bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid disassembly of cold-stable microtubules by calmodulin.
Authors: Job D , Fischer E H , Margolis R L
Published: May 2006

Purified cold-stable microtubules from the rat brain are insensitive to podophyllotoxin and to millimolar concentrations of free calcium. However, in the presence of calmodulin at concentrations substoichiometric to that of tubulin, calcium causes rapid microtubule disassembly. The half-maximal effective calcium concentration in the presence of calmodulin is 100 microM. With 800 microM free calcium, the half-maximal effective concentration of calmodulin is 1.0 microM (or one-tenth the tubulin concentration). Calmodulin is without effect in the absence of calcium. Troponin C is approximately one-fifth as effective as calmodulin, and parvalbumin is totally ineffective. Troponin I partially inhibits the calcium/calmodulin-induced disassembly of microtubules in the crude extract and blocks the calcium/calmodulin effect on purified cold-stable microtubules. A 5-fold excess of trifluoperazine does not inhibit the calcium/calmodulin-induced disassembly.
7-Methylguanine in poly(dG-dC).poly(dG-dC) facilitates z-DNA formation.
Authors: Möller A , Nordheim A , Nichols S R , Rich A
Published: May 2006

Poly(dG-dC).poly(dG-dC) was methylated at the N-7 position of guanine through the use of dimethyl sulfate. The conversion of poly(dG-dC).poly(dG-dC) from B-DNA to the Z-DNA form was followed by measuring both the circular dichroic spectra and changes in the absorbance ratio A295/A260. Increasing methylation steadily decreases the amount of NaCl or MgCl2 that is required to convert the polymer from B-DNA to Z-DNA. At 100% methylation of the guanine residues, the modified polymer is fully converted to Z-DNA in a physiological salt solution. Kinetic experiments show that methylation markedly accelerates the speed of the conversion from B-DNA to Z-DNA in the presence of added salt. These effects may be partly due to the positive charge on guanine that accompanies N-7 methylation.
On the molecular mechanisms of transposition.
Authors: Galas D J , Chandler M
Published: May 2006

We present a model for transposition that allows a choice between cointegrate formation (replicon fusion) and direct transposition. We propose that initiation of the process occurs by invasion of the target DNA by a single-stranded end of the transposable element. This leads to nicking of one of the DNA strands of the target molecule and ligation of this strand to that of the invading transposon. Transposition then occurs in a processive way by replication of the element from the invading end into the target site in a looped rolling-circle mode similar to replication of phage phi X174 replicative form to viral strand. The choice between cointegrate formation and direct transposition occurs at the nick-ligation step, which terminates the process. We suggest that the choice is determined by the topology of the transposition enzymes and could be related to whether the element generates five- or nine-base-pair repeats in the target DNA on insertion.
Positive cooperativity of the estrogen receptor.
Authors: Notides A C , Lerner N , Hamilton D E
Published: May 2006

The equilibrium [3H]estradiol binding by the partially purified estrogen receptor from calf uteri was measured at 25 degrees C. The Scatchard plot of the binding data showed a convex curve characteristic of positive cooperativity and a Hill coefficient of 1.58 +/- 0.21, at receptor concentrations of 1 to 10 nM. Below a receptor concentration of approximately 0.3 nM the Scatchard plot approached linearity, suggesting that the cooperative interactions are dependent upon a monomer--dimer equilibrium. Trypsin pretreatment of the receptor resulted in a loss of dimer formation and of the cooperative interactions. The positive cooperative characteristics of the estrogen receptor were shown not to be produced by receptor inactivation, failure to complete the [3H]estradiol--receptor equilibrium reaction, or radioimpurity of the [3H]estradiol. These findings indicate that the activated 5S estrogen receptor is a homodimer and that its formation is associated with a positive cooperative estradiol-binding reaction.
Increase in proliferative markers after inhibition of transglutaminase.
Authors: Birckbichler P J , Orr G R , Patterson M K , Conway E , Carter H A
Published: May 2006

Cystamine inhibited transglutaminase activity (R-glutaminyl-peptide:amine gamma-glutamyltransferase, EC 2.3.2.13) of proliferating WI-38 cells in a dose-dependent manner over the concentration range 0.005-0.25 mM when added to the culture medium. The epsilon-(gamma-glutamyl)lysine content in the cells was decreased and several proliferation markers were enhanced. "Non-mitotic" cells were stimulated by cystamine (about 25% of that observed with 10% fetal bovine serum) to undergo DNA synthesis with subsequent increases in nuclei number. Numerous other disulfides, thiols, and amines were ineffective when added to culture medium. The findings are supportive of the concept that growth control involves a relationship between isopeptide crosslinks and proliferation.
Identification and characterization of recombinant plasmids carrying the complete qa gene cluster from Neurospora crassa including the qa-1+ regulatory gene.
Authors: Schweizer M , Case M E , Dykstra C C , Giles N H , Kushner S R
Published: May 2006

