on Hfr), is located in this region, and is essential for these plasmids to replicate in Hfr cells. The most interesting explanation, among others, would be that the poh+ site represents the replication origin of the bacterial chromosome.
Carbohydrate moieties of procollagen: incorporation of isotopically labeled mannose and glucosamine into propeptides of procollagen secreted by matrix-free chick embryo tendon cells.
Authors: Clark C C , Kefalides N A
Published: May 2006

Cells obtained from chick embryo tendons incorporate isotopically labeled glucosamine and mannose into the pro-alpha1 and pro-alpha2 chains of procollagen as judged by sodium dodecyl sulfate-gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The label was further localized to the propeptides of pro-alpha1 and pro-alpha2 by its chromatographic behavior after digestion with bacterial collagenase or alpha-chymotrypsin. Carbohydrate analysis of isolated pro-alpha chains showed the presence of labeled galactosamine in addition to mannose and glucosamine. Resistance to mild alkaline hydrolysis suggested that greater than 90% of the oligosaccharide units are not linked to the propeptide backbone by either serine or threonine.
Radioimmunoassay of bovine heart protein kinase.
Authors: Fleischer N , Rosen O M , Reichlin M
Published: May 2006

Immunization of guinea pigs with bovine cardiac cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) resulted in the development of precipitating antibodies to the cAMP-binding subunit of the enzyme. Both the phosphorylated and nonphosphorylated cAMP-binding protein of the protein kinase reacted with the antiserum. A radioimmunoassay was developed that detects 10 ng of holoenzyme and permits measurement of enzyme concentrations in bovine cardiac muscle. Bovine liver, kidney, brain, and skeletal muscle contain protein kinases which are immunologically identical to those found in bovine cardiac muscle. However, the proportion of immuno-reactive enzyme activity differed for each tissue. All of the immunologically nonreactive enzyme in skeletal muscle and heart was separable from immunoreactive enzyme by chromatography on DEAE-cellulose. Rat tissues and pig heart contained protein kinase activity that crossreacted immunologically in a nonparallel fashion with bovine cardiac enzyme. These results indicate that cAMP-dependent protein kinases within and between species are immunologically heterogeneous.
Amino-acid sequence of fragment A, an enzymically active fragment from diphtheria toxin.
Authors: DeLange R J , Drazin R E , Collier R J
Published: May 2006

The amino-acid sequence of Fragment A from diphtheria toxin is reported. Fragment A (molecular weight, Mr, 21,145) is the major enzymically active fragment produced upon activation of the intact toxin (Mr about 60,000) by limited tryptic digestion and reduction. It, or a similar fragment, is believed responsible for the inhibition of protein synthesis in animal cells exposed to the toxin. Fragment A, which corresponds to the amino terminus of the toxin, is shown here to consist of three major forms (190, 192, and 193 residues) resulting from cleavage by trypsin adjacent to any of three closely spaced arginine residues. All three forms are enzymically active.
Primary structure of the gamma-carboxyglutamic acid-containing protein from bovine bone.
Authors: Price P A , Poser J W , Raman N
Published: May 2006

The amino-acid sequence of the gamma-carboxyglutamic acid-containing protein of bovine bone is presented. The sequence of 43 of the 49 residues was determined automatically on the intact protein and on a tryptic peptide. The remainder was determined by conventional procedures on tryptic and chymotrytic peptides. Residue 9 in the sequence has been identified as 4-hydroxyproline. The protein contains three gamma-carboxyglutamic acid residues, which are located at positions 17, 21, and 24. The role of these unusual amino acids in the binding interaction between the protein and hydroxyapatite crystals is discussed.
Novel structure at 5'-ends of nascent DNA chains.
Authors: Siegmann D W , Werner R
Published: May 2006

Because of their association with protein short nascent DNA chains in Escherichia coli can be separated from other cellular DNA by chromatography on hydroxylapatite. Protein-free DNA chains of less than 500 nucleotides in length are resistant to degradation from the 5'-end by alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1] and spleen phosphodiesterase (oligonucleate 3'-nucleotidohydrolase; EC 3.1.4.18). In contrast, DNA chains containing more than 500 nucleotides are degradable. From these results we conclude that short nascent DNA chains are structurally modified at their 5'-ends. The nature of this structure and its possible functions are discussed.
On the mechanism of delayed inhibition of protein synthesis in heme-defecient rabbit reticulocyte lysates.
Authors: Cherbas L , London I M
Published: May 2006

