bluuebunny/crossref_metadata_2025 huggingface dataset:
Essential role for mevalonate synthesis in DNA replication
Authors: Quesney-Huneeus Valeria , Wiley Millie Hughes , Siperstein Marvin D.
Published: May 2006

The relationship between 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity [mevalonate:NADP
+
oxidoreductase (CoA-acylating), EC 1.1.1.34] and DNA synthesis was studied in synchronized cultures of BHK-21 cells. During a 24-hr period of cell replication, two phases of accelerated thymidine incorporation into DNA corresponding to two S phases of the cell cycle occurred. A marked increase in activity of HMG CoA reductase was consistently observed at or just prior to each of these peaks of DNA synthesis. Moreover, when HMG CoA reductase activity was suppressed by the competitive inhibitor compactin, the normal S-phase burst of DNA synthesis was specifically and totally prevented. Finally, the compactin-induced inhibition of DNA synthesis could be completely reversed within minutes by the addition of mevalonate, the product of the HMG CoA reductase reaction. By contrast, addition of cholesterol-rich lipoproteins had no effect upon DNA synthesis in compactin-treated cells. These data demonstrate that HMG CoA reductase activity, and therefore the production of mevalonate, plays an essential role in the synthesis of DNA specifically during the S phase of the cell cycle. Moreover, the results indicate that this function of mevalonate in regulating DNA replication is independent of its conversion to cholesterol.
Protein synthesis in rabbit reticulocytes: mechanism of protein synthesis inhibition by heme-regulated inhibitor.
Authors: Das A , Ralston R O , Grace M , Roy R , Ghosh-Dastidar P , Das H K , Yaghmai B , Palmieri S , Gupta N K
Published: May 2006

Partially purified Met-tRNAf binding factor, eIF-2, was phosphorylated by using heme-regulated inhibitor (HRI). Phosphorylated eIF-2 was freed from HRI by phosphocellulose column chromatography. Analysis by isoelectric focusing showed 100% phosphorylation of the 38,000-dalton subunit of eIF-2. Both eIF-2 and eIF-2(P) formed ternary complexes with Met-tRNAf and GTP with almost the same efficiency, and in both cases the ternary complex formation was drastically inhibited by prior addition of Mg2+. However, whereas the ternary complexes formed with eIF-2 could be stimulated by Co-eIF-2C at 1 mM Mg2+ and dissociated by Co-eIF-2B at 5 mM Mg2+, the ternary complexes formed with eIF-2(P) were unresponsive to both Co-eIF-2B and Co-e-IF-2C. Also, under conditions of eIF-2 phosphorylation, HRI drastically inhibited AUG-dependent Met-tRNAf binding to 40S ribosomes. However, HRI (in the presence of ATP) had no effect on the joining of preformed Met-tRNAf . 40S . AUG complex to the 60S ribosomal subunit to form Met-tRNAf-80S . AUG complex. These studies suggest that HRI inhibits protein synthesis initiation by phosphorylation of the 38,000-dalton subunit of eIF-2. HRI-phosphorylated eIF-2 does not interact with at least two other protein factors, Co-eIF-2B and Co-eIF-2C, and is thus inactive in protein synthesis initiation.
A response of protein synthesis to temperature shift in the yeast Saccharomyces cerevisiae.
Authors: Miller M J , Xuong N H , Geiduschek E P
Published: May 2006

When Saccharomyces cerevisiae are subjected to a sudden increase in temperature (22 degrees C to 37 degrees C) they undergo extensive and, in some cases, extreme alterations in their rates of synthesizing individual polypeptides. These changes were monitored by pulse-labeling cells with [35S]methionine and separating the total soluble proteins by two-dimensional gel electrophoresis. Incorporation of 35S into individual proteins was measured by a computer-coupled autoradiogram-scanning method. The rates of synthesis of most proteins are transiently changed; 10-fold or greater induction or respression is common. This temperature response has also been studied in a mutant strain that is temperature sensitive for the nucleus-to-cytoplasm transport of RNA. In this mutant, not only the induction, but also a part of the repression in response to temperature upshift is largely inhibited. Conceivable mechanisms are discussed.
Gene mutations (de novo) found in electrophoretic studies of blood protein of infants with anomalous development.
Authors: Dubinin N P , Altukhov Y P
Published: May 2006

Twelve proteins of enzymic and nonenzymic nature in blood samples of infants that deviate from the average population in physical development (50 premature and 177 full-term infants with rough and multiple developmental defects) were studied by electrophoresis in polyacrylamide and starch gels. The control group consisted of 500 normal newborns. In infants with developmental disorders, the frequency of rare electrophoretic protein variants was found to be about one order of magnitude higher than in the control. It has been shown for at least five cases that such variants are de novo mutations. According to these data the mutation rate is approximately 2 x 10(-3) per locus per generation for the group selected and approximately 6 x 10(-5) for the total population. Despite the fact that further specification of the estimations found is required, we consider the results obtained as evidence in favor of the efficiency of the earlier substantiated monitoring model of gene mutations in the human population [Dubinin, N.P. & Altukhov, Yu. P. (1977) in Genetic Consequences of Environmental Pollution, ed. Dubinin, N.P. (Mysl, Moscow), pp. 14-45]. This approach, which infers electrophoretic screening of blood proteins in a specially selected group of newborns, makes it possible to reduce the size of samples needed for statistically reliable estimations of the alteration of mutation rate.
Organization of the multiple genes for the 70,000-dalton heat-shock protein in Drosophila melanogaster.
Authors: Mirault M E , Goldschmidt-Clermont M , Artavanis-Tsakonas S , Schedl P
Published: May 2006

