bluuebunny/crossref_metadata_2025 huggingface dataset:
Human monoclonal IgM with autoantibody activity against intermediate filaments.
Authors: Dellagi K , Brouet J C , Perreau J , Paulin D
Published: May 2006

Monoclonal IgMs from two patients with Waldenström macroglobulinemia were found to react with intermediate filaments. This was shown by (a) immunostaining of various tissues and cultured cells and (b) immunological characterization of the reactive antigen after blotting of polypeptides separated from total cell extracts by gel electrophoresis or purified intermediate filaments on nitrocellulose sheets. One monoclonal IgM had an activity directed only against vimentin, whereas the other reacted with four different classes of intermediate filaments--vimentin, desmin, glial fibrillary protein, and keratins. All of the reactivity of the latter IgM was absorbed by purified vimentin, suggesting that different classes of proteins of intermediate filaments share common antigenic determinant(s). The significance of such autoantibody activity of human monoclonal IgM is discussed in the light of the startling frequency of IgM anti-intermediate filaments antibodies in various diseases.
Simulated and empiric wind pollination patterns of conifer ovulate cones
Authors: Niklas Karl J.
Published: May 2006

Wind tunnel analyses of conifer ovulate cones indicate that the total geometry of the cone enhances the probability of pollen entrapment. Aerodynamic characteristics of cone scale-bract complexes are such that suspended pollen is directed toward the micropyles of attached ovules. Within the taxa examined, there appears to be a preferential entrapment by ovulate cones of pollen of the same species. The data are interpreted as evidence for an aerodynamic reciprocity between wind-suspended pollen and the structure of ovulate cones which increases the frequency of pollination and the potential for fertilization.
Inverted repeat nucleotide sequences in the genomes of Marek disease virus and the herpesvirus of the turkey.
Authors: Cebrian J , Kaschka-Dierich C , Berthelot N , Sheldrick P
Published: May 2006

The DNAs of two herpesvirus, the oncogenic Marek disease virus and the serologically related herpesvirus of the turkey, were studied by electron microscopy. On the basis of fold-back molecules observed in single-stranded DNA from both viruses, structures have been derived from the overall nucleotide sequence arrangement in their genomes. Although differing in molecular weight, the genomes of Marek disease virus and turkey herpesvirus are both constructed according to the same plan--two regions of unique nucleotide sequence, each enclosed by inverted repeat sequence. The genome structure of these viruses therefore closely resembles that of herpes simplex virus rather than the biologically more similar herpesvirus Epstein--Barr virus, H. saimiri, and H. ateles.
In vitro
immune responses to a T cell-dependent antigen by cultures of disassociated murine Peyer's patch
Authors: Kiyono Hiroshi , McGhee Jerry R. , Wannemuehler Michael J. , Frangakis Maria V. , Spalding David M. , Michalek Suzanne M. , Koopman William J.
Published: May 2006