In the absence of added hemin, protein synthesis in a rabbit reticulocyte lysate declines abruptly (shuts off) after about 5 min at 30 degrees. In these studies we have examined the basis for the lag period preceding shut-off. The initiation factor that binds Met-tRNAf, previously shown to be rate-limiting in inhibited, heme-deficient lysates, is found to be used stoichiometrically in the presence of excess inhibitor. We suggest that a principal effect of the inhibitor is to impair the recycling of the Met-tRNAf-binding factor; the lag period is attributable largely to the presence of a pool of excess Met-tRNAf-binding factor, which, once used in initiation, cannot be recycled because of the action of the inhibitor.
Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate.
Authors: Mizuuchi K , Nash H A
Published: May 2006

A novel assay has been developed for in vitro genetic recombination of DNA. Substrate and product DNAs are cleaved with a restriction endonuclease and the resulting fragments are separated by electrophoresis in agarose gels. The substrate DNA has been chosen so that the recombination to be studied deletes a segment of DNA. The remaining DNA gives rise to a unique restriciton fragment, as does the DNA segment that has been removed. The method provides a convenient and physical, rather than genetic, assessment of the conversion of parental to recombinant DNA. This method has been applied to an in vitro system that carries out integrative recombination of bacteriophage lambda. We find that, different molecular forms of DNA tested, closed circular DNA is the only efficient substrate. Linear DNA and three kinds of circular DNA containing interruptions are at best very poor substrates. The implications of this surprising result are discussed. In addition, we show that the in vitro recombination system completes the breaking and rejoining steps of recombination. No stable DNA intermediates involving chiasmata or broken end structures are found.
Partial purification and characterization of the mRNA for rat preproinsulin.
Authors: Duguid J R , Steiner D F , Chick W L
Published: May 2006

Electrophoretically homogeneous messenger RNA for rat preproinsulin has been prepared from an islet cell tumor by the use of oligo(dT(-cellulose chromatography and sucrose density gradient centrifugation. The molecular weight of the mRNA is about 210,000, as determined by polyacrylamide gel electrophoresis in formamide, and its sedimentation coefficient is 9.3 S in sucrose gradients containing 0.2 M NaCl. These results indicate that the synthesis of preproinsulin is directed by a monocistronic mRNA 600 nucleotides in length and requires approximately 55% of the molecule's coding capacity.
Reversion from transformed to normal phenotype by inhibition of protein synthesis in rat kidney cells infected with a temperature-sensitive mutant of Rous sarcoma virus.
Authors: Ash J F , Vogt P K , Singer S J
Published: May 2006

By the use of a rat kidney cell line infected with a temperature-sensitive Rous sarcoma virus, we have shown that, at permissive temperatures where the cells are transformed, concanavalin A induces a clustering of its cell membrane receptors into patches, and the intracellular smooth muscle myosin-like protein is in a disordered state. By contrast, with infected cells grown at nonpermissive temperatures, the addition of concanavalin A does not alter the uniform distribution of its receptors, and the smooth muscle myosin-like protein is arranged in an ordered filamentous structure. These results are consistent with the hypothesis that the myosin protein is part of an intracellular aggregating-disaggregating complex. In the normal cell it is in its aggregated state and inhibits the lateral mobility of the concanavalin A receptors in the membrane; in the transformed cell the complex is relatively disaggregated and permits the concanavalin A receptors to be mobile. The addition of protein synthesis inhibitors to infected cells grown at the permissive temperature causes the cell to change from the transformed phenotype to the normal. Removal of the reversible inhibitors causes the cells to revert to the transformed phenotype. These results show that (i) protein synthesis, presumably of an unstable product of the transforming gene of the temperature-sensitive virus, is required to maintain the transformed state in these infected cells at the permissive temperature; and (ii) protein synthesis is not required for the intracellular myosin-containing complex to revert from its disordered transformed state to its ordered normal state. This suggests that the product of the transforming gene directly or indirectly causes the disaggregation of the myosin-containing complex in the process of transformation.
Characterization of heteropolymeric hexosaminidase A in human X mouse hybrid cells.
Authors: Chern J , Beutler E , Kuhl W , Gilbert F , Mellman W J , Croce C M
Published: May 2006

