bluuebunny/crossref_metadata_2025 huggingface dataset:
Variations of DNA polymerase-alpha and -beta during prolonged stimulation of human lymphocytes.
Authors: Bertazzoni U , Stefanini M , Noy G P , Giulotto E , Nuzzo F , Falaschi A , Spadari S
Published: May 2006

Stimulation of human lymphocytes with phytohemagglutinin is known to induce an increase in overall DNA polymerase activity (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7). Previous work [Pedrali Noy, G., Dalprà, L. Pedrini, A. M., Ciarrocchi, G., Giulotto, E., Nuzzo, F. & Falaschi, A. (1974) Nucleic Acids Res. 1, 1183] has shown that two subsequent waves of induction of DNA polymerase can be observed in this system; a first wave occurs in parallel with the increase in DNA replication rate; a second one occurs when the DNA synthesis rate is returned to minimal levels; the second peak is parallel to a maximum in DNA ligase and DNase levels. In the present work we have measured the levels of the DNA polymerases-alpha and -beta in phytohemagglutinin-stimulated lymphocytes during a 12-day period; both enzymes are present at detectable levels at time zero; in correspondence to the peak of DNA synthesis rate (between the fourth and fifth day) a peak of DNA polymerase-alpha is observed, increasing by a factor of approximately 20-fold over the zero time value; subsequently, the level of DNA polymerase-alpha decreases in parallel with DNA synthesis rate. The DNA polymerase-beta is also increased in correspondence to the peak in DNA synthesis rate, but reaches its maximum at later times, between the eighth and tenth day of incubation. The capacity of stimulated lymphocytes to perform repair synthesis following UV damage was measured in the same cells used for the enzyme activity determinations; this capacity also shows two maxima: a first one correlated with the peak in DNA replication rate, and a second one correlated with the peak of DNA polymerase-beta. These data suggest a certain tendency to the specialization of functions in human cell DNA polymerases; the alpha-enzyme seems mainly correlated with DNA replication, whereas the beta-enzyme seems more correlated with the ability of the cell to perform repair type synthesis.
Biological activity of catecholamines covalently linked to synthetic polymers: proof of immobilized drug theory.
Authors: Verlander M S , Venter J C , Goodman M , Kaplan N O , Saks B
Published: May 2006

l-Isoproterenol was covalently coupled via an azo linkage to soluble copolypeptides of molecular weight 1500 and 10,000 containing an aromatic amine. The polymeric azo-isoproterenol derivatives were purified by gel chromatography which reduced contamination by the parent isoproterenol to undetectable levels (i.e., less than 0.01%) and by 6-aminoisoproterenol to less than 0.4%. Both polymeric isoproterenol derivatives were found to elicit positive chronotropic responses in isolated perfused guinea pig hearts. The mean effective doses (ED50S) for the 1500 and 10,000 molecular weight derivatives were within 1.3 and 2.0 orders of magnitude, respectively, of the ED50 of l-isoproterenol. The responses cannot be attributed to free isoproterenol because this drug could not be detected in our preparations. Neither can the observed biological activity be attributed to 6-aminoisoproterenol, since this compound's dose-response is shifted 3 orders of magnitude to the right of l-isoproterenol and 1-2 orders of magnitude to the right of the polymeric derivatives. Inotropic response decay times in isolated cat papillary muscles following washouts indicate that the polymer-bound drug does not diffuse into the muscle tissues. We feel that our findings demonstrate that under controlled conditions the catecholamines can retain biological activity while covalently bound to a polymeric support.
Molecular structure of yeast RNA polymerase III: demonstration of the tripartite transcriptive system in lower eukaryotes.
Authors: Valenzuela P , Hager G L , Weinberg F , Rutter W J
Published: May 2006

Homogeneous RNA polymerase III (RNA nucleotidyltransferase III) has been obtained from yeast. The subunit composition of the enzyme was examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is composed of 12 putative subunits with molecular weights 160,000, 128,000, 82,000, 41,000, 40,500, 37,000, 34,000, 28,000, 24,000, 20,000, 14,500, and 11,000. The high-molecular-weight subunits and several of the smaller subunits of yeast RNA polymerase III are clearly different from those of enzymes I and II, indicating a distinct molecular structure. However, the molecular weights of some of the small subunits (41,000, 28,000, 24,000, and 14,500) appear to be identical to those of polymerases I and II. Thus, it is possible that the three classes of enzymes in yeast have some common subunits. As in other eukaryotes, yeast polymerase II is inhibited by relatively low concentrations of alpha-amanitin; however, contrary to what has been found in higher eukaryotes, yeast polymerase III is resistant (up to 2 mg/ml) to alpha-amanitin, while yeast polymerase I is sensitive to high concentrations of the drug (50% inhibition at 0.3 mg/ml). These results establish the existence of RNA polymerase III in yeast and provide a structural basis for the discrimination of the three functional polymerases in eukaryotes.
Distribution of envelope-specific and sarcoma-specific nucleotide sequences from different parents in the RNAs of avian tumor virus recombinants.
Authors: Wang L H , Duesberg P , Mellon P , Vogt P K
Published: May 2006