The early reactions in the catabolism of quinic acid in Neurospora crassa are controlled by at least four genes which are clustered on linkage group VII. Three of the loci (qa-2, qa-4, and qa-3) encode enzymes that convert quinic acid to protocatechuic acid. The fourth gene (qa-1) encodes a positive regulatory protein which, in the presence of quinic acid, leads to the de novo synthesis of the other proteins in the qa cluster. This communication describes a series of recombinant plasmids that span 36.5 kilobases of linkage group VII and contain the coding sequences for qa-2, qa-4, qa-3, and the qa-1 regulatory protein. The plasmids were obtained by partial digestion of wild-type N. crassa DNA with EcoRI and ligation into the cosmid cloning vehicle pHC79. Two independently derived plasmids (pMSK331 and pMSK335), each containing 36.5-kilobase inserts, were shown by transformation back into N. crassa to contain the entire qa gene cluster. A preliminary physical organization of the gene cluster is presented. An improved procedure for the transformation of N. crassa with plasmid DNA is also described.
Reduced secretion of structurally abnormal type I procollagen in a form of osteogenesis imperfecta.
Authors: Barsh G S , Byers P H
Published: May 2006

Osteogenesis imperfecta is a clinically and genetically heterogeneous group of inherited connective tissue disorders in which bone fragility is the predominant feature. Cultured dermal fibroblasts from one patient with the lethal perinatal form of osteogenesis imperfecta secrete type I procollagen at a rate half that of normal cells. Short-term labeling experiments and treatment with alpha,alpha'-dipyridyl (which prevents posttranslational prolyl and lysyl hydroxylation) demonstrated that these cells produce two distinct pro alpha 1(I) chains, which are synthesized at the same rate. Analysis of cyanogen bromide peptides indicated that the two chains differ in their primary structures. Thus, structural abnormalities in type I procollagen prevent this molecule from being secreted normally, resulting in an anomalously low ratio of type I procollagen to other extracellular matrix molecules. While the lethal perinatal form of osteogenesis imperfecta may be heterogeneous, we propose that the underlying pathogenesis of at least one form is decreased secretion of type I procollagen.
Spikes and fimbriae: alpha-helical proteins form surface projections on microorganisms.
Authors: Cohen C , Phillips G N
Published: May 2006

Two basic alpha-helical plans appear to characterize portions of the proteins projecting from the surface of many microorganisms. Structure A is related to that of the antibody-binding protein A of Staphylococcus aureus, in which a single polypeptide chain doubles back to form an Anti-parallel domain; the other, structure M, is related to the M proteins of group A streptococci, in which Multiple parallel alpha-helical chains interlock in a coiled-coil conformation. A projection about 100 A long may be organized on either plan, but very long projections are likely to be of the M pattern. The alpha-helical motif may account for the length of these spikes, with other functions such as anchoring or antiphagocytic activity located in domains of different structure.
Structure of the promoter for chicken alpha 2 type I collagen gene.
Authors: Vogeli G , Ohkubo H , Sobel M E , Yamada Y , Pastan I , de Crombrugghe B
Published: May 2006

The chicken alpha 2 type I collagen gene is 38 kilobases long and its coding information is subdivided into more than 50 exons. In the current study, we used primer extension and S1 nuclease mapping to determine the sequence of the 5' end of alpha 2 collagen mRNA and to locate the start site for transcription of the alpha 2 collagen gene. The DNA sequence around the start site for transcription shows a typical Goldberg-Hogness sequence, 5' T-A-T-A-A-A-T 3', between -33 and -26 and a 5' G-C-C-C-A-T-T 3' sequence ("CAT" box) between -84 and -78. Three AUGs are found in the initial portion of the mRNA, the first from +54 to +56, the second from +117 to +119, and the third from +134 to +136. The first two AUGs are followed by short coding sequences that could specify a hexapeptide a tetrapeptide, respectively. Only the third AUG is followed by an open reading frame coding for a sequence that presents considerable homology with the previously determined amino acid sequence of prepro alpha 1 collagen. In the promoter region sequence there are several extensive dyads of symmetry. Three of these inverted repeats which precede the start site for transcription overlap each other and may have a role in the developmental regulation of this gene.
Primary structure of porcine heart citrate synthase.
Authors: Bloxham D P , Parmelee D C , Kumar S , Wade R D , Ericsson L H , Neurath H , Walsh K A , Titani K
Published: May 2006

The sequence of 437 amino acid residues of porcine heart citrate synthase [citrate oxaloacetate-lyase (pro-3S-CH2COO leads to acetyl-CoA), EC 4. 1. 3. 7] has been determined by the alignment of fragments generated by cleavage with cyanogen bromide and with trypsin. Isolation of the peptides was facilitated by recent developments in the high-performance liquid chromatography of peptide mixtures. The alignment of these peptides was consistent with that previously deduced from fragments derived by restricted cleavage of citrate synthase by limited proteolysis and cleavage of aspartyl-prolyl bonds and asparaginyl-glycyl bonds. The enzyme contains a modified amino acid, trimethyllysine, at residue 368, showing that the enzyme is subjected to post-translational modification.
beta-meso-Phenylbiliverdin IX alpha and N-phenylprotoporphyrin IX, products of the reaction of phenylhydrazine with oxyhemoproteins.
Authors: Saito S , Itano H A
Published: May 2006