The organization and number of 70,000-dalton heat-shock protein genes of Drosophila melanogaster has been investigated in a wild-type Oregon R fly stock and in a KC cell line. Six copies were found in the KC cells, and slightly more were found in the Oregon R population examined. In both cases, the basic gene element consisting of the mRNA coding region plus a short 5' "noncoding" sequence element was conserved. Two gene variants distinguished by specific restriction sites were found in both genomic DNAs. Restriction maps of the six genes in KC cells showed that these two gene variants are arranged differently. Restriction analysis of Oregon R embryonic DNA revealed polymorphism in the organization of the genes, which is not observed in KC cells. The data suggest that the arrangement as well as the number of genes for the 70,000-dalton heat-shock protein in D. melanogaster is subject to variations at both the 87A and 87C cytogenetic loci.
Effective tumor immunization induced by cells of elevated membrane-lipid microviscosity.
Authors: Shinitzky M , Skornick Y , Haran-Ghera N
Published: May 2006

The immunogenicity of a series of mouse tumor lines propagated in vivo (T and B lymphomas and mammary adenocarcinoma) was tested after alteration of the cell membrane-lipid microviscosity. Tumor cells used for immunization were first treated to alter the lipid content, then irradiated and injected intraperitoneally into syngeneic mice. A second identical immunization was performed 14 days later. The degree of immunization in the treated mice was assessed by survival time after challenge with untreated viable tumor cells of the same origin as the immunizing cells. For all tumors tested, enrichment of the immunizing cells with cholesterol or cholesteryl hemisuccinate, which increased the membrane-lipid microviscosity significantly, afforded a marked increase in immunization, compared to that obtained with cells that were only irradiated. Furthermore, in over 90% of the mice that were pretreated with cholesteryl hemisuccinate-enriched cells, tumor growth after the challenge was not detectable. Because the lipid-modifying treatments of the immunizing cells involve no toxic substances, these results may provide the basis for a potent approach to immunotherapy of human cancer.
Tissue sites of degradation of low density lipoprotein: Application of a method for determining the fate of plasma proteins
Authors: Pittman Ray C. , Attie Alan D. , Carew Thomas E. , Steinberg Daniel
Published: May 2006

A method for determining tissue sites of plasma protein degradation is described as applied to studies of low density lipoprotein (LDL) catabolism in swine. The method is based on the fact that sucrose is not degraded by lysosomal enzymes and thus accumulates in lysosomes. [
14
C]Sucrose was activated with cyanuric chloride and covalently coupled to the LDL protein. Studies in cultured fibroblasts have established that the sucrose
14
C accumulates intracellularly in degradation products at a rate equal to the rate of degradation of
125
I-labeled LDL simultaneously measured.
In vivo
the fractional catabolic rate of [
14
C]sucrose-LDL was the same as that of
125
I-labeled LDL.
14
C-Labeled degradation products in all major tissues were determined 24 hours after injection of [
14
C]sucrose-LDL. About 75% of the LDL degraded (calculated from analysis of the plasma decay curve) was accounted for in the
14
C-labeled degradation products accumulated in the tissues examined; only 4% appeared in the urine. In three studies, 37.9, 39.6, and 37.8% of the LDL degraded was recovered in the liver. Results were similar at 48 hr (38.7 and 39.9% hepatic degradation), but urinary losses were then about 10% and about 4% was lost in bile. All extrahepatic tissues examined contained
14
C-labeled degradation products. The concentration was highest in the adrenal glands—2 to 5 times that in liver and 10 times that in the next most active tissues. In principle this approach should be applicable to studies of the tissue sites of degradation of any of the plasma proteins.
Specific [3H]phencyclidine binding in rat central nervous system.
Authors: Zukin S R , Zukin R S
Published: May 2006

[3H]Phencyclidine (PCP) bound specifically and with high affinity (Kd = 0.15 microM at pH 7.4) to a single saturable class of binding sites in rat brain membrane preparations. Specific binding constituted approximately 70% of total binding at 0 degrees C and 33% of total binding at 37 degrees C (at 10 nM [3H]PCP). Bound [3H]PCP could be displaced by nonradioactive PCP, a series of its derivatives, and the psychotomimetic opiate N-allylnorcyclazocine (SKF 10,047) with relative potencies that closely paralleled those determined in animal behavioral tests. Muscarinic cholinergic ligands inhibited [3H]PCP binding, but only at 0.1 mM and in rank order at variance with that for binding to muscarinic sites or for pharmacological potencies. Other drugs, including opiates other than SKF 10,047, were unable to displace specifically bound [3H]PCP at 0.1 mM. [3H]PCP binding was most enriched in crude synaptosomal subcellular fractions, and was about three times higher in hippocampus (region of highest density) than in cervical spinal cord (region of lowest density). Trypsin and Pronase reduced specific [3H]PCP binding. Thus, PCP may exert its effects on the central nervous system via binding to specific brain receptor sites.
Sulfur base ligation to iron(II) and cobalt(II) porphyrins
Authors: Ellis Paul E. , Jones Robert D. , Basolo Fred
Published: May 2006