The distribution of leukosis-virus- and sarcoma-virus-specific oligonucleotide sequences was investigated in the RNAs of viral recombinants selected for an envelope gene (env) from a leukosis parent and a sarcoma gene (src) from a sarcoma parent. For this purpose 20 to 30 RNase-T1-resistant oligonucleotides were chemically analyzed and mapped within the 10,000 nucleotides of each viral RNA relative to the 3'-poly(A) end. The resulting oligonucleotide maps were compared. Proceeding from the 3' to the 5' end, the maps of four recombinants contained: (i) in a segment of 2000 nucleotides, three to four src-specific oligonucleotides, so identified because they were shared only with the sarcoma parent; and (ii) in a segment of 8000 nucleotides, 20 oligonucleotides shared with the leukosis parent, of which six to seven were also shared with the sarcoma parent. Two other recombinants contained: (1) in a segment of 2000 (one) or 3000 (the other) nucleotides, three src-specific oligonucleotides; (ii) in a segment of 3000 (one) or 2000 (the other) nucleotides, five (one) or four (the other) oligonucleotides, all or some of which are env-specific, because they were shared with the leukosis parent; (iii) in a segment of 5000 nucleotides (both), 11 functionally unidentified sarcoma-virus-derived oligonucleotides, of which seven were also shared with the leukosis parent. The map locations of parental oligonucleotides were not changed in recombinants and all viral strains tested shared six to eight highly conserved oligonucleotides at equivalent map locations. The partial map -env-src-poly(A) emerged from the analyses of these recombinants.
Variant forms of mitochondrial translation products in yeast: evidence for location of determinants on mitochondrial DNA.
Authors: Douglas M G , Butow R A
Published: May 2006

Products of mitochondrial protein synthesis in yeast have been labeled in vivo with 35SO42-. More than 20 polypeptide species fulfilling the criteria of mitochondrial translation products have been detected by analysis on sodium dodecyl sulfate-exponential polyacrylamide slab gels. A comparison of mitochondrial translation products in two wild-type strains has revealed variant forms of some polypeptide species which show genetic behavior consistent with the location of their structural genes on mtDNA. Our results demonstrate the feasibility of performing genetic analysis on putative gene products of mtDNA in wild-type yeast by direct examination of the segregation and recombination behavior of specific polypeptide species.
Two forms of simian-virus-40-specific T-antigen in abortive and lytic infection.
Authors: Ahmad-Zadeh C , Allet B , Greenblatt J , Weil R
Published: May 2006

Simian-virus-40-specific T-antigen was isolated by immunoprecipitation. From other studies we have proof that the T-antigen described in this work is coded by the viral DNA. The molecular weight estimated from electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels of T-antigen isolated from nonpermissive mouse cells in abortive infection is 86,000 and from permissive monkey cells in lytic infection is 82,000. The 86 kilodalton T-antigen is readily converted in vitro into an 82 kilodalton form by incubation with extracts from permissive monkey cells but not with extracts from nonpermissive mouse or hamster cells. This and the results of fingerprinting analysis of tryptic peptides suggest that T-antigen may be processed in permissive cells.
Control of protein synthesis in reticulocyte lysates: effects of 3':5'-cyclic AMP, ATP, and GTP on inhibitions induced by hemedeficiency, double-stranded RNA, and a reticulocyte translationa inhibitor.
Authors: Ernst V , Levin D H , Ranu R S , London I M
Published: May 2006

Protein chain initiation in reticulocyte lysates is inhibited by (a) heme-deficiency, (b) low levels of double-stranded RNA, and (c) a purified translational inhibitor isolated from heme-deficient lysates. Previous studies have shown that the inhibitions produced by heme-deficiency and double-stranded RNA are prevented by 3': 5'-cyclic AMP, and that GTP, but not ATP, prevents the inhibition of heme-deficiency. In view of the recent finding that the inhibitor purified from heme-deficient lysates is associated with a protein kinase which appears to be involved in the mechanism of inhibition, the effects of cyclic AMP, GTP, and ATP on the three modes of inhibition were examines. In all three types of inhibition, cyclic AMP or GTP (a) prevents the onset of inhibition when added at zero time, and (b) restores protein synthesis in inhibited lysates. In contrast to these effects, ATP potentiates each inhibition, and blocks reversal of inhibition by cyclic AMP or GTP. On the basis of these and earlier findings, we propose that (a) these inhibitions involve the phosphorylation by protein kinases of the Met-tRNAf binding factor and/or a related site(s) on the 40S ribosomal subunit; and (b) cyclic AMP, GTP, and ATP exert their effects by their actions on this phosphorylation mechanism.
Transcription-translation and translation-messenger RNA decay coupling: separate mechanisms for different messengers.
Authors: Walker A C , Walsh M L , Pennica D , Cohen P S , Ennis H L
Published: May 2006

Antibiotics were used to inhibit protein synthesis at specific steps in the biosynthetic pathway. In this way, it was possible to study the coupling of protein synthesis to the accumulation of biologically active mRNA in T4-infected Escherichia coli. Functional mRNA for the phage enzymes deoxynucleotide kinase (EC 2.7.4.4; ATP: nucleoside monophosphate phosphotransferase or nucleosidemonophosphate kinase) and alpha-glucosyltransferase (EC 2.4.1.5; 1, 4-alpha-D-glucan: 1, 6-alpha-D-glucan 6-alpha-glucosyltransferase or dextrin dextranase) accumulated during inhibition of protein synthesis irrespective of the step in the synthesis of protein that was blocked. Under these conditions, however, the rate of mRNA synthesis for both enzymes was significantly inhibited. In contrast, the rate of degradation of these mRNAs was markedly dependent on the step in protein synthesis that was inhibited. That is, the site for mRNase action was different for each message. The most important step in protein synthesis required for the stability of deoxynucleotide kinase mRNA is the initiation step. A single ribosome bound to the 5' end of the deoxynucleotide kinase mRNA can stabilize the molecule. On the other hand, the initiation event does not seem to be important for stabilizing the alpha-glucosyltransferase mRNA. Instead, a high ribosome denisty on the alpha-glucosyltransferase messenger is required to achieve significant stability. Therefore, in studying messenger metabolism, it is important to focus on the functional stability of specific mRNAs instead of on total messenger since each mRNA can be metabolized differently.
Intracellular localization of mouse DNA polymerase-alpha.
Authors: Herrick G , Spear B B , Veomett G
Published: May 2006