Oxyhemoglobin and oxymyoglobin were allowed to react aerobically with phenylhydrazine and p-tolylhydrazine. The chloroform extract of each reaction mixture, after treatment with H2SO4/methanol, yielded a blue pigment and a green pigment, which were identified by electronic absorption, mass, and proton NMR spectroscopy as the dimethyl esters of beta-meso-arylbiliverdin IX alpha and N-arylprotoporphyrin IX, respectively. N-Phenylprotoporphyrin IX dimethyl ester formed complexes with Zn2+, Cd2+, and Hg2+ but not with other cations. The proton NMR spectrum of the zinc complex suggested binding of the phenyl group to one of the two pyrrole rings of protoporphyrin IX with a propionic acid substituent. The effectiveness of phenylhydrazine as an inducer of Heinz body formation may be due to destabilization of the hemoglobin molecule by the replacement of heme with phenyl adducts of biliverdin and protoporphyrin.
Localization of 3' ends of 5S and 23S rRNAs in reconstituted subunits of Escherichia coli ribosomes.
Authors: Stöffler-Meilicke M , Stöffler G , Odom O W , Zinn A , Kramer G , Hardesty B
Published: May 2006

Periodate-oxidized 3' ends of 5S, 23S, and 16S rRNAs from Escherichia coli were allowed to react with fluorescein thiosemicarbazide, then labeled rRNAs were reconstituted into active ribosomal subunits. The fluorescein moiety on each of the rRNAs when reconstituted into ribosomal subunits was accessible to anti-fluorescein IgG as determined by fluorescence quenching and by sucrose gradient centrifugation. The region at which an antibody molecule bond to the labeled ribosomal subunits was determined by immunoelectron microscopy. The 3' end of the 5S RNA was localized on the central protuberance of the 50S subunit. The corresponding region for the 3' end of the 23S RNA was below the stalk on the noninterfacing surface. The 3' end of the 16S RNA was localized to the upper edge of the large lobe of 30S subunits, as reported previously.
Mg
2+
/KCl-ATPase of plant plasma membranes is an electrogenic pump
Authors: Sze Heven , Churchill Kathleen A.
Published: May 2006

The function of the Mg
2+
-requiring KCl-stimulated ATPase (ATP phosphohydrolase, EC 3.6.1.3) of higher plants in active ion transport was investigated by using a purified microsomal fraction containing sealed plasma membrane vesicles. (Sze, H. (1980)
Proc. Natl. Acad. Sci. USA
77, 5904-5908). A transmembrane electrical potential (+30 to +44 mV), monitored by uptake of a permeant anion (
35
SCN
-
), was generated specifically by ATP in purified microsomal vesicles of tobacco callus. ATP-dependent
35
SCN
-
uptake required Mg
2+
, was optimal at pH 6.75, and showed similar ATP concentration dependence as the Mg
2+
-requiring KCl-stimulated ATPase activity. Plasma membrane ATPase inhibitors (
N,N
′-dicyclohexylcarbodiimide and vanadate) inhibited generation of the ATP-dependent electrical potential. A proton conductor (carbonyl cyanide
m
-chlorophenylhydrazone), but not a K
+
ionophore (valinomycin), completely collapsed the electrical potential. The results provide
in vitro
evidence that the Mg
2+
/KCl-ATPase of higher plants is an electrogenic pump. These results are consistent with the hypothesis that an electrogenic H
+
pump is catalyzed by the plasma membrane ATPase of plants.
Fast responses of bacterial membranes to virus adsorption: a fluorescence study.
Authors: Bayer M E , Bayer M H
Published: May 2006

After collision with their host cells, virus particles may remain mobile on cell surfaces until they become attached at firm binding sites. We propose that a virion will arrive within a typical median time at such a site, generating a membrane signal such as an increased membrane fluorescence in cells labeled with the voltage-sensitive dyes 8-anilino-1-naphthalene-sulfonate (Mg-salt) (ANS), N-phenylnaphthylamine (NPA), or 3,3'-dipentyl-2,2'-oxacarbocyanine (di-O-C5[3]). We found that the time span between virus adsorption and fluorescence response varies widely among phages and also depends on bacterial strain, metabolic state, and type of dye. di-O-C5[3]-labeled cells react within 1 sec to uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells labeled with ANS and NPA react to CCCP in 4-6 sec. Bacteriophages T4, T5, chi, and BF23, added to ANS-labeled cells, change the fluorescence in 9-15 sec. T-even ghosts cause a response at identical times. Baseplate-defective phage mutant T412- and isolated adsorption organelles of smaller viruses fail to cause an effect. di-O-C5[3]-labeled cells respond to T4 at a multiplicity of infection greater than or equal to 40 within 1 sec. A longer time (8 sec) is required at lower multiplicities. The receptor-degrading phages epsilon 15, epsilon 34, c 341, and K29 need the longest time (1 min for ANS) to cause a fluorescence increase. We suggest that the delayed fluorescence response is concomitant with the surface "walk" of the virion, which is terminated at an injection site. T4 tail sheath contraction coincides with the onset of the membrane fluorescence response.
Measurement of rapidly exchangeable cellular calcium in the perfused beating rat heart.
Authors: Hunter D R , Haworth R A , Berkoff H A
Published: May 2006