Equilibrium constants for the binding of sulfur bases to cobalt(II) porphyrins were measured in toluene solution by a spectrophotometric method. The order of decreasing binding strength of sulfur ligands to cobalt(II) porphyrins was found to be mercaptide ions » thioethers > mercaptans. It is suggested that a similar stability order of these sulfur ligands should exist towards iron(II) porphyrins, but formation constants could be obtained only for the mercaptide ions.
Properties and localization of beta-endorphin receptor in rat brain.
Authors: Law P Y , Loh H H , Li C H
Published: May 2006

Stereospecific high-affinity binding sites for beta h-[3H]endorphin could be demonstrated in the P2 pellet of rat brain homogenate. Scatchard analysis of the binding data revealed binding sites with Kd values of 0.81 and 6.8 nM and density of 120 and 240 fmol/mg of protein. Distribution of beta h-[3H]endorphin binding in various brain regions parallels that of opiate receptor:striatum greater than thalamus greater than amygdala greater than hypothalamus, septum greater than cortex greater than midbrain, brainstem. Similar to their effect on 3H-labeled agonist binding, Na+ and other monovalent cations, GTP, trypsin, chymotrypsin, phospholipase A2, and N-ethylmaleimide all inhibited the specific binding of beta h-[3H]endorphin. In contrast to their action on alkaloid and enkephalin binding, Ca2+, Mg2+, and Mn2+ also inhibited beta h-[3H]endorphin binding. These data suggest a difference between beta h-endorphin and alkaloid/enkephalin binding sites.
Extracts of Drosophila embryos mediate chromatin assembly in vitro.
Authors: Nelson T , Hsieh T S , Brutlag D
Published: May 2006

Extracts of Drosophila embryos can mediate the assembly of a chromatinlike structure from histones and DNA under physiological conditions. The histone-DNA complex formed in vitro contains micrococcal nuclease-sensitive sites spaced at 200-base pair intervals. More extensive digestion of the complex by micrococcal nuclease generates 11S particles which cosediment with nucleosome core particles isolated from native chromatin. These particles contain 140-base pair DNA fragments which upon further cleavage with micrococcal nuclease give rise to a pattern of discretely sized DNA fragments characteristic of nucleosome core particles. We have assayed the chromatin assembly process both qualitatively by measuring the induction of supertwists into a relaxed circular DNA (a process requiring a nicking-closing enzyme) and quantitatively by measuring the formation of micrococcal nuclease-resistant DNA fragments from radioactively labeled linear DNA. The amount of chromatin formed depends primarily on the amount of histones, whereas the rate of assembly depends on the amount of extract protein added. The factors in the extract that mediate chromatin assembly appear to interact first with the DNA because preincubation of the DNA with the extract markedly increases the extent of assembly.
Control of phosphoenolpyruvate-dependent phosphotransferase-mediated sugar transport in Escherichia coli by energization of the cell membrane.
Authors: Reider E , Wagner E F , Schweiger M
Published: May 2006

The phosphoenolpyruvate-dependent phosphotransferase-mediated sugar transport in Escherichia coli is inhibited by the energized of the membrane. This was shown in intact cells as well as in membrane vesicles. Relaxation of the proton gradient by uncouplers stimulated the uptake of sugars via the phosphotransferase system in aerobically cultured cells. No such effect was seen in anaerobic cells, apparently because the cell membrane of these cells is poorly energized. Energization by respiration of D-lactate or ascorbate inhibited the phosphotransferase uptake system in membrane vesicles. This inhibition was reversed by the addition of cyanide. Oxamate, a specific inhibitor of lactate dehydrogenase, prevented the inhibitory effect of D-lactate. Membrane vesicles prepared from a cytochrome-less mutant were not energized by D-lactate oxidation and the phosphotransferase uptake system was not inhibited.
Ricin linked to monophosphopentamannose binds to fibroblast lysosomal hydrolase receptors, resulting in a cell-type-specific toxin.
Authors: Youle R J , Murray G J , Neville D M
Published: May 2006

The receptor specificity of the plant seed toxin ricin, which ordinarily binds to galactose-containing receptors, has been altered by coupling monophosphopentamannose residues to ricin by reductive amination and by reversibly binding lactose to the modified ricin. The added monophosphopentamannose residues provide ricin with the recognition factor common to fibroblast lysosomal hydrolases and enable the modified ricin (Man6P-ricin) to bind to the fibroblast Man6P receptor and inhibit protein synthesis in the cells via this receptor. The addition of lactose to Man6P-ricin saturates the galactose site on Man6P-ricin and prevents the binding of Man6P-ricin to cells via galactose-containing ricin receptors. The Man6P receptor-mediated toxicity of Man6P-ricin, identified in human fibroblasts by competition by Man6P and blockade by alkaline phosphatase treatment, was not detected in HeLa cells or human amnion cells. Consequently, in the presence of lactose, the fibroblasts were 8 and 13 times more sensitive than amnion and HeLa cells, respectively. These results show that highly toxic cell-type-specific reagents can be made by the proper alteration of toxin receptor specificities. An attempt to construct a highly toxic altered toxin by adding Man6P residues to diphtheria toxin fragment A was unsuccessful. A possible explanation is that in Man6P-ricin the ricin B chain performs some entry function, even though the initial binding step occurs at the Man6P receptor.
Rotational diffusion of cell surface components by time-resolved phosphorescence anisotropy.
Authors: Austin R H , Chan S S , Jovin T M
Published: May 2006