Although DNA polymerase-alpha (DNA nucleotidyltransferase; deoxynucleoside triphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) probably functions in the nucleus, it is usually found predominantly in the nonnuclear fraction of disrupted cells. We have reexamined the intracellular location of this enzyme using cytochalasin-B-induced enucleation, a technique which avoids exposure of nuclei to extra-cellular conditions during cell fractionation. In conditions where viability of separated cell parts is high and recovery is quantitative, we find greater than 85% of total DNA polymerase-alpha (and DNA polymerase-beta) activity in the nucleated cell fragments (karyoplasts), from which we conclude that the location in vivo of DNA polymerase-alpha is either nuclear or perinuclear. On the other hand, thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 5.7.1.75) is found primarily in the enucleated cell fragments (cytoplasts). The enucleation procedure used in this work should be of general use for intracellular location studies.
Fluorometric assay of vasopressin and oxytocin: a general approach to the assay of peptides in tissues.
Authors: Gruber K A , Stein S , Brink L , Radhakrishnan A , Udenfriend S
Published: May 2006

A fluorometric method for the quantitative assay of vasopressin and oxytocin in individual rat pituitaries has been developed. Acid extracts of pituitaries are freed of amino acids and polyamines by passage over a copper-Sephadex column, and the peptides fraction is then labeled by reaction with fluorescamine. The resulting peptide fluorophors are separated by chromatography on a reverse-phase bonded column. Specificity of the procedure was ascertained by several criteria, including bioassay and amino-acid analysis of the eluted peptide fluorophors. The procedure serves as a model system for the assay of tissue peptides in the picomole range.
Molecular abnormality of human alpha1-antitrypsin variant (Pi-ZZ) associated with plasma activity deficiency.
Authors: Yoshida A , Lieberman J , Gaidulis L , Ewing C
Published: May 2006

A human alpha1-antitrypsin variant protein was purified to homogeneity from homozygous variant subjects (Pi-ZZ) who had a deficiency of plasma trypsin inhibitory capacity. Molecular weight, specific trypsin inhibitory capacity, and immunologic activity of the variant protein were identical to those of normal. Amino acids, N-acetylglucosamine, and hexose contents were closely similar in the normal and variant proteins, but the sialic acid content in the variant protein was significantly lower than normal. The structural difference between the normal and the variant alpha1-antitrypsin was elucidated by fingerprinting of their tryptic peptides. Two amino acid substitutions, i.e., glutamic acid in the normal protein to lysine in the variant protein, and glutamic acid in the normal protein to glutamine in the variant protein, were found.
Ploy and counterploy in predator-prey interactions: Orb-weaving spiders versus bombardier beetles
Authors: Eisner Thomas , Dean Jeffrey
Published: May 2006

Bombardier beetles (
Brachinus
spp.) offered to orb-weaving spiders are either captured or lost, depending on the attack strategy of the spider.
Nephila clavipes
grasps a beetle directly and attempts to bite it outright, but is repelled by the beetle's defensive spray. As the spider recovers from the spray, the beetle makes its escape from the web.
Argiope
first imprisons the beetle by wrapping it delicately in silk, without causing it to spray. When the spider then proceeds to bite, the wrapping protects it against the full effects of the spray. The wrapping strategy may be generally effective against chemically protected insects, and it is suggested that this may be one of its principal adaptive justifications.
Spectroscopic studies and a structural model for blue copper centers in proteins.
Authors: Solomon E I , Hare J W , Gray H B
Published: May 2006

Low temperature absorption, circular dichroism, and magnetic circular dichroism spectral studies of the blue copper proteins Rhus vernicifera stellacyanin, bean plastocyanin, and Pseudomonas aeruginosa azurin have been made. Low energy bands attributable to the d-d transitions 2B2 leads to 2E and 2B2 leads to 2B1 in a flattened tetrahedral (D 2d) copper-(II) center are observed in these proteins at about 5000 and 10,000 cm-1, respectively. The band positions accord well with ligand field calculations based on a tetrahedral structure that is distorted approximately 6 degrees toward a square plane. The ligands in this flattened tetrahedral coordination unit in bean plastocyanin are identified from various spectroscopic experiments as His-38, Cys-85, His-88, and a deprotonated peptide nitrogen (N) a few residues above His-38.
Uncoupling of local field spectra in nuclear magnetic resonance: determination of atomic positions in solids.
Authors: Waugh J S
Published: May 2006

Solid state nuclear magnetic resonance as a method of determining crystal structure has had limited success. Three distinct reasons for this failure can be identified when the resonance spectrum is of a dilute spin species in the presence of another abundant species. Two of these difficulties, and in part the third, can be mitigated by a family of coherent averaging techniques in double resonance, with considerably improved prospects for locating the rare spins with respect to their nearby neighbors. A particularly advantageous procedure is described and possible applications are discussed.
Nuclear magnetic resonance studies of slowly exchanging peptide protons in cytochrome c in aqueous solution.
Authors: Patel D J , Canuel L L
Published: May 2006