Although Ca2+ has long been known to play a vital role in excitation--contraction coupling in the heart, investigation of the details of this role has been hampered by the experimental difficulty of measuring Ca2+ movements through the different compartments of the cell. A major problem has been to distinguish the relatively small amount of rapidly exchangeable cellular Ca2+ from the large amount of vascular and interstitial Ca2+. We report here a method that overcomes this problem. Rat hearts were labeled by perfusion at 37 degrees C with medium containing 45Ca2+. They were then cooled, and extracellular 45Ca2+ was removed by perfusion at 6 degrees C with Ca2+-free medium. Cellular 45Ca2+ that had been trapped in the hearts by cooling was then released by reperfusion at 37 degrees C with medium containing unlabeled Ca2+. The cellular origin of this 45Ca2+ was confirmed by using [3H]sucrose: When hearts were also labeled with [3H]sucrose, very little [3H]-sucrose was released with the 45Ca2+ peak. The amount of exchangeable cellular Ca2+ in hearts labeled for 5 min was 125 +/- 13 nmol/g of wet weight. The half-time for its release was less than 1 min. This cellular Ca2+ contained at least two pools: a rapidly exchanging pool that required extracellular Ca2+ for release (pool A, 38% of total), and a more slowly exchanging pool that did not (pool B, 62% of total). Hearts stimulated with isoproterenol during the 5-min labeling period showed an increase of 46% for the total amount of exchangeable cellular Ca2+; this increase was entirely located in pool A.
Delivery of hexosaminidase A to the cerebrum after osmotic modification of the blood--brain barrier.
Authors: Neuwelt E A , Barranger J A , Brady R O , Pagel M , Furbish F S , Quirk J M , Mook G E , Frenkel E
Published: May 2006

The present studies were undertaken to evaluate the possibility that hexosaminidase A, the enzyme deficient in Tay--Sachs disease, could be effectively delivered to brain. Previous studies from our laboratory have shown that hypertonic mannitol can be used to osmotically produce reversible disruption of the blood--brain barrier in animals (rat and dog) and man without significant neurotoxicity and that such barrier modification significantly increases the delivery of cytoreductive chemotherapy agents to selected areas of brain. By using the rat model of blood--brain barrier modification and radiolabeled enzyme, increased hexosaminidase A delivery to brain has been demonstrated in more than 85 animals. The time of injection of hexosaminidase A after blood--brain barrier disruption is critical for maximum delivery. Rapid (over 30 sec) intra-arterial administration of hexosaminidase A immediately after blood--brain barrier disruption resulted in a marked increase in enzyme delivery to the brain when compared with controls without prior barrier disruption. When the enzyme was administered 15-20 min after barrier disruption, approximately 50% less hexosaminidase A was delivered; when given 60-120 min after barrier modification, the amount delivered was the same as in control animals. This critical time course is very different than that seen in trials of low molecular weight chemotherapeutic agents (methotrexate and adriamycin). These preliminary studies suggest that hexosaminidase A can be delivered to the brain by blood--brain barrier modification and may be indicative of the potential for enzyme replacement in patients who hae Tay--Sachs disease.
Mitochondria are associated with microtubules and not with intermediate filaments in cultured fibroblasts.
Authors: Ball E H , Singer S J
Published: May 2006

Triple-immunofluorescence experiments with antibodies to cytochrome c oxidase, tubulin, and vimentin have been used to immunolabel the mitochondria, microtubules, and intermediate filaments inside the same cultured fibroblasts. In particular, fibroblasts were immunolabeled after they had either been transformed by infection with Rous sarcoma virus or given long-term treatment with cycloheximide. These treatments induced redistribution of the intermediate filaments into a perinuclear arrangement, segregated away from the microtubules, which remained extended to the cell periphery. In such cells, many labeled mitochondria were observed to be codistributed with the peripherally located microtubules. From these results, we infer that an association, probably involving some type of chemical linkage(s), between mitochondria and microtubules exists in these cells that is independent of the intermediate filaments.
Inhibition of actomyosin ATPase by vanadate.
Authors: Goodno C C , Taylor E W
Published: May 2006

Actin-myosin subfragment-1 (SF-1) or actin-heavy meromyosin is dissociated by the binding of ADP and vanadate (Vi) under conditions such that ADP alone does not dissociate the complex. The association constant of the stable complex M.ADP.Vi, in which M indicates myosin [Goodno, C. C. (1979) Proc. Natl. Acad. Sci. USA 76, 2620-2624] with actin is smaller than the average association constant of the intermediate states of the actin-SF-1 ATPase cycle. Actin-SF-1 ATPase activity is 90% inhibited by ADP plus vanadate. The reaction of actin with M.ADP.Vi produces a slow release of ADP and vanadate and quantitative recovery of ATPase activity. The rate of dissociation of ligands was almost linear in actin concentration; consequently, the rate constant of dissociation could only be roughly estimated as 0.5-1 sec-1. The rate of dissociation of ADP and vanadate is thus increased by a factor of 10(5) compared to M.ADP.Vi. The rate of release of ligands by regulated actin (actin-tropomyosin-troponin) was reduced to 1/10th to 1/20th by removal of calcium ion. Therefore the M.ADP.Vi complex has the properties of a more stable analogue of the myosin-ADP-phosphate complex that is generated in the normal ATPase cycle. The activation of ligand release (ratio of rate of dissociation of ADP and vanadate from actomyosin relative to myosin) is much larger than the activation of myosin ATPase by actin, whereas the actual rates of the reactions are much slower.
Rat preprocarboxypeptidase A: cDNA sequence and preliminary characterization of the gene.
Authors: Quinto C , Quiroga M , Swain W F , Nikovits W C , Standring D N , Pictet R L , Valenzuela P , Rutter W J
Published: May 2006