Expression of heteropolymeric hexosaminidase A activity is reported in a human X mouse hybrid cell line that contains an X/15 translocation chromosome but lacks human chromosome 5 and has no detectable human hexosaminidase B activity. (Hexosaminidase is beta-N-acetylglucosaminidase; EC 3.2.1.30; 2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase.) That the "hexosaminidase A" enzyme of these cells contains the human hexosaminidase-alpha subunit and not the human hexosaminidase-beta subunit is indicated by the cells' reactions to specific antisera prepared against the alpha subunit and against the beta subunit. Our results indicate that the "hexosaminidase A" activity in this hybrid cell line is the expression of a hybrid molecule composed of human hexosaminidase-alpha subunit and a mouse hexosaminidase subunit. These results are consistent with the hypothesis that the human hexosaminidase A enzyme is formed from alpha and beta subunits coded for by genes on chromosomes 15 and 5, respectively.
Polyclonal activation of bone-marrow-derived lymphocytes from human peripheral blood measured by a direct plaque-forming cell assay.
Authors: Fauci A S , Pratt K R
Published: May 2006

A culture and assay system for the stimulation of human peripheral blood lymphocytes with polyclonal activators of bone-marrow-derived lymphocytes (B cells), such as pokeweed mitogen and Escherichia coli lipopolysaccharide, and subsequent measurement of single cell antibody production by a hemolysis-in-bel direct plaque-forming cell assay against sheep erythrocytes has been established. The critical culture requirements have been delineated and a new highly sensitive ultrathin gel assay method has been described. Under these conditions a substantial and highly reproducible plaque-forming cell response was detected in normal human peripheral blood. This system can be readily used to explore the complex events associated with activation of human B cells.
Nuclear magnetic resonance description of molecular motion and phase separations of cholesterol in lecithin dispersions.
Authors: Opella S J , Yesinowski J P , Waugh J S
Published: May 2006

Proton-enhanced 13C nuclear magnetic resonance is used to obtain signals from labeled cholesterols in lecithin dispersions. The [26-(13)C]cholesterol resonance indicates that the aliphatic tail of the molecule undergoes reorientation fast enough to average completely the chemical shift anisotropy. In contrast, [4-(13)C]cholesterol signals are characteristic of limited anisotropic reorientation. The resonances from the 4 position are sensitive to the temperature-concentration phase diagram. A phase boundary is observed at about 20 mole percent cholesterol.
Regulation of dopamine stimulation of striatal adenylate cyclase by an endogenous Ca++ -binding protein.
Authors: Gnegy M E , Uzunov P , Costa
Published: May 2006

Membranes of rat caudate nucleus contain a dopamine-dependent adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and a Ca++ binding protein that activates phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17). This activator can be released from the membranes by a phosphorylation with a 3':5' cAMP-dependent protein kinase (ATP-protein phosphotransferase, EC 2.7.1.37). Under the conditions of membrane phosphorylation and activator release, dopamine fails to activate striatal adenylate cyclase. The basal activity of this enzyme is not decreased by the release of the protein activator but the activation by NaF is reduced. Adenylate cyclase is not phosphorylated when the dopamine activation is blocked after the release of the activator, but other membrane proteins are phosphorylated. It is postulated that the endogenous protein stored in striatal membranes can regulate the intracellular concentration of cAMP by an activation of adenylate cyclase while stored in striatal membrane, and by an activation of phosphodiesterase when released into the cytosol after membrane phosphorylation.
Purification and characterization of a putative sigma factor from Chalamydomonas reinhardi.
Authors: Surzycki S J , Shellenbarger D L
Published: May 2006

Two proteins with sigma-like activity have been isolated from the alga, Chlamydomonas reinhardi. One protein, sigma 2, has been partially purified and appears to have a molecular weight of 51,000. The interaction of this protein with a heterologous (Escherichia coli) and homologous (Chlamydomonas, chloroplast rifampicin-sensitive) core RNA-polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was studied. Sigma 2 protein appears to stimulate the formation of open (rapid starting) binary complexes by both of the core enzymes. Stimulation of transcription by sigma 2 on chloroplast DNA was greater when Chlamydomonas core enzyme was used. Moreover, in vitro transcription on a variety of templates using RNA polymerases I and II from Chlamydomonas was not stimulated by this protein.
Changes in microfilament organization and surface topogrophy upon transformation of chick embryo fibroblasts with Rous sarcoma virus.
Authors: Wang E , Goldberg A R
Published: May 2006