The slowly exchanging protons in oxidized and reduced horse heart cytochrome c (D20, uncorrected pH meter reading 6.5 room temperature) have been monitored by recording the 270 and 360 MHz proton nuclear magnetic resonance spectra of the reduced protein between 5 and 11 parts per million downfield from 2,2-dimethyl-2-silapentane-5-sulfonate.
A maximal function associated to the curve (
t, t
2
)
Authors: Nagel Alexander , Riviere Nestor , Wainger Stephen
Published: May 2006

The authors prove that [Formula: see text] is bounded from

L
P

(
R
2
) to

L
P

(
R
2
) for 1 <
p
≤ ∞.
Induction of vitellogenin synthesis by estrogen in avian liver: relationship between level of vitellogenin mRNA and vitellogenin synthesis.
Authors: Mullinix K P , Wetekam W , Deeley R G , Gordon J I , Meyers M , Kent K A , Goldberger R F
Published: May 2006

We have investigated the estrogen-mediated induction of vitellogenin synthesis in rooster liver. We compared the concentrations of vitellogenin messenger RNA (mRNA) in the liver with the concentrations of vitellogenin in the sera of roosters that had recieved various treatments with estrogen. We found no vitellogenin mRNA in the livers of the unstimulated roosters. An initial injection of estrogen was attended by de novo synthesis of vitellogenin mRNA in the liver and accumulation of vitellogenin in the serum. When vitellogenin was no longer present in the serum or liver (the "post-estrogen-serum-negative" state), the liver was found to contain appreciable amounts of vitellogenin mRNA. This mRNA was of the same size as that found in the liver of the rooster actively synthesizing vitellogenin in response to estrogen. Whereas vitellogenin mRNA was in large polysomes in the livers of the roosters actively synthesizing vitellogenin, the vitellogenin mRNA in the liver of the post-estrogen-serum-negative rooster was not associated with polysomes. The possible relevance of these findings to the fact that the rooster responds differently to a primary stimulation with estrogen than to subsequent stimulations is discussed.
A mutant of Escherichia coli with an altered elongation factor Tu.
Authors: Pedersen S , Blumenthal R M , Reeh S , Russell L B , Lemaux P , Laursen R A , Nagarkatti S , Friesen J D
Published: May 2006

A previously isolated mutant of E. coli K12 HAK 88 [Kuwano, M., Endo, h & yamamoto, M. (1972) J. Bacteriol, 112, 1150-1156], contains a new protein that in two-dimensional gel electropherorgrams has the same molecular weight as normal elongation factor Tu, but whose isoelectric point is altered approximately 0.1 pH unit in the acidic direction. Peptide mapping, purification properties and the ratio of leucyl plus isoleucyl to methionyl plus cysteinyl residues of the normal elongation factor Tu protein and the new protein show a close similarity between the two. The mutation causing the altered electrophoretic mobility is located between argH and rif (79 min on the E. coli genetic map). These biochemical and genetic data indicate that strain HAK 88 has a mutationally altered tufB gene.
Differential effect of phosphonoacetic acid on the expression of Epstein-Barr viral antigens and virus production.
Authors: Nyormoi O , Thorley-Lawson D A , Elkington J , Strominger J L
Published: May 2006

The effects of phosphonoacetic acid on cell growth, expression of Epstein-Barr virus antigens, and virus production in human and marmoset lymphoblastoid cell lines have been studied. The drug had no significant effect at concentrations up to 100 mug/ml on cell growth or total cell DNA synthesis. Higher doses induced not only a drastic decrease in DNA synthesis and cell grwoth, but also a dramatic cell enlargement. Immunofluorescence studies showed that greater than or equal to 30 mug/ml of phosphonoacetic acid inhibited viral capsid antigen synthesis without affecting the expression of the nuclear antigen or the spontaneous and 5-iodo-2'-deoxyuridine-induced early antigens. Production of transforming Epstein-Barr virus was also blocked.
Ribosomal proteins of Escherichia coli that stimulate stringent-factor-mediated pyrophosphoryl transfer in vitro.
Authors: Christiansen L , Neirhaus K H
Published: May 2006

Guanosine tetra- and pentaphosphate, (p)ppGpp, can be synthesized in vitro in a reaction containing only the enzyme (stringent factor), salts, and substrates (nonribosomal system). This reaction is greatly stimulated upon addition of methanol (methanol system) or by ribosomes, mRNA, and tRNA (ribosome system). Here we show that several ribosomal proteins alone stimulate the synthesis of (p)ppGpp in the presence of stringent factor (protein system). The optimal ionic conditions for the ribosome and protein systems are identical. The concentration of ribosomes or any stimulating ribosomal protein required for saturation of a given concentration of stringent factor is similar. Fifty of 54 ribosomal proteins were tested for stimulation in the protein system; 15 proteins showed high activity, seven of these from the 30S ribosomal subunit and eight from the 50S subunit. The physiological relevance of this finding is discussed.
Combined effects of dissociable and undissociable local anesthetics upon ATP-induced firefly bioluminescence.
Authors: Kamaya H , Ueda I , Eyring H
Published: May 2006