Rat carboxypeptidase A cDNA clones have been isolated from a cDNA library prepared from pancreatic mRNA. An almost complete mRNA sequence has been deduced that predicts a polypeptide having 78% amino acid sequence homology with bovine carboxypeptidase A. The amino acid sequence of the activation and signal peptides of the carboxypeptidase A precursor were inferred from the nucleotide sequence. The cDNA was used as a probe to identify DNA fragments containing carboxypeptidase A sequences in a bacteriophage lambda library of rat genomic DNA. Heteroduplexes revealed that the DNA coding sequence occupies 5.5 kilobases and is interrupted by nine intervening sequences. The nucleotide sequence of the 5' end of the gene and the adjacent flanking region provides information on the site of initiation of transcription and the putative control regions. There is no evident relationship between the localization of intervening sequences in the gene and functional/structural domains of the protein.
Direct photoaffinity labeling of an allosteric site on subunit protein M1 of mouse ribonucleotide reductase by dTTP.
Authors: Eriksson S , Caras I W , Martin D W
Published: May 2006

The protein M1 subunit of ribonucleotide reductase contains at least two allosteric nucleotide binding sites that control the capacity of the enzyme to reduce ribonucleotides to the deoxyribonucleotides required for DNA synthesis. Direct photoaffinity labeling of partially purified protein M1 from mouse T-lymphoma (S49) cells was observed after UV irradiation in the presence of dTTP at 0 degrees C. The relative molar incorporation of nucleotide per subunit was 4-8%. Competition experiments showed that the dTTP was bound to an allosteric domain genetically and kinetically defined as the substrate specificity site of the enzyme. An altered protein M1 isolated from a thymidine-resistant mutant cell line showed significantly decreased photoincorporation of dTTP, consistent with the fact that its CDP reductase activity is resistant to feedback inhibition by dTTP. Specific photolabeling of several other proteins with pyrimidine and purine nucleotides was also found, indicating the general usefulness of direct photoaffinity labeling in the study of enzymes involved in nucleotide and nucleic acid metabolism.
Embryonic brain extract induces collagen biosynthesis in cultured muscle cells: involvement in acetylcholine receptor aggregation.
Authors: Kalcheim C , Vogel Z , Duksin D
Published: May 2006

The involvement of extracellular matrix components in induction of the aggregation of acetylcholine (AcCho) receptors by factor(s) present in embryonic brain extract was investigated. Embryonic brain extract induced a three-fold increase in the number of AcCho receptor aggregates on the surface of cultured myotubes and a 5- to 10-fold increase in the synthesis of procollagen, which was secreted into the medium and converted to collagen. Adult brain extract, embryonic serum, and embryonic liver extract were less active in stimulating both collagen synthesis and AcCho receptor aggregation. A physiological connection between the two processes is suggested, since the number of AcCho receptor aggregates could be reduced to control levels by treating brain extract-stimulated myotubes with purified bacterial collagenase. In addition, stimulation of collagen secretion by ascorbic acid (50 micrograms/ml) promoted a 1.6-fold increase in AcCho receptor aggregation. When ascorbic acid was added together with the brain extract, further increases in both collagen synthesis and AcCho receptor aggregation were observed.
Sodium channels induced by depolarization of the Xenopus laevis oocyte.
Authors: Baud C , Kado R T , Marcher K
Published: May 2006

An electrically gated Na+ channel can be made to appear in the membrane of the Xenopus laevis oocyte by simple depolarization. This membrane normally responds passively to imposed transmembrane currents with resting potentials around -60 mV, but when it is held depolarized to more than about +30 mV it becomes possible to obtain long-lasting regenerative depolarizations up to +80 mV; these depolarizations can last as long as 20 min. This potential is due to an "induction" of a Na+-dependent channel that is electrically gated open and closed. Its threshold for opening is about -20 mV and it is selective for Na+ over Cs+ and choline+ but is blocked by relatively small quantities of Li+. When a long voltage clamp step to a positive potential under ENa (+70 to +90 mV) is applied, an inward current is observed for many minutes, implying that this channel does not have an inactivation mechanism. The inward Na+ current is blocked by 0.50 mM tetrodotoxin. When the membrane is held at or near resting potential, the excitability will disappear with time, but it can be made to reappear by again depolarizing the membrane.
Human transferrin receptor: expression of the receptor is assigned to chromosome 3.
Authors: Enns C A , Suomalainen H A , Gebhardt J E , Schröder J , Sussman H H
Published: May 2006

Human chromosome 3 has been identified as responsible for expression of the transferrin receptor in mouse-human lymphocyte hybrids. The receptor was detected by immunoprecipitation with anti-human receptor antibody of 125I-labeled cells. This method also detected a similar 94,000-dalton protein in mouse cells. A radioimmunoassay developed for the human transferrin receptor measured 10% crossreactivity with the mouse protein. The two proteins were distinguished by NaDodSO4/polyacrylamide gel patterns of partial proteolytic digests of the immunoprecipitated proteins. Mouse-human hybrids were generated by fusing a mouse thymoma (BW5147) cell line to either concanavalin A- or pokeweed mitogen-activated human peripheral blood lymphocytes or a mouse myeloma (NS-1) to uncultured human peripheral blood lymphocytes. Each hybrid was karyotyped with respect to both mouse and human chromosomes. In every case, expression of the human transferrin receptor correlated only with human chromosome 3.
Understanding the chemistry and physics of coal structure (A Review)
Authors: Levine Duane G. , Schlosberg Richard H. , Silbernagel Bernard G.
Published: May 2006