The rotational diffusion of concanavalin A receptors of viable Friend erythroleukemia cells and the band 3 anion transport system of human erythrocytes has been measured via the time-dependent phosphorescence emission intensity and anisotropy of triplet probes excited by a 5-ns laser pulse. High-quality phosphorescence decay curves with a 10-microseconds time resolution were obtained at concentrations of the eosin probe down to 20 nM and in aqueous media at temperatures of 4-38 degrees C. A strong temperature dependence in the rotational behavior was observed for the band 3 anion transport protein, but the lectin receptors of the Friend erythroleukemia cells were found to be immobile on the time scale of 1-4000 microseconds at either 4 degrees C or 37 degrees C. The technique is applicable to other triplet probes and membrane components of living cells under conditions that do not destroy viability.
Assignment of human beta-, gamma-, and delta-globin genes to the short arm of chromosome 11 by chromosome sorting and DNA restriction enzyme analysis.
Authors: Lebo R V , Carrano A V , Burkhart-Schultz K , Dozy A M , Yu L C , Kan Y W
Published: May 2006

Normal human metaphase chromosomes isolated from fibroblasts were resolved into 14 peaks based on total Hoechst 33258 fluorescence and sorted with the fluorescence-activated cell sorter. The chromosomal DNA was extracted and characterized by EcoRI analysis. As expected, analysis of the peak containing chromosomes 16 and 18 detected the alpha-globin genes and of the peak containing chromosomes 9, 10, 11, and 12 detected the beta-, gamma-, and delta-globin genes. Translocations were then used to localize further the beta-, gamma-, and delta-globin genes. The first translocation t(11;22)(q25;q11), which moved nearly all of chromosome 11 to a different peak, confirmed that the beta-, gamma-, and delta-globin genes are on this chromosome. The second, t(4;11)(q25;q13), which moved the distal portion of the long arm of chromosome 11 to a new peak, showed that the genes are not in this segment. The third, t(X;11)(q11;p13), moved the distal region of the short arm of chromosome 11 to a peak which now contained the beta-, gamma-, and delta-globin genes. Therefore, the beta-, gamma-, and delta-globin genes residue on the distal portion of the chromosome 11 short arm including bands p13, p14, and p15. This sorting method may be used generally to assign other genes to chromosomal segments of the entire chromosome complement.
Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane
Authors: Fearon Douglas T.
Published: May 2006

An activity that is inhibitory to the properdin-stabilized amplification C3 convertase (C3b,Bb,P) was solubilized from human erythrocyte (E
hu
) membranes by Nonidet P-40 and purified to homogeneity. The inhibitory membrane glycoprotein had an apparent
M
r
of 1-1.2×10
6
on gel filtration in the presence of Nonidet P-40. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis it presented a single stained band with an apparent
M
r
of 205,000, with or without prior reduction of disulfides. The inhibitory protein of the E
hu
membrane produced a dose-related, first-order decay of C3b,Bb,P function on sheep erythrocytes (E
s
) and released
125
I-labeled Bb from these sites, indicating a mechanism of inhibition by decay-dissociation of the amplification C3 convertase. The 50% inhibitory dose of the E
hu
membrane protein was not altered by removal of sialic acid from the E
s
bearing C3b,Bb,P sites. E
hu
membrane protein also serves as a cofactor for C3b inactivator-induced cleavage of the α polypeptide chain of C3b. Thus, the inhibitory membrane protein can abrogate the activity of amplification convertase sites that have formed and also can prevent generation of such sites by augmenting irreversible inactivation of C3b.

Discrimination between cells by the alternative complement pathway occurs after initial deposition of C3b and is related to the modulation by surface constituents of the capacity of bound C3b to function as a subunit of the amplification C3 convertase. The existence in the E
hu
membrane of a protein that can impair the functions of membrane-bound C3b and C3b,Bb,P could represent a molecular basis for preventing inappropriate self-recognition.
Triene prostaglandins: Prostaglandin D
3
and icosapentaenoic acid as potential antithrombotic substances
Authors: Whitaker Mark O. , Wyche Angela , Fitzpatrick Frank , Sprecher Howard , Needleman Philip
Published: May 2006

Addition of the 3-series fatty acid precursor (icosapentaenoic acid, IPA), its endoperoxide [prostaglandin (PG)H
3
], or thromboxane A
3
to human platelet-rich plasma (PRP) does not result in aggregation of the platelets. In fact, preincubation of human PRP with exogenous PGH
3
actually inhibited aggregation by increasing platelet cyclic AMP concentrations. PGH
3
undergoes rapid spontaneous degradation to PGD
3
in human PRP. The PGD
3
so formed is adequate to account for the increase of platelet cAMP and inhibition of aggregation. Furthermore, addition of PGD-specific antisera to human PRP blocked the platelet inhibitory activity of exogenous PGH
3
. PGD
3
has considerable potential as a circulating antithrombotic agent. Pretreatment of human PRP with the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine blocked the increase of platelet cyclic AMP and the inhibition of aggregation normally produced by PGI
2
, PGE
1
, PGD
2
, PGH
3
, and PGD
3
. Furthermore, the dideoxyadenosine unmasked a direct but moderate reversible aggregatory effect in response to the subsequent addition of PGH
3
. Similarly, the dideoxyadenosine markedly enhanced the aggregation produced by exogenous PGH
2
. IPA is readily incorporated into tissue lipids but proved to be a poor substrate for kidney, blood vessel, or heart cyclooxygenase. IPA was previously shown to be a poor substrate for platelet cyclooxygenase. IPA is readily deacylated from the renal phospholipid pool in response to bradykinin, a substance that also stimulates the release of arachidonic acid. A diet that relies primarily on cold-water fish, as in the case of the Greenland Eskimos, lowers endogenous arachidonic acid and markedly increases the IPA content of tissue lipids. Thus, because IPA has the potential to act as an antagonist with arachidonic acid for platelet cyclooxygenase and lipoxygenase, the simultaneous release of IPA could suppress any residual arachidonic acid conversion to its aggregatory metabolites.
Blockage of narcotic-induced dopamine receptor supersensitivity by cyclo(Leu-Gly).
Authors: Ritzmann R F , Walter R , Bhargava H N , Flexner L B
Published: May 2006