The first line of defense against pathogens that enter the host by the oral route appears to involve the gut-associated lymphoreticular tissue—e.g., Peyer's patches (PP). Although animals can readily be immunized by orally administered antigen that mobilizes the secretory immune system, there is a total lack of local antibody synthesis in the PP and the cellular basis for this deficiency remains a mystery. A lymphoreticular cell population, obtained when murine PP were treated with a neutral protease (Dispase), consisted of accessory cells [macrophages (MΦ)] and T and B lymphocytes.
In vitro
cultures of these PP cell preparations with the thymic-dependent antigen sheep erythrocytes (SRBC) resulted in good anti-SRBC plaque-forming cell (PFC) responses. The time courses of these responses were identical to those seen with spleen cell cultures. Submitogenic concentrations of concanavalin A (Con A) and optimal doses of
N
-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and lipopolysaccharide (LPS) enhanced
in vitro
responses of PP cell cultures to SRBC. PP possess fully functional antigen-presenting MΦ because incubation of optimal proportions of splenic T and B cells with purified populations of PP MΦ supported good
in vitro
immune responses. Murine PP possess all of the necessary elements for an IgA immune response because PP cell cultures derived from mice orally primed with SRBC and immunized with SRBC
in vitro
gave high IgA anti-SRBC PFC responses. All of the adjuvants tested (LPS, MDP, and Con A) enhanced IgA responses in PP cell cultures from orally primed mice; however, Con A induced the greatest enhancement. These results demonstrate that murine PP possess MΦ capable of accessory cell functions for
in vitro
immune responses and that oral priming with antigen induces the precursor T- and B-cell populations necessary for IgA responses, that are potentiated by adjuvants, in PP cell cultures. Thus, murine PP possess the lymphoreticular cells required for antibody responses; however, the tissue architecture likely prevents local responses
in vivo
. The finding that enzymatically dissociated PP contain all of the necessary cellular components for antibody synthesis, whereas the
in vivo
tissue architecture prevents the complex interactions necessary for this response, suggests that the initial inductive events take place
in situ
, and additional cell interactions are required for final differentiation of IgA-synthesizing plasma cells to occur at distant mucosal sites.
Effect of a conjugate of daunomycin and antibodies to rat alpha-fetoprotein on the growth of alpha-fetoprotein-producing tumor cells.
Authors: Tsukada Y , Bischof W K , Hibi N , Hirai H , Hurwitz E , Sela M
Published: May 2006

Daunomycin was covalently attached via a dextran bridge to specific antibodies against rat alpha-fetoprotein produced in a horse. The effect of this conjugate on an alpha-fetoprotein-producing tumor was investigated in terms of cytotoxicity and inhibition or retardation of tumor development. Under the experimental conditions used, the covalent conjugate was by both criteria more efficient than either daunomycin alone or a mixture of daunomycin and specific antibodies or a conjugate of daunomycin with horse immunoglobulin. These results show that the conjugate may be useful as a specific cytotoxic agent against alpha-fetoprotein-producing tumors.
n-Tetradecanoyl is the NH2-terminal blocking group of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle.
Authors: Carr S A , Biemann K , Shoji S , Parmelee D C , Titani K
Published: May 2006

The unusual NH2-terminal blocking group of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein was found to be amide-linked n-tetradecanoic acid by gas chromatographic-, direct chemical ionization-, and fast atom bombardment-mass spectrometry. In addition, fast atom bombardment mass spectrometry revealed the presence of an additional alanine which had been overlooked when the original sequence was determined. The corrected and completed NH2-terminal sequence of the 350-amino acid catalytic subunit is CH3(CH2)12CONH-Gly-Asn-Ala-Ala-Ala-Ala-Lys.
Corticotropin-releasing factor stimulates phospholipid methylation and corticotropin secretion in mouse pituitary tumor cells.
Authors: Hook V Y , Heisler S , Axelrod J
Published: May 2006

The 41-residue synthetic ovine corticotropin-releasing factor (CRF; corticoliberin) has been shown to stimulate release of corticotropin (adrenocorticotropic hormone; ACTH) and beta-endorphin from AtT-20/D16-16 mouse pituitary tumor cells. Phospholipid methylation of phosphatidylethanolamine to phosphatidylcholine with S-adenosylmethionine as methyl donor has been suggested as a possible membrane transduction mechanism for some receptor-induced events. CRF increased phospholipid methylation in pituitary tumor cells at concentrations that also stimulated immunoreactive ACTH secretion, and both processes increased linearly and in parallel with time. The methionine sulfoxide derivative of CRF was less potent than CRF was in stimulating both phospholipid methylation and hormone secretion, and the COOH-terminal free acid analogue of CRF had no effect on either process. CRF-induced increases in phospholipid methylation and ACTH secretion were reduced when cells were treated with the phospholipid methyltransferase inhibitors 3-deazaadenosine and L-homocysteine thiolactone. These CRF-stimulated effects were also blocked by the glucocorticoid dexamethasone. It is suggested that phospholipid methylation may be a CRF receptor-mediated event associated with ACTH release in pituitary tumor cells.
Properties of a cell-free system for synthesis of citrus exocortis viroid
Authors: Flores Ricardo , Semancik J. S.
Published: May 2006