This article outlines our current understanding of the structure of coal in terms of both the chemical and physical bonding processes responsible for its structural integrity and the extensive network of pores that permeate the organic material. Information on the microscopic chemistry of coal and its relationship to coal's physical structure and reactivity is an essential component in the successful development of the next generation of coal conversion technologies. Although coal is an extremely complex and heterogeneous material, many of its fundamental properties can be determined by the coordinated efforts of organic and physical chemists, solid state physicists, and chemical engineers. The scientific questions that emerge from these efforts lie at the frontiers of chemistry and physics research.
Methylation of elongation factor 1 alpha from the fungus Mucor.
Authors: Hiatt W R , Garcia R , Merrick W C , Sypherd P S
Published: May 2006

A basic protein from the dimorphic fungus Mucor racemosus, found to be highly methylated, is shown to be protein synthesis elongation factor 1 alpha. This protein is the most abundant protein in hyphal cells but is less abundant in yeast cells. It is post-translationally methylated with the formation of mono-, di-, and trimethyllysine at as many as 16 sites. Nearly 20% of the 44 lysine residues of elongation factor 1 alpha from mycelia are modified while those from sporangiospores are virtually unmethylated.
Post-translational modification of neuronal proteins: evidence for transglutaminase activity in R2, the giant cholinergic neuron of Aplysia.
Authors: Ambron R T , Kremzner L T
Published: May 2006

[3H]Putrescine injected into the cell body of the giant neuron R2 of Aplysia was readily converted to gamma-aminobutyric acid, acetylputrescine, spermidine, and spermine. In addition, labeled putrescine and spermidine were found covalently linked to protein through the action of an intracellular transglutaminase. This was shown by exhaustively treating the acid-insoluble fraction from injected cells with Pronase, aminopeptidase M, and carboxypeptidases A and B. High-performance liquid chromatography of the digest revealed labeled gamma-glutamylputrescine and gamma-glutamylspermidine, the products expected from the transglutaminase-catalyzed post-translational modification of intracellular proteins. In vitro assays of Aplysia nervous tissue showed the presence of transglutaminase as well as gamma-glutaminyl cyclotransferase, an enzyme that cleaves the gamma-glutamylpolyamine bond. Incorporation of polyamine into proteins in R2 is a specific process because only a few 3H-labeled polypeptides were found after injection.
Putative mediators of insulin action: regulation of pyruvate dehydrogenase and adenylate cyclase activities.
Authors: Saltiel A R , Siegel M I , Jacobs S , Cuatrecasas P
Published: May 2006

Recent evidence suggests that certain actions of insulin may be mediated by the selective generation of chemically undefined intracellular substances. Incubation of rat liver particulate fraction with low concentrations of insulin enhances the release into the supernatant of a substance that stimulates mitochondrial pyruvate dehydrogenase. Higher concentrations of insulin release less stimulating activity. It is possible to resolve activities that stimulate and inhibit pyruvate dehydrogenase by differential ethanol extraction of the supernatant solutions. The elaboration of both factors is dependent upon the presence of insulin in a dose-dependent manner. Moreover, fractions that contain the pyruvate dehydrogenase-inhibiting activity also inhibit adipocyte basal and hormonally stimulated adenylate cyclase. The production of this adenylate cyclase inhibitory activity is also stimulated by insulin. Cyclase inhibition is virtually abolished when the nonhydrolyzable ATP analog, 5'-adenylyl imidodiphosphate, is included in the assay. These results indicate that the bimodal effects of insulin on certain functions may be ascribed to the generation of at least two distinct chemical substances that show opposing activities, which may operate by regulating phosphorylation reactions.
Tissue specificity and clustering of methylated cystosines in bovine satellite I DNA.
Authors: Sano H , Sager R
Published: May 2006

The positions of all 5-methylcytosine (mC) residues in bovine satellite I DNA were determined by sequence analysis of native purified satellite I DNAs from three bovine tissues as well as from cloned DNA. The EcoRI cleavage units from thymus and liver were found to contain 1,402 residues; that from brain contained 1,401 residues. Satellite I DNA from thymus contained a total of 5.0% mC, whereas that from liver and brain contained 4.4% and 2.6% mC, respectively. Thus, the extent of methylation of this DNA is tissue-specific. So is the location. In each tissue, the location of mCs is nonrandom, consisting of three clusters of heavily methylated regions, each of about 200 bases. However, the extent of methylation within each cluster is tissue-specific. The mCs are located entirely in C-G doublets and primarily in palindromic sequences, C-C-G-G sequences (10 methylatable sites) are almost completely methylated in all tissues examined, but T-G-G-A sequences (16 methylated in all tissues examined, but T-G-G-A sequences (16 metylatable sites) are methylated to different extents in each tissue. Neither the tissue specificity of methylation nor the clustering pattern is detectable by examining only G-C-G-G sites, leading us to emphasize the importance of total sequence determination for genomic DNAs in studies of methylation. The clustering pattern, which is preserved despite a 2-fold difference in mC content between brain and thymus, may indicate a role for DNA methylation in chromatin structure.
Cell-free translation of a biologically active, antigen-specific suppressor T cell factor.
Authors: Wieder K J , Araneo B A , Kapp J A , Webb D R
Published: May 2006