We have previously reported that the administration of cyclo(Leu-Gly) to mice prior to morphinization blocked the development of tolerance to the analgesic effects of morphine as well as the development of some signs of physical dependence. In the present series of experiments, the effect of the same peptide treatment on changes in dopamine receptor sensitivity induced by chronic morphine treatment were determined. Changes in dopamine receptor sensitivity were determined by measuring (i) the effect of the dopamine agonist apomorphine on locomotor activity and (ii) the hypothermic response to another dopamine agonist, piribedil. Mice that had received the chronic morphine treatment were found to require significantly less apomorphine to produce an increase in locomotor activity, and they exhibited a significantly greater hypothermic response to piribedil than did morphine-naive mice. The injection of 0.2 mumol of cyclo(Leu-Gly) per mouse 2 hr prio to morphine treatment prevented this increased response to both dopamine agonists. Administration of the peptide after the tolerance and dependence had developed did not alter morphine tolerant and dependent states states or the enhanced response to apomorphine or piribedil. It is concluded that dopamine receptor supersensitivity may be involved in the development of narcotic tolerance and physical dependence.
Interrelationships between ganglionic acetylcholinesterase and nonspecific cholinesterase of the cat and rat.
Authors: Koelle G B , Rickard K K , Ruch G A
Published: May 2006

When homogenates of cat or rat superior cervical ganglia in Krebs-Ringer solution were incubated at 37 degrees C, the ensuing decrease in acetylcholinesterase (acetylcholine acylhydrolase, EC 3.1.1.7) activity was increased significantly by prior administration in vivo of tetramonoisopropylpyrophosphotetramide at doses that produced selective alkylphosphorylation of butyrylcholinesterase or propionylcholinesterase. These findings are consistent with the proposal that the latter enzymes are posttranscriptional precursors of acetylcholinesterase. Results of similar studies with homogenates of ganglia in water or in M NaCl/1% Triton X-100 were inconclusive, as were those of heat-inactivation studies and immunoprecipitation of the enzymes.
Dynamics of technological evolution: Random walk model for the research enterprise
Authors: Montroll Elliott W. , Shuler Kurt E.
Published: May 2006

Technological evolution is a consequence of a sequence of replacements. The development of a new technology generally follows from model testing of the basic ideas on a small scale. Traditional technologies such as aerodynamics and naval architecture involved feasibility experiments on systems characterized by only one or two dimensionless constants. Technologies of the “future” such as magnetically confined fusion depend upon many coupled dimensionless constants. Research and development is modeled and analyzed in terms of random walks in appropriate dimensionless constant space.
Carbon monoxide binding to iron porphyrins.
Authors: Collman J P , Brauman J I , Doxsee K M
Published: May 2006

The carbon monoxide affinities of iron complexes of meso-tetra (alpha, alpha, alpha, alpha-o-pivalamidophenyl)porphyrin (the "picket fence" porphyrin) and of a "picket fence" porphyrin derivative with an appended axial base have been measured in solution and compared with the CO affinities of various hemoproteins. The model complexes bind CO with much greater affinity than normal hemoproteins; the role of the steric bulk of distal residues in lowering the CO affinities of the hemoproteins is discussed. The significance of this lowered CO affinity is described with regard to endogenous CO. A discussion of mutant hemoglobins lacking distal residues that sterically inhibit the binding of CO is presented. The use of pressure units versus concentration units in equilibrium expressions is analyzed.
A topoisomerase from Escherichia coli related to DNA gyrase.
Authors: Brown P O , Peebles C L , Cozzarelli N R
Published: May 2006

We have identified a topoisomerase activity from Escherichia coli related to DNA gyrase (topoisomerase II): we designate it topoisomerase II'. It was constructed of two subunits, which were purified separately. One is the product of the gyrA (formerly nalA) gene and is identical to subunit A of DNA gyrase. The other is a 50,000-dalton protein, which we have purified to homogeneity and call v. v may be a processed form of the much larger gyrase subunit B or may be derived from a transcript of part of the subunit B structural gene, because preliminary peptide maps of the two subunits are similar. Topoisomerase II' relaxes negatively supercoiled DNA and, uniquely among E. coli topoisomerases, relaxes positive supercoils efficiently. It is the only topoisomerase that can introduce positive supercoils; these are stoichiometric with enzyme molecules. Topoisomerase II' resembles gyrase in its sensitivity to oxolinic acid, the wrapping of DNA in an apparent positive supercoil around the enzyme, and the introduction in an aborted reaction of site-specific double-strand breaks in the DNA with concomitant covalent attachment of protein to both newly created 5' ends. Unlike DNA gyrase, topoisomerase II' has no negative supercoiling activity. Functional chimeric topoisomerases were constructed with the alpha subunit of the Micrococcus luteus gyrase and v or gyrase subunit B from E. coli. We discuss the implications of the dual of the gyrA gene product.
Unusual nucleotide sequences at the 5' end of actin genes in Dictyostelium discoideum.
Authors: Firtel R A , Timm R , Kimmel A R , McKeown M
Published: May 2006