Combined effects of two drugs present simultaneously are usually expressed as summation, synergism or potentiation, and antagonism. When the sum of the effects of each drug present separately equals the combined effect of the two drugs present simultaneously, the action is called additive or summation. However, the expected value of the sum of each effect of drugs present alone has not been well defined. In this report, the thearetical value of the expected sum of each effect of two inhibitors is given and a graphical method is presented to visualize summation, synergism, and antagonism. The inhibitory effects of a dissociable local anesthetic, tetracaine, and an undissociable local anesthetic, benzyl alcohol, upon a soluble firefly luminescent system were analyzed according to the above theory. The results clearly indicate that the action of these two classes of local anesthetics is pure additive or summation.
The electrochemical gradient of protons and its relationship to active transport in Escherichia coli membrane vesicles.
Authors: Ramos S , Schuldiner S , Kaback H R
Published: May 2006

Membrane vesicles isolated from E. coli generate a trans-membrane proton gradient of 2 pH units under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure the proton gradient (deltapH) and the distribution of the lipophilic cation triphenyl-methylphosphonium to measure the electrical potential across the membrane (delta psi), the vesicles are shown to generate an electrochemical proton gradient (deltamuH+) of approximately-180 mV at pH 5.5 in the presence of ascorbate and phenazine methosulfate, the major component of which is a deltapH of about -110mV. As external pH is increased, deltapH decreases, reaching 0 at pH 7.5 and above, while delta psi remains at about-75 mV and internal pH remains at pH 7.5. Moreover, the ability of various electron donors to drive transport is correlated with their ability to generate deltamuH+. In addition, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin. These data and others (manuscript in preparation) provide convincing support for the role of chemiosmotic phenomena in active transport.
Ribonuclease P substrate specificity: cleavage of a bacteriophage phi80-induced RNA.
Authors: Bothwell A L , Stark B C , Altman S
Published: May 2006

RNase P can cleave in vitro a bacteriophage phi80-induced RNA which is 62 nucleotides long [M3 RNA, G. Pieczenik et al. (1972) Arch. Biochem. Biophys. 152, 152-165] to yield two specific fragments 25 and 37 nucleotides long. As is the case for another substrate of RNase P; the precursor to Escherichia coli 4.5S RNA, the cleavage site in M3 RNA is at the end of a long double-stranded region immediately adjacent to a single-stranded segment. Similar nucleotide sequences span the cleavage site in both substrates. These and other features of the reaction of RNase P with M3 and 4.5S precursor RNA are different from some aspects of the reaction of this enzyme with tRNA precursor molecules. A qualitative scheme is presented that is directed towards the understanding of the differences in RNase P cleavage site specificity for these substrates.
Conformational flexibility and protein folding: rigid structural fragments connected by flexible joints in subtilisin BPN.
Authors: Honig B , Ray A , Levinthal C
Published: May 2006

Conformational energy calculations are used to analyze the interactions of structural substructures in subtilisin BPN. These substructures are kept fixed or "rigid" so that the only variables in the calculations are the backbone segments that separate them. The flexible segments are assumed to be free turns. Using this representation of the protein it is possible to predict both a likely order of events along a folding pathway and preferred modes of conformational changes of the native protein. Moreover, when the native structure has been perturbed by moving the substructures apart, it is possible to assess the range of interactions that return the protein, upon energy minimization, to its original conformation. These results suggest an approach to the folding problem based on the piecemeal formation of tertiary structure from smaller prefolded fragments.
Affinity isolation of cultured tumor cells by means of drugs and hormones covalently bound to glass and Sepharose beads.
Authors: Venter B R , Venter J C , Kaplan N O
Published: May 2006

Isoproterenol, corticotropin (ACTH), and triodothyronine immobilized on glass and Sepharose beads by diazotization procedures have been shown to interact with cultured tumor cells of "target tissue" origin. Cells used were rat glioma cells (C6), rat adrenal tumor cells (Y-1), and rat pituitary tumor cells (GH3). The rat glioma cells bound principally to immobilized isoproterenol, whereas the rat adrenal tumor cells bound to immobilized corticotropin, and rat pituitary tumor cells bound to immobilized triiodothyronine. Binding was inhibited by preincubation of the cells in soluble drug or hormone. With C6 cells there was a positive correlation between adenylate cyclase [ATP pyrophosphate-lyase (cyclizing, EC 4.6.1.1] stimulation and the degree of binding to the immobilized isoproterenol. Norepinephrine, bound through the ethanolamine side chain via an amide linkage, did not bind cells, demonstrating specific structural requirements for drug-cell interactions. HeLa cells were shown to bind tightly to diphtheria toxin coupled to Sepharose beads via an amide bond. This binding was inhibited by prior incubation of the Sepharose toxin with purified antitoxin. Toxin bound to Sepharose via an azo bond did not bind cells. These data suggest that the cell affinities are due to cell surface receptors interacting with the immobilized drugs and hormones, and that the observed affinities possibly reflect the relative receptor complement of these cells.
Temperature-sensitive changes in surface modulating assemblies of fibroblasts transformed by mutants of Rous sarcoma virus.
Authors: Edelman G M , Yahara I
Published: May 2006