In vitro synthesis of an antigen-specific T cell suppressor factor (TsF) has been accomplished by using partially purified poly(A)-containing RNA in a rabbit reticulocyte lysate cell-free translation system. The poly(A)-containing mRNA was isolated from a cloned T cell hybridoma that constitutively produces a TsF specific for the synthetic polypeptide antigen poly-(LGlu60LAla30LTyr10) (GAT). The RNA was fractionated by size and translated in vitro. The 16S RNA fraction stimulated synthesis of a biologically active protein that specifically suppressed both the GAT-specific antibody response by spleen cells in vitro and the proliferation response to GAT by lymph node T cells from GAT-primed mice. Further, the suppressor factor had a binding site for GAT, a determinant encoded by the I subregion of the major histocompatibility complex (MHC), and an apparent Mr 19,000 estimated by functional assays on protein separated by NadodSO4/polyacrylamide gel electrophoresis. These results indicate that virtually no posttranslational modifications (other than proteolytic cleavage) are necessary to obtain biologically active TsF. Hence, the presence of carbohydrate or other chemical groups does not contribute to either the serological properties of GAT-TsF or its biological properties.
Specific recognition of the product of a transferred major histocompatibility complex gene by cytotoxic T lymphocytes.
Authors: Woodward J G , Orn A , Harmon R C , Goodenow R S , Hood L , Frelinger J A
Published: May 2006

Mouse L cells transfected with a genomic clone containing the H-2Ld gene (8-5 cells) were shown to function as targets for H-2Ld-specific cytotoxic T lymphocytes (CTL). The CTL-mediated lysis of 8-5 cells was shown to be H-2Ld specific by the use of (i) CTL with restricted reactivity, (ii) unlabeled target inhibiton, and (iii) monoclonal antibody inhibiton. We also demonstrated that 8-5 cells could function as targets for antibody-plus-complement-mediated cell lysis. Specificity was confirmed by using H-2Ld-specific monoclonal antibodies. These experiments demonstrate that the gene products of a major histocompatibility complex genomic clone can be functionally expressed in a foreign cell and can mediate immunologically specific cellular interactions.
Identification in culture of a class of hemopoietic colony-forming units with extensive capability to self-renew and generate multipotential hemopoietic colonies.
Authors: Nakahata T , Ogawa M
Published: May 2006

Mouse marrow and spleen cells formed colonies consisting of 40-1,000 blast cells after 16 days of incubation in methylcellulose culture in the presence of medium conditioned by pokeweed mitogen-stimulated mouse spleen cells. These colonies could be distinguished from other hemopoietic colonies in situ by the complete absence of signs of terminal differentiation. Replating of these colonies (tentatively named stem cell colonies) revealed their self-renewal capacity and the extensive ability to generate secondary colonies, many of which were multipotential hemopoietic colonies. Some of the colonies revealed 100% replating efficiencies. Analyses of individual stem cells colonies revealed concurrent and high incidences of spleen colony-forming units and the macroscopic granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming units (CFU-GEMM) in culture. Replating comparison between the stem cell colonies and GEMM colonies strongly indicated that the progenitors for the stem cell colonies are higher in the hierarchy of stem cell differentiation than are CFU-GEMM. Quantitation of stem cell colonies provides an assay for the class of primitive hemopoietic progenitors described here.
Purification of polyoma virus medium-size tumor antigen by immunoaffinity chromatography.
Authors: Walter G , Hutchinson M A , Hunter T , Eckhart W
Published: May 2006

We have used antibodies against the synthetic peptide Lys-Arg-Ser-Arg-His-Phe, corresponding to the six COOH-terminal amino acids of the polyoma virus medium tumor (T) antigen, to purify the medium T antigen by affinity chromatography. Release of the medium T antigen from the anti-peptide antibody was achieved under mild conditions by using a large excess of the peptide in an isotonic buffer at neutral pH containing mixed detergents. This procedure yielded a 2,500-fold purification of the medium T antigen in a single step. The protein kinase activity associated with the medium T antigen was also released and was studied in this active state in solution. Sedimentation analysis showed that the bulk of the purified medium T antigen was in a monomeric form (Mr about 42,000) not associated with protein kinase activity. A small fraction of the medium T antigen was found in a rapidly sedimenting form (Mr about 200,000) that possessed protein kinase activity.
Bovine papillomavirus vector that propagates as a plasmid in both mouse and bacterial cells.
Authors: DiMaio D , Treisman R , Maniatis T
Published: May 2006

We report the construction of a bovine papillomavirus (BPV)-derived recombinant plasmid that propagates as an extrachromosomal element in both mouse and bacterial cells. Plasmids composed of a subgenomic transforming fragment of BPV DNA, a deletion derivative of pBR322, and a 7.6-kilobase fragment of DNA from the human beta-globin gene cluster efficiently induce focus formation on mouse C127 cells. BPV-beta-globin hybrids are maintained in the transformed cells as plasmids with a copy number of about 10-30 per cell. Plasmids indistinguishable from the input DNA have been recovered by transformation of bacteria with low molecular weight DNA from transformed mouse cells. The human beta-globin gene linked to BPV DNA is transcribed from its own promoter at a high level in these cells. The expression of BPV-linked cellular genes in conjunction with the ability to shuttle DNA between bacteria and mammalian cells may provide a rapid means of analyzing and recovering genes that confer an identifiable phenotype upon mammalian cells.
Soybean leghemoglobin gene family: normal, pseudo, and truncated genes.
Authors: Brisson N , Verma D P
Published: May 2006