The nucleotide sequences at the 5' end of one actin cDNA and six actin genomic clones from Dictyostelium have been determined. The amino acid sequences derived from the nucleotide sequences show strong conservation for six of the seven genes relative to the NH2-terminal region of Physarum actin. The region 5' to the AUG initiating codon is greater than 90% A+T residues in all of the genes.
Cloning and expression of the Escherichia coli replication origin in a single-stranded DNA phage.
Authors: Kaguni J , LaVerne L S , Ray D S
Published: May 2006

The Escherichia coli DNA replication origin (oriC) and the adjacent asparagine synthetase gene (asnA) have been inserted into the duplex replicative form DNA of the single-stranded phage vector M13Goril. By in vitro recombination, the entire oriC asnA-containing plasmid pJS5 was inserted into M13Gori1 in both possible orientations. Both phage types transduce the asnA gene and confer upon the M13 vector the ability to replicate as a plasmid in the E. coli mutant rep3. In rep+ hosts, these phages undergo single-stranded DNA synthesis and viral morphogenesis.
Amplification and characterization of the proline transport carrier of Escherichia coli K-12 by using proT+ hybrid plasmids.
Authors: Motojima K , Yamato I , Anraku Y , Nishimura A , Hirota Y
Published: May 2006

A previous report [Motojima, K., Yamato, I. & Anraku, Y. (1978) J. Bacteriol. 136, 5-9) described the characteristics of mutants (proT-) of Escherichia coli K-12 that are defective in proline transport carrier activity. Two hybrid plasmids from the Clarke and Carbon colony bank were found to complement the mutation by conjugation and transformation. Analysis with restriction endonucleases showed that both plasmid DNAs carried the same fragment of the E. coli chromosome. A polypeptide with a molecular weight of 24,000, specifically coded for by the proT+ plasmid, was identified in the cytoplasmic membrane by using a double-isotope labeling method in a minicell system. The strain harboring the proT+ plasmid has 6 times as much proline transport carrier as the wild strain. This amplification of the carrier makes it possible to measure proline binding to the carrier by microequilibrium dialysis. Detailed analysis of binding indicated that the maximal amount of proline bound to the carrier is 0.70 nmol/mg of protein of the cytoplasmic membrane of the amplified strain. From this value and the assumption that the carrier has one binding site per minimal molecular weight of 24,000, the content of the proline transport carrier in the cytoplasmic membrane was estimated to be 1.7%.
The biosynthetic precursor of epidermal growth factor and the mechanism of its processing.
Authors: Frey P , Forand R , Maciag T , Shooter E M
Published: May 2006

The biosynthesis of epidermal growth factor (EGF) was studied in mouse submaxillary glands incubated with L-[35S]cystine. EGF and EGF-like proteins were isolated from the gland homogenates by immunoprecipitation with anti-EGF antiserum. The major species appearing after short labeling periods is significantly larger (Mr, 9000) than EGF. The label in the Mr 9000 species plateaus after 1 hr whereas tha in EGF continuously increases. When glands are chased with unlabeled L-cystine after a brief period of labeling, the Mr 9000 peak decreases and a corresponding amount of label appears in EGF. The Mr 9000 species was isolated from boiled homogenates in which it accounts for approximately 1% of the total EGF content. It contains five of the six chymotryptic peptides of EGF and a sixth peptide which is a modified form of the COOH-terminal chymotryptic peptide of EGF. Of the arginyl esteropeptidases, gamma subunit of 7S nerve growth factor, beta-endopeptidase, trypsin, and EGF-binding protein, only the latter converts the isolated Mr 9000 species to EGF. The extrapeptide material released in the conversion comes from the COOH terminus of the Mr 9000 species. These results suggest that the Mr 9000 species is a biosynthetic precursor of EGF and that the EGF-binding protein is the specific intracellular cleaving enzyme that converts the precursor to EGF. In the process, the stable high molecular weight complex of EGF is formed.
Enucleation of differentiated murine erythroleukemia cells in culture.
Authors: Tsiftsoglou A S , Barrnett R J , Sartorelli A C
Published: May 2006

Friend murine leukemia cells induced to undergo erythrocytic differentiation by dimethyl sulfoxide give rise to progeny resembling ortho- or polychromatic normoblasts, which usually do not complete the maturation process to yield forms analogous to erythrocytes. Treatment of these differentiated cells with cytochalasin B can lead to a high proportion (i.e., 80-85%) of enucleated cells in vitro. Nuclear extrusion in cells induced to differentiate by dimethyl sulfoxide and subsequently treated with cytochalasin B began within 24-36 hr of exposure to the antibiotic, with the appearance of a pre-enucleated stage in which the cell nucleus became pycnotic and eccentrically located. Maximum enucleation occurred after 72-96 hr of exposure to cytochalasin B and was significantly enhanced when dimethyl sulfoxide was included in the culture medium during the period of treatment with cytochalasin B. Enucleation appeared to take place only in differentiated progeny, because nondifferentiated cells treated with cytochalasin B alone yielded a population of multinucleated cells. The findings indicate that highly tumorigenic nondifferentiated Friend erythroleukemia cells can be converted in high yield to mature enucleated forms that are unable to proliferate in vitro.
Absence of filipin-sterol complexes from large coated pits on the surface of culture cells.
Authors: Montesano R , Perrelet A , Vassalli P , Orci L
Published: May 2006