Partially purified nuclei from citrus exocortis viroid (CEV)-infected
Gynura aurantiaca
are able to synthesize linear and circular viroid molecules. Pretreatment of the nuclei with actinomycin D or deoxyribonuclease did not affect viroid synthesis, whereas the synthesis of other cellular RNAs was severely reduced. These observations support the essential role of CEV complementary RNA sequences in viroid replication. However, when α-amanitin was included in this
in vitro
synthesis system, CEV replication was markedly reduced by concentrations of 10 nM or greater. Taken together, these data support the proposition that viroid synthesis is catalyzed by a DNA-dependent RNA polymerase acting on a RNA template.
Normal coordinate analysis of the copper center of azurin and the assignment of its resonance Raman spectrum.
Authors: Thamann T J , Frank P , Willis L J , Loehr T M
Published: May 2006

Normal coordinate analysis that utilizes a general valence force field and the Wilson FG matrix method has been applied to several structural models representing the active site of the blue copper protein, azurin. The models included tetrahedral and square planar CuN2SS', trigonal CuN2S, and trigonal bipyramidal CuN2SS'O structures in which the Ns are imidazole nitrogens of histidines, S is the thiolate sulfur of cysteine, S' is the thioether sulfur of methionine, and O is a peptide carbonyl oxygen. For constant Cu--ligand bond lengths and initial force constants, the force field was refined against the most intense of the observed frequencies (424, 404, 369, and 261 cm-1) in the resonance Raman spectrum of Pseudomonas aeruginosa azurin. The most satisfactory fit between observed and calculated frequencies occurs for tetrahedral and trigonal structures. The calculations provide detailed assignments for the resonance Raman spectrum of azurin and reveal considerable mixing of Cu--S(Cys) and Cu--N(His) vibrational modes. The trigonal model is favored because it is shown that the approximately equal to 260-cm-1 vibration is an invariant feature in the resonance Raman spectra of blue copper proteins, even those lacking a methionine in the vicinity of the copper atom. The present analysis ascribes the high frequencies of the Cu--ligand stretching modes and the resonance enhancement to the coupled nature of their vibrations and the Franck-Condon overlaps with predominant (Cys)S leads to Cu(II) charge transfer bands in the visible region.
Characterization of the Mr difference between secreted murine fourth component of complement and the major plasma form: evidence for carboxyl-terminal cleavage of the alpha chain.
Authors: Karp D R , Shreffler D C , Atkinson J P
Published: May 2006

The alpha-chain of murine fourth component of complement (C4) secreted by cells in vitro and in vivo has a Mr that is larger by approximately equal to 4,000 than that of the alpha-chain of the principal form of C4 in plasma. By using in vivo labeling of C4 with [35S]methionine, C4 was shown to be first synthesized with the higher Mr ("secreted") alpha-chain, which was then quickly processed (t1/2 approximately equal to 1 hr) extracellularly to the mature ("plasma") C4 possessing the lower Mr alpha-chain. Both forms of C4 were functional as assayed by the ability of their alpha-chains to be cleaved by the protease C1, to bind methylamine, and to undergo denaturation-dependent autolysis. When secreted C4 and plasma C4 were activated to C4b, the Mr difference of 4,000 was maintained in the alpha'-chains. The Mr difference was localized to the carboxyl-terminal autolytic fragment of the alpha-chain and was unaffected by the removal of carbohydrate. C4 from resident peritoneal macrophage cultures could be converted to the plasma form by incubation with heparin/plasma. This conversion could be blocked by EDTA or 1,10-phenanthroline. These data suggest that an enzyme, presumably a neutral proteinase present in mouse plasma, cleaves the carboxyl terminus of newly synthesized C4 alpha-chains, thereby creating the major form of C4 in plasma.
Initial synaptic transmission at the growth cone in Xenopus nerve-muscle cultures.
Authors: Kidokoro Y , Yeh E
Published: May 2006