The hypothesis that surface modulating assemblies containing microfilaments and microtubules and altered after cellular transformation was tested on cells infected with temperature-sensitive mutants of avian sarcoma virus. Untransformed cells (mouse 3T3 and chick fibroblasts), cells transformed by simian virus 40 (SV 3T3), and chick fibroblasts infected with Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV-A-infected cells) were first compared for differences in microfilament and microtubule patterns after treatment with fluorescein-labeled antibodies to actin and tubulin. Transformed cells showed disappearance of ordered stress microfilaments and thickened or diffuse alterations of microtubular arrays. At restrictive temperatures (41 degrees), chick fibroblasts infected with a temperature-sensitive mutant (ts 68) of Rous sarcoma virus showed normal patterns of stress fialments and radial microtubular arrays originating in 1 or 2 centrioles. At permissive temperatures (37 degrees), these patterns were disordered and resembled those of SR-RSV-A-infected cells. After a shift from 41 degrees to 37 degrees, the changes in microtubules were observed in the majority of cells within 1 hr. These changes were reversible and did not result from the inability of tubulin to polymerize. In ts 68-infected cells at permissive temperatures, concanavalin A induced much less surface modulation (inhibition of receptor mobility) than at restrictive temperatures. These results suggest that cellular transformation alters both the structure and function of surface modulating assemblies and prompt the hypothesis that products of viral transforming genes may affect these assemblies with a consequent loss of growth control.
Characterization of 2-mum DNA of Saccharomyces cerevisiae by restriction fragment analysis and integration in an Escherichia coli plasmid.
Authors: Hollenberg C P , Degelmann A , Kustermann-Kuhn B , Royer H D
Published: May 2006

Electrophoretic analysis of EcoRI and HindIII restriction fragments of 2-mum supercoiled DNA of Saccharomyces cerevisiae indicated that this class of DNA is heterogeneous and probably consists of two types of molecules. Integration of the 2-mum yeast DNA in E. coli plasmid pCR1 directly showed that existence of two types of molecules as each of these could be individually inserted into separate bacterial plasmids. The difference between the two types of 2-mum circles is due to an inversion of about 1.6 X 10(6) daltons. The inversion is flanked by a reversed duplicated sequence of 0.45 X 10(6) daltons. Possible implications of this structure are dicussed.
Mechanism of human lymphocyte stimulation by concanavalin A: role of valence and surface binding sites.
Authors: Wands J R , Podolsky D K , Isselbacher K J
Published: May 2006

A monovalent form of concanavalin A (m-Con A) has been prepared to determine the importance of valence for human lymphocyte surface binding and subsequent lymphocyte stimulation as measured by blast transformation and cytotoxicity. Concanavalin A (Con A) was fragmented by a proteolytic process and the m-Con A) derivative was isolated by elution with an ascending D-glucose gradient on a Sephadex G-200 column. The molecular weight of m-Con A was 18,000 by sodium dodecyl sulfate-polyacrylamide electrophoresis. Equilibrium dialysis with alpha-methyl D-glucoside and subsequent Scatchard plot analysis revealed an association constant (Ka) of 1.2 X 10(3) liters/mol and a valence of 1.1. Incubation of lymphocytes with 125I-labeled m-Con A demonstrated surface binding at 1.21 X 10(6) molecules per cell, which was comparable to the binding of [3H] Con A (1.02 X 10(6) molecules per cell). However, in contrast to the intact lectin, m-Con A had a markedly reduced capacity to agglutinate rabbit erythrocytes and human lymphocytes and did not stimulate lymphocyte blast transformation or cytotoxicity at 1 and 10 mug/ml. Finally, pretreatment of lymphocytes with m-Con A blocked blast transformation induced by Con A. These observations demonstrate that m-Con A binds to lymphocyte surface receptors but does not stimulate blast transformation or cytotoxicity, suggesting that Con A must bridge binding sites on the lymphocyte surface to induce lymphocyte activation.
Structure of L-arabinose-binding protein from Escherichia coli at 5 A resolution and preliminary results at 3.5 A.
Authors: Phillips G N , Mahajan V K , Siu A K , Quiocho F A
Published: May 2006

The three-dimensional crystal structure of the L-arabinose-binding protein from E. coli, an essential component in the active transport of L-arabinose, has been solved at 5 A resolution using the method of multiple isomorphous replacement. Five heavy atom derivatives were used. A preliminary 3.5 A electron density map has also been calculated. The results indicate that the molecule is ellipsoidal with approximate dimensions 68 A X 38 A X 30 A. Two similar domains within the molecule (which is a single polypeptide chain) are related by an approximate noncrystallographic rotation-translation axis. This relationship involves approximately 20% of the structure.
Chloroplast coupling factor 1: A species-specific receptor for tentoxin
Authors: Steele John A. , Uchytil Thomas F. , Durbin Richard D. , Bhatnagar Pradip , Rich Daniel H.
Published: May 2006

Tentoxin, a cyclic tetrapeptide, induces chlorosis in certain plant species. It inactivated photophosphorylation and coupling factor 1 (CF
1
) ATPase in lettuce, a sensitive species. This effect was due to binding of tentoxin with CF
1
at a single site (affinity constant 1.3 to 20 × 10
7
M
-1
). Neither AMP nor adenyl-5′-yl imidodiphosphate appeared to bind to this site. In radish, an insensitive species, 20 times more tentoxin was required for 50% inhibition of photophosphorylation. In this species CF
1
ATPase was unaffected by tentoxin, and its CF
1
bound tentoxin only weakly (affinity constant less than 1 × 10
4
M
-1
). Sensitivity of photophosphorylation to tentoxin was correlated with chlorosis sensitivity in six other species examined.
Temporal relationships of chromatin protein synthesis, DNA synthesis, and assembly of deoxyribonucleoprotein.
Authors: Seale R L
Published: May 2006

Chromatin assembly has been investigated in terms of the sites on DNA where newly synthesized chromatin proteins associate. Chromatin from cells labeled with [14C]-BrdUrd and [3H]lysine was fixed with formaldehyde and resolved in CsCl gradients. By varying the spacing of the labeling intervals of the two isotopes so as to encompass all possible periods in S-phase, the association of labeled, newly synthesized proteins on newly synthesized (BrdUrd-substituted) or preexisting chromatin DNA was determined. In all experiments it was found that newly synthesized chromatin proteins predominantly associated with nonreplicating DNA. Possible mechanisms by which cells recycle preexisting chromatin proteins to restore the protein content of newly synthesized DNA are discussed.
DNA sequence organization in the lepidopteran Antheraea pernyi.
Authors: Efstratiadis A , Crain W R , Britten R J , Davidson E H , Kafatos F C
Published: May 2006