Monolayer cultures of normal or transformed fibroblasts and of liver cells fixed in a glutaraldehyde solution containing 300 microM filipin, a sterol-specific polyene antibiotic, were freeze-fractured to study the distribution of cholesterol within their plasma membranes. Filipin-sterol complexes, recognizable as 25- to 30-nm protuberances scattered in the fracture face of plasma membrane, were absent from invaginations corresponding to large, bristle-coated pits (and possibly also from small, flask-shaped invaginations). These results suggest that invaginating regions on the cell surface are specialized plasma membrane domains with a lower cholesterol content than the surrounding membrane. The localized change in membrane fluidity due to the low cholesterol concentration could play a role in endocytosis.
Enhanced phosphorylation of many endogenous protein substrates in human fibroblasts transformed by simian virus 40
Authors: Epstein J. , Breslow J. L. , Fontaine J. H.
Published: May 2006

Protein phosphorylation in normal and in simian virus 40-transformed human skin fibroblasts was assessed by two different methods: incubation of whole-cell homogenates with [γ-
32
P]ATP or labeling of intact cells with Na
2
H
32
PO
4
. Phosphorylated proteins were detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. With both methods, the Coomassie-blue-stained protein patterns of the three transformed cell lines studied were similar to the patterns of the nontransformed normal human cells. However, although the phosphoprotein autoradiograms of the three transformed cell lines were nearly identical, their patterns were strikingly different from those of the nontransformed cells. Each of the three transformed lines tested showed approximately 25-30 phosphoprotein bands that were significantly enhanced when compared to the patterns of the nontransformed cells. Quantitation of 12 of the enhanced phosphoprotein bands in one of the transformed cell lines showed an average of 4.4 times as much phosphorylation as in the normal cells. The enhanced phosphorylation observed in the transformed cell lines was not dependent on the growth rate of the cells or on cyclic AMP. Furthermore, when homogenates of transformed and nontransformed cells were mixed prior to incubation with [γ-
32
P]ATP, the resultant phosphoprotein patterns resembed those obtained with transformed cells alone. In addition, an evaluation of the time course of protein phosphorylation revealed that the initial reaction rate was greater in the transformed than in the normal cells, although in both cell types the reaction was complete after 1 min. The results suggest that the simian virus 40-transformed human fibroblasts possess an increased ability to phosphorylate proteins rather than that the normal cells possess a diffusible inhibitor. There appear to be many endogenous cellular substrates for this increased activity.
Endothelial plasmalemmal vesicles as elements in a system of branching invaginations from the cell surface.
Authors: Bundgaard M , Frøkjaer-Jensen J , Crone C
Published: May 2006

In electron microscopy studies of the endothelial vesicles in frog mesenteric capillaries, an accidental observation was made concerning vesicular organization. When tannic acid was added to already fixed tissue, the mordant reached apparently free vesicles in the cytoplasm under conditions in which vesicular movement was excluded and in which the impermeability of the cell membranes was preserved. This indicates a spatial continuity between the vesicles and the cell exterior. It is proposed that cytoplasmic vesicles in endothelial cells are elements of branching, permanent or semipermanent invaginations of the plasmalemma.
Lysosomal composition in cultured vascular smooth muscle cells: electron probe analysis.
Authors: James-Kracke M R , Sloane B F , Shuman H , Somlyo A P
Published: May 2006

Spherical electron-dense organelles in the perinuclear region of cultured guinea pig aortic smooth muscle cells were identified as lysosomes by their ability to accumulate acridine orange and by cytochemical demonstration of their acid phosphatase content. The number and size of lysosomes increased in subcultured cells. The elemental composition of the lysosomes was quantitated by electron probe analysis of whole freeze-dried cells and of cryosections. In lysosomes at this stage in their development, the sulfur concentration was higher than that in the cytoplasm and the K/Na concentration ratio was similar to that in the cytoplasm.
Induction of glutamine synthetase in embryonic neural retina: localization in Müller fibers and dependence on cell interactions.
Authors: Linser P , Moscona A A
Published: May 2006

The cellular localization of glutamine synthetase [GSase; L-glutamate:ammonia ligase(ADP)-forming), EC 6.3.1.2] induced by cortisol in the neural retina of chicken embryos was investigated by immunostaining with GSase-specific antiserum and indirect immunofluorescence. In organ cultures of retina tissue, and in the retina in vivo, hormone-induced GSase was found to be confined only to the Müller fibers (retinoglia). Also, in mature chicken retina, which contains a very high level of GSase, the enzyme was detected solely in Müller fibers. In short-term monolayer cultures of dispersed embryonic retina cells, there was no GSase induction and no immunodetectable increase in enzyme level. However, when the dispersed cells were reaggregated and they restituted retinotypic cell associations, GSase could be induced and it was localized in Müller fibers. The results suggest that, in addition to the hormonal stimulus, contact-dependent interactions between Müller glia cells and retina neurons are involved in the mechanism of GSase induction in the retina.
Is trisomy cause or consequence of murine T cell leukemia development? Studies on Robertsonian translocation mice.
Authors: Spira J , Wiener F , Ohno S , Klein G
Published: May 2006

Trypsin-Giemsa banding studies on T cell leukemias induced in Robertsonian translocation mice by dimethylbenz[a]anthracene and Moloney leukemia virus show a trisomy of chromosome 15 even in cases in which chromosome 15 has undergone centromeric fusion with chromosomes 1, 5, or 6. These results suggest that the duplication of gene(s) located on chromosome 15 is of critical importance for murine T cell leukemia development.
Immunofluorescent localization of angiotensin converting enzyme in epithelioid and giant cells of sarcoidosis granulomas.
Authors: Silverstein E , Pertschuk L P , Friedland J
Published: May 2006