Leghemoglobin (Lb) genes in soybean represent a small family of closely related genes. Three Lb sequences isolated from a genomic library were analyzed at the nucleotide sequence level. A Lb gene present on an 11.5-kilobase (kb) EcoRI genomic fragment spans approximately 1,200 nucleotides and is interrupted at amino acid positions 32 to 33, 68 to 69, and 103 to 104. The intervening sequences, as well as the 5' and 3' flanking regions of this gene, contain the consensus sequences found in other eukaryotic genes. The length of the 5'-untranslated region is 49 bases as determined by nuclease S1 mapping. R-loop analysis of the DNA from the recombinant phage containing the 11.5-kb EcoRI genomic fragment showed that another Lb gene is located 2.5 kb away. The nucleotide sequence of the second gene showed that this gene is incomplete, containing only exons 3 and 4. The deduced amino acid sequence of this gene, although showing 76% homology with the corresponding region of the other Lb gene, is not represented in any of the known Lb proteins. Both genes are oriented in the same direction with respect to the coding strand. Analysis of the sequence present on a second genomic clone containing a 4.2-kb EcoRI fragment revealed a truncated Lb gene showing homology with the last exon and the noncoding region at the 3' end of the two other Lb genes.
Human DNA sequence homologous to the transforming gene (mos) of Moloney murine sarcoma virus.
Authors: Watson R , Oskarsson M , Vande Woude G F
Published: May 2006

We describe the molecular cloning of a 9-kilo-base-pair BamHI fragment from human placental DNA containing a sequence homologous to the transforming gene (v-mos) of Moloney murine sarcoma virus. The DNA sequence of the homologous region of human DNA (termed humos) was resolved and compared to that of the mouse cellular homolog of v-mos (termed mumos) [Van Beveren, C., van Straaten, F., Galleshaw, J.A. & Verma, I.M. (1981) Cell 27, 97-108]. The humos gene contained an open reading frame of 346 codons that was aligned with the equivalent mumos DNA sequence by the introduction of two gaps of 15 and 3 bases into the mumos DNA and a single gap of 9 bases into the humos DNA. The aligned coding sequences were 77% homologous and terminated at equivalent opal codons. The humos open reading frame initiated at an ATG found internally in the mumos coding sequence. The polypeptides predicted from the DNA sequence to be encoded by humos and mumos also were found to be extensively homologous, and 253 of 337 amino acids were shared between the two polypeptides. The first five NH2-terminal and last two COOH-terminal amino acids of the humos gene product were in common with those of mumos. In addition, near the middle of the polypeptide chains, four regions ranging from 19 to 26 consecutive amino acids were conserved. However, we have not been able to transform mouse cells with transfected humos DNA fragments or with hybrid DNA recombinants containing humos and retroviral long terminal repeat (LTR) sequences.
An amplified chromosomal sequence that includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments.
Authors: Heintz N H , Hamlin J L
Published: May 2006

We have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) containing a 500-fold amplification of a 135-kilobase chromosomal DNA sequence. This sequence includes the gene for dihydrofolate reductase (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3). The high copy number of the amplified sequence permits it to be visualized as a distinct series of restriction fragments in genomic digests separated on ethidium bromide-stained agarose gels. Initiation of DNA replication in the amplified sequence was studied by radiolabeling DNA synthesized during the onset of S phase in synchronized CHOC 400 cells. Autoradiography of Southern blots of labeled genomic digests shows that DNA synthesis initiates in a small subset of the EcoRI fragments derived from the amplified units. These early labeled fragments are not synthesized at later times during S phase, when different subsets of fragments are synthesized. Regardless of the drug used to collect cells at the beginning of S phase, the replication pattern observed remains the same. These data suggest that replication of the amplified sequence initiates at specific sites within each repeated unit and proceeds in nonrandom order throughout the remainder of the sequence--i.e., that initiation of DNA synthesis in the chromosomes of mammalian cells is sequence specific.
Effects of electromagnetic stimuli on bone and bone cells
in vitro:
Inhibition of responses to parathyroid hormone by low-energy low-frequency fields
Authors: Luben Richard A. , Cain Christopher D. , Chen Monica Chi-Yun , Rosen David M. , Adey W. Ross
Published: May 2006

Low-energy electromagnetic fields pulsed at frequencies of 10-90 Hz significantly increase healing of chronic fracture nonunions in man. These fields are effective at tissue current levels several orders of magnitude lower than those required for transmembrane depolarization of normal cells. We have examined the effects of two clinically used pulsed electromagnetic fields on cultures of the osteoblast-like mouse bone cell line MMB-1. Both fields significantly reduced cellular production of cAMP in response to parathyroid hormone and osteoclast activating factor. Neither basal nor fluoride-activated levels of adenylate cyclase were altered in membranes from cells cultured in the fields; however, the same membrane preparations exhibited markedly inhibited responses to parathyroid hormone. The fields blocked the inhibitory effects of the hormone on collagen synthesis by MMB-1 cells. However, there was no effect on the inhibition of collagen synthesis by 1,25-dihydroxyvitamin D
3
, which is believed to act primarily by a nuclear, rather than by a membrane-dependent, mechanism. No significant differences were noted between effects of the two fields, one generating continuous pulse trains (72 Hz) and the other generating recurrent bursts (15 Hz) of shorter pulses. We hypothesize that these field effects are mediated primarily at the plasma membrane of osteoblasts, either by interference with hormone-receptor interactions or by blocking of receptor-cyclase coupling in the membrane. These responses occurred with induced extracellular fields of 1 mV/cm or less, even though transmembrane potential gradients are typically 10
5
V/cm.