A large fraction of the genome of the lepidopteran Antheraea pernyi consists of interspersed single-copy sequences and repetitive DNA sequences of 300 nucleotide average length. Most of the single-copy sequences are about 800 nucleotides long and a minority are considerably longer. Thus, the organization of the DNA sequences of this insect is similar to that of most higher organisms and different from that of Drosophila.
Tubulin aggregation and disaggregation: mediation by two distinct vinblastine-binding sites.
Authors: Bhattacharyya B , Wolff J
Published: May 2006

Rat brain tubulin possesses two distinct binding sites for vinblastine per molecule: a high-affinity site with an affinity constant of 6.2 x 10(6) M-1 and a low-affinity site with an affinity constant of 8 x 10(4) M-1. The high-affinity site is labile, with a t1/237 degrees of 3.5 hr, is protected by colchicine, and is unaffected by salt, whereas the low-affinity site is stable but is inhibited by salt. Binding to both sites is rapid. The high-affinity binding constant of vinblastine to tubulin (6.2 x 10(6) M-1) corresponds to the half-maximal concentration of vinblastine needed to prevent polymerization of tubulin in vitro, whereas the low-affinity binding constant (8 x 10(4) M-1) corresponds to the half-maximal concentration of vinblastine required to aggregate tubulin. We conclude that vinblastine binding to the high- and low-affinity sites, respectively, accounts for the depolymerization and aggregation behavior of tubulin.
Maximal functions: Poisson integrals on symmetric spaces
Authors: Stein Elias M.
Published: May 2006

Let
u
be a harmonic function on a symmetric space which is the Poisson integral of a function
f
in

L
p

, 1 ≤
p
≤ ∞. Then
u
converges restrictedly and admissibly to
f
almost everywhere. This result is proved by obtaining an appropriate maximal theorem which takes into account the structure of the Poisson kernel.
Purification and characterization of two initiation factors required for maximal activity of a highly fractionated globin mRNA translation system.
Authors: Safer B , Adams S L , Kemper W M , Berry K W , Lloyd M , Merrick W C
Published: May 2006

Two additional initiation factors (IF-M4 and IF-M5) have been purified and characterized both physically and biologically. IF-M4 is active as a single polypeptide chain with a molecular weight of 48,000. In contrast, IF-M5 is active as a complex with a molecular weight of about 500,000 and consists of seven major and several minor polypeptide components. Analysis of IF-M5 in two polyacrylamide gel electrophoresis systems indicated that one of the major polypeptide chains of IF-M5 was the 35,000 dalton subunit of IF-MP. This analysis also revealed that IF-M2A, IF-M3, and elongation factor 2 were present as minor components. Both IF-M4 and IF-M5 are required to achieve maximal activity in an assay system dependent on exogenous globin mRNA, but neither factor has been observed to stimulate model reactions that utilize artificial templates [poly(U) or AUG].
Enzymatic conversion of benzo(a)pyrene leading predominantly to the diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene through a single enantiomer of r-7, t-8-dihydroxy-7,8-dihydrobenzo(a)pyrene.
Authors: Yang S K , McCourt D W , Roller P P , Gelboin H V
Published: May 2006

Benzo(a)pyrene is metabolically and stereospecifically converted by mixed-function oxidases of rat liver microsomes and epoxide hydratase (glycol hydro-lyase (epoxide-forming), EC 4.2.1.63)to the single enantiomer (-)r-7,t-8-dihydroxy-7,8-dihydrobenzol (A) pyrene. This enantiomer is further metabolized stereoselectively by the mixed-function oxidases to predominantly the diol-epoxide, r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzol(a)pyrene in which the 7-hydroxyl and the 9,10-epoxide are trans. Other unidentified metabolites are also formed from the r-7,t-8-dihydroxy-7,8-dihydrobenzo(a)pyrene. Racemic r-7,t-8-dihydroxy-7,8-dihydrobenzo(a)-pyrene is converted metabolically to both r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene and r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene. The diol-epoxides are unstable in aqueous medium, and their identification and characterization as r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene and r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene were accomplished by the identity of their tetrahydroxytetrahydrobenzo(a)pyrenes hydrolysis products with those of the authentic synthetic compounds with respect to mobility on high-pressure liquid chromatography and mass and ultraviolet absorption spectral analysis. The diol-epoxides were also reduced in the presence of NADPH to distinct trihydroxypentahydrobenzo(a)pyrenes. Since the synthetic racemic r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene is very highly mutagenic in mammalian cells, we suggest that it is the metabolically formed diol-epoxide that may be an ultimate carcinogenic form of benzo(a)pyrene.
Electron microscopy of DNA crosslinked with trimethylpsoralen: test of the secondary structure of eukaryotic inverted repeat sequences.
Authors: Cech T R , Pardue M L
Published: May 2006