A series of morphological changes occurred when chick embryo fibroblasts infected with the NY68 mutant of Rous sarcoma virus were shifted from nonpermissive temperature (41degrees) to permissive temperature (37 degrees). We observed three distinct stages in cell morphology and surface topography that were correlated with a reduction in the organization and assembly of actin-containing microfilament bundles. Our observations suggest that control of microfilament organization and surface topography are responsive to the presence of a functioning transforming gene (src) product of Rous sarcoma virus.
Active cell edge and movements of concanavalin A receptors of the surface of epithelial and fibroblastic cells.
Authors: Vasiliev J M , Gelfand I M , Dominina L V , Dorfman N A , Pletyushkina O Y
Published: May 2006

Movements of the receptors of concanavalin A on various parts of the surfaces of substrate-attached cells were compared. Cultured mouse embryo cells of several types were used: epithelial kidney cells and normal and transformed fibroblasts. Initial distribution of receptors was random on the cells of all types. Binding of concanavalin A induced patching of its receptors on all the cell parts. In contrast, directional centripetal movement of receptors was observed only on the surface of certain cell parts, namely, only the surface of peripheral lamellar cytoplasm was cleared of the receptors. Clearing was always initiated in the zone of lamellar cytoplasm located near active cell edges. In epithelial sheets, clearing was not observed on the surface of central cells that had no lamellar cytoplasm. Concanavalin A receptors on the cleared areas of cell surface were gradually restored after the end of incubation. It is suggested that anchoring of the patches of membrane receptors by cortical microfilaments is possible only on the surface of pseudopods and of lamellar cytoplasm but not on the surface of other cell parts. Besides receptor movements, this hypothesis may be explain differences in the adhesive properties of various parts of the cell surface.
Alterations in surface glycoproteins and level of sialyltransferase of cells transformed by a temperature-sensitive mutant of simian virus 40.
Authors: Onodera K , Yamaguchi N , Kuchino T , Aoi Y
Published: May 2006

Mouse cells transformed by a temperature-sensitive mutant of simian virus 40 belonging to complementation group A lost their ability to regulate cell growth when grown at the permissive temperature (35 degrees) but showed the low saturation density of cell growth at the restrictive temperature (39.5 degrees) that is characteristic of normal cells in vitro. Biochemical analysis of the membranes of cells grown under the restrictive and the permissive conditions demonstrated no qualitative temperature-dependent differences either in neutral glycolipids or in acidic glycolipids of the cells. Plasma membrane glycoproteins labeled with radioactive glucosamine showed significantly different patterns on both polyacrylamide gel electrophoresis and electrofocusing. When the levels of glycoprotein glycosyltransferases of the cells were examined, the level of sialyltransferase (CMP-N-acetylneuraminytransferase,EC 2.4.99.1) of the cells grown at the restrictive temperature was low compared with that of cells grown at the permissive temperature. Our results indicate that the level of sialyltransferase is under the control of the gene A function of simian virus 40 and consequently is related to alterations in the cell surface glycoproteins.
Growth of human diploid fibroblasts in the absence of glucose utilization.
Authors: Zielke H R , Ozand P T , Tildon J T , Sevdalian D A , Cornblath M
Published: May 2006

Normal human diploid fibroblasts were able to undergo one to two cell divisions without glucose utilization in Eagle's minimum essential medium plus 10% dialyzed fetal calf serum if the medium was supplemented with hypoxanthine, thymidine, and uridine (supplemented medium termed HTU-MEM). Under these conditions, the added purine and pyrimidines were required for nucleic acid synthesis, as shown by the inability of Lesch-Nyhan fibroblasts to grow in HTU-MEM. Normal human diploid fibroblasts continued to produce lactate in HTU-MEM, but at a greatly reduced rate. Since cells grew in HTU-MEM without glucose utilization, the probable energy and carbon source was glutamine, which is present in relatively high concentration. Furthermore, the rate of glutamine utilization per cell division was 2-fold greater in HTU-MEM than in medium with 5.5 mM glucose. These results suggest that glutamine can be a major energy source for cells grown in vitro.