Angiotensin converting enzyme (ACE) (dipeptidyl carboxypeptidase; peptidyldipeptide hydrolase, EC 3.4.15.1) was localized in epithelioid and giant cells within ACE-rich sarcoidosis granulomas, but not control granulomas, by immunofluorescence by using a rabbit anti-human ACE immunoglobulin for localization and fluorescein-labeled goat anti-rabbit immunoglobulin for detection. ACE was frequently not detected in the very central epithelioid cells, perhaps due to a paucity of cellular or molecular stimulatory interaction in this region. The results suggest that marked elevation of ACE in the granulomas and often in the serum in sarcoidosis may be due to intense induction of ACE in most epithelioid and giant cells of the sarcoidosis granulomas.
Immunoprecipitation and partial characterization of diphtheria toxin-binding glycoproteins from surface of guinea pig cells.
Authors: Proia R L , Hart D A , Holmes R K , Holmes K V , Eidels L
Published: May 2006

125I-Labeled membrane glycoproteins that specifically interact with diphtheria toxin and CRM197 protein--but not with diphtheria toxoid, fragment A of diphtheria toxin, or cholera toxin--were detected by use of the lactoperoxidase labeling technique followed by an immunoprecipitation system. These glycoproteins, which adhere to lentil lectin-Sepharose columns, are present on the surface of diphtheria toxin-sensitive guinea pig lymph node cells but are completely lacking on the surface of diphtheria toxin-resistant mouse L cells. The major 125I-labeled glycoprotein that interacts with diphtheria toxin exhibits anomalous behavior, characteristic of glycoproteins, when analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. This demonstration of the biochemical nature of specific diphtheria toxin binding membrane components raises the possibility that the detected components are diphtheria toxin receptors.
alpha-Fetoprotein and albumin genes of rats: no evidence for amplification-deletion or rearrangement in rat liver carcinogenesis.
Authors: Sala-Trepat J M , Sargent T D , Sell S , Bonner J
Published: May 2006

Full-length radiolabeled albumin and alpha-fetoprotein (AFP) cDNAs were synthesized from pure albumin and AMP mRNA preparations by using avian myeloblastosis virus reverse transcriptase (RNA-dependent DNA polymerase). The cDNAs have been used to quantitate the number of albumin and AFP genes in different rat tissues by two independent methods, both of which yielded similar results. First, the kinetics of the association of these cDNAs with nuclear DNA from rat liver, rat kidney, and Morris hepatoma 7777 under conditions of vast DNA excess indicated that the albumin and AFP mRNA's are transcribed from "nonrepetitive DNA." Second, saturation hybridization experiments in which a constant amount of rat liver DNA or Morris hepatoma 7777 was hybridized with increasing amounts of cDNA to albumin mRNA have shown the presence of 1--2 albumin genes per rat haploid genome. The number of AFP genes obtained in similar titration experiments was approximately 2--3. This was true whether rat liver DNA or hepatoma 7777 DNA was used in the reassociation experiments. When high molecular weight DNA preparations from both these tissues were digested with the restriction endonuclease EcoRI and the fragments were transferred to a nitrocellulose filter, the albumin and AFP [32P]cDNA probes hybridized to different sets of DNA fragments. However, each probe gave the same hybridization pattern whether Buffalo rat liver DNA or hepatoma 7777 DNA was utilized.
Interesting and unusual features in the sequence of Neurospora crassa mitochondrial tyrosine transfer RNA.
Authors: Heckman J E , Alzner-Deweerd B , RajBhandary U L
Published: May 2006

The mitochondrial tyrosine tRNA from Neurospora crassa has been sequenced and found to have several interesting features: (i) It resembles prokaryotic rather than eukaryotic tyrosine tRNAs in that it possesses a large variable loop (loop III); moreover, it can be quantitatively aminoacylated by Escherichia coli tyrosyl-tRNA synthetase but not by yeast tyrosyl-tRNA synthetase. (ii) This tRNA differs from all tRNA's sequenced to date in lacking the A residue at position 14 and the constant purine residue at position 15, two nucleosides that have been found so far in loop I of all tRNA's and that have been implicated in base-base tertiary interactions, respectively, with the universal U residue at position 8 and the constant pyrimidine residue at the end of loop III. (iii) Unlike the N. crassa mitochondrial initiator tRNA, this tRNA contains the usual TpsiC sequence in loop IV and the highly conserved GG sequence in loop I common to other tRNAs.
Apparent dependence of sporulation on synthesis of highly phosphorylated nucleotides in
Bacillus subtilis
Authors: Rhaese H. J. , Groscurth R.
Published: May 2006

Bacillus subtilis
contains an enzyme that synthesizes the highly phosphorylated nucleotide adenosine 5′,3′(2′)-bis(triphosphate), abbreviated p
3
Ap
3
. This enzyme can be demonstrated to be present in the cytoplasmic membrane of
B. subtilis
at all stages of growth and development. During vegetative growth its enzymatic activity is inhibited by phosphorylated metabolites of energy-rich carbon sources, as was shown with the aid of conditional sporulation mutants. The finding that a temperature-sensitive sporulation mutant with a mutation in the
spoOF
gene, an early sporulation gene, shows a temperature-sensitive activity of p
3
Ap
3
-synthetase indicates that p
3
Ap
3
plays an important role in sporulation, probably initiation of sporulation.