It has been suggested that inverted repeat (palindrome) sequences, which are widespread in eukaryotic genomes, exist in two alternate configurations, a linear form and a cruciform. To investigate the relative frequency of these forms, the DNA of intact mouse tissue culture cells was covalently crosslinked with 4,5',8-trimethylpsoralen (me3-psoralen) in order to prevent rearrangement of the DNA secondary structure during DNA isolation. The distribution of me3-psoralen crosslinks was determined by electron microscopy after denaturation of the DNA in the presence of glyoxal. Because of the high frequency and the relatively uniform distribution of the me3-psoralen crosslinks, it could be concluded that almost all of the inverted repeat sequences had been crosslinked. In spite of this, no significant number of cruciforms was detected by electron microscopy. To determine whether the me3-psoralen might itself be disrupting cruciform structures, cruciforms were first produced in isolated Tetrahymena rDNA by heat treatment and then crosslinked in vitro. The crosslinking was found to stabilize rather than disrupt these cruciforms. We conclude that the inverted repeat sequences of the mouse tissue culture cells we tested are predominantly in linear forms rather than in cruciform structures inside the cell.
Dual role of Zn2+ as inhibitor and activator of fructose 1,6-bisphosphatase of rat liver.
Authors: Tejwani G A , Pedrosa F O , Pontremoli S , Horecker B L
Published: May 2006

At neutral pH, Zn2+ is a potent and specific inhibitor of rat liver fructose 1,6-bisphosphatase (EC 3.1.3.11; D-fructose-1,6-bisphosphate 1-phosphohydrolase). Inhibition by Zn2+ is uncompetitive with respect to the activating cations Mg2+ and Mn2+, and the kinetic data suggest that the enzyme possesses a distinct high-affinity binding site for Zn2+, with Ki of approximately 0.3 muM. At higher concentrations (about 10(-5) M) Zn2+, and to a lesser extent Co2+, function as activating cations. Binding studies show that the enzyme binds two equivalents of Zn2+ per subunit; one equivalent is partially displaced by Mg2+ and is presumably bound to the site for activating cations. A second equivalent binds to the high-affinity site, presumably identical to the inhibitory site. The results suggest that Zn2+ functions as an allosteric regulator, and that the commonly observed activation of fructose 1,6-bisphosphatase at neutral pH by EDTA, histidine, and other chelators is due to removal of endogenous Zn2+ by these agents.
1,N6-etheno-AMP and 1,N6-etheno-2'-deoxy-AMP as probes of the activator site of glycogen phosphorylase from rabbit skeletal muscle.
Authors: Vandenbunder B , Morange M , Buc H
Published: May 2006

Both 1,N6-etheno-AMP and 1,N6-etheno-2'-deoxy-AMP bind at the AMP site of phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1). Etheno-AMP induces the same activation as AMP, about 30-fold higher than the activation induced by etheno-dAMP. The fluorescence of etheno-AMP and etheno-dAMP is associated with the base moiety; therefore, when free in solution, the two derivatives have identical fluorescence properties. However, when bound to phosphorylase, the fluorescence of etheno-AMP is quenched more efficiently than the fluorescence of etheno-dAMP. This difference between the fluorescence properties of the bound nucleotides suggests that a modification in the ribose ring affects the position of the adenine in the AMP site of phosphorylase b. The observed quenching may be due to a stacking interaction between an aromatic residue and the base moiety of the bound nucleotide.
Detection of mRNA sequences in nuclear 30S ribonucleoprotein subcomplexes.
Authors: Kinniburgh A J , Martin T E
Published: May 2006

RNA from nuclear 30S ribonucleoprotein (RNP) complexes of mouse ascites cells has been shows to contain sequences homologous to poly(A) + mRNA by its ability to hybridize with complementary DNA prepared from poly(A) + mRNA template. Analysis of the hybridization kinetics of poly(A) + mRNA with its own complementary DNA revealed several abundancy classes. The total complexity of poly(A) + mRNA from ascites cells was estimated to be approximately 30,000 sequences of average molecular weight (6 X 10(5)). When the hybridization reaction of 30S RNP-RNA with mRNA-specific cDNA was compared to the homologous reaction the majority, and most probably all, of the poly(A) + mRNA sequences were found to be present in the RNA. The kinetics of hybridization suggest that 10-15% of the RNA in this RNP complex is homologous to poly(A) + mRNA. The 30S RNP subcomplexes therefore contain nuclear poly(A) + mRNA sequences as well as the bulk of heterogeneous RNA.
Bacteriophage T3 and T7 early RNAs are translated by eukaryotic 80S ribosomes: active phage T3 coded S-adenosylmethionine cleaving enzyme is synthesized.
Authors: Anderson C W , Atkins J F , Dunn J J
Published: May 2006

RNA transcribed in vitro from the early region of bacteriophage T3 or T7 was translated by cytoplasmic ribosomes which synthesized protein in cell-free systems prepared from mammalian cells and wheat germ. The proteins synthesized in vitro and their counterparts prepared from infected Escherichia coli comigrate by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and are similarly affected by deletion or amber bacteriophage mutations. Bacteriophage T3 codes for an enzyme that cleaves S-adenosylmethionine and this activity was detected among the products of the mammalian cell-free system. Bacteriophage T3 or T7 RNA, after endoribonuclease III (EC 3.1.4.24) cleavage, gave higher levels of incorporation into phage T3 or T7 polypeptides than when an equivalent amount of the uncleaved RNA was added to the eukaryotic cell-free systems. Methylation of phage T3 or T7 RNAs is apparently not required for translation in either the wheat germ or mammalian cell-free system. The ability of T3 and T7 RNA to be translated in the presence of saturating amounts of natural eukaryotic mRNAs suggests that many prokaryotic genes introduced into mammalian cells might be expressed if they were transcribed in an appropriate form.