The excellent visibility of cultured cells allows the early events during formation of the neuromuscular junction to be suitably studied. It has been shown in various culture systems that synaptic transmission occurs early after nerve-muscle contact. Early synaptic potentials are small in amplitude and slow in time course reflecting a low acetylcholine receptor density at the site of nerve contact. Acetylcholine receptors accumulate later at the contact region. We have examined initial synaptic transmission at the growth cone-muscle contact in Xenopus nerve-muscle cultures. The approaching growth cone was observed under a phase-contrast microscope while the membrane potential of its target muscle cell was continuously monitored by using an intracellular microelectrode. The innervating neuron was stimulated extracellularly at the cell body. No synaptic potential was evoked when the growth cone was contacting the muscle only at the tip of filopodia. However, as soon as the main portion of the growth cone contacted the muscle membrane, nerve-evoked synaptic potentials were detected after stimulation of the nerve. This immediate appearance of synaptic potentials raises the possibility that acetylcholine could be released at the growth cone even prior to contact with muscle cells. As the area of contact enlarged during the observation period the amplitude of end-plate potentials also increased. Spontaneous synaptic potentials (miniature end-plate potentials) were rarely observed in these early growth cone-muscle contacts. Although there were several inherent difficulties, quantal analysis of the end-plate potentials was attempted by using binomial statistics. This analysis suggests that nerve-evoked transmitter release at the growth cone occurs in a quantal fashion.
Activation and desensitization of Torpedo acetylcholine receptor: evidence for separate binding sites.
Authors: Dunn S M , Raftery M A
Published: May 2006

The acetylcholine receptor from Torpedo californica was labeled by reaction with the fluorescent probe 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenz-2-oxa-1,3-diazole without apparent effect on its in vitro ligand binding and functional properties. Addition of acetylcholine or carbamoylcholine to the labeled-receptor preparations enhanced the fluorescence of the bound probe, and this effect was specific for agonists and inhibited by prior incubation with excess alpha-bungarotoxin. Equilibrium fluorescence titrations gave apparent dissociation constants of 0.86 +/- 0.14 mM for carbamoylcholine and 79 +/- 11 microM for acetylcholine, in good agreement with the dissociation constants measured for the permeability response of the receptor. Stopped-flow experiments showed that the fluorescence change was a single exponential process whose rate increased with ligand concentration, reaching a saturating value for carbamoylcholine of approximately 400 s-1. The equilibrium binding of carbamoylcholine was not significantly affected by prior incubation of the receptor with d-tubocurarine or histrionicotoxin and the dissociation constant was only slightly increased in the presence of lidocaine. These inhibitory ligands do not, therefore, compete directly with agonists for this low-affinity binding site, suggesting that their mode of action may be indirect.
Small nuclear ribonucleoprotein complexes of Drosophila melanogaster.
Authors: Wooley J C , Cone R D , Tartof D , Chung S Y
Published: May 2006

We report here on the isolation and characterization of small nuclear ribonucleoproteins (snRNPs) and corresponding small nuclear RNA (snRNA) species from nuclei of Drosophila melanogaster. Velocity sedimentation in sucrose gradients was used to partially fractionate the RNPs; analysis of fractions so obtained suggests that, in general, one snRNP contains one snRNA. At least 11 species of snRNA are present in Drosophila nuclei; among them we identify a potential mammalian U1 homolog based on sequence homology. Autoimmune antiserum designated anti-Sm from patients with systemic lupus erythematosus recognizes nuclear antigens in Drosophila and precipitates seven species of snRNPs. The antigens in HeLa and Drosophila nuclei recognized by anti-Sm antibodies have been identified and compared. Anti-Sm antibodies at least bind to a 26,000-dalton polypeptide in HeLa extracts and to two polypeptides, one of 18,000 daltons and one of 26,000 daltons, in Drosophila extracts. This suggests that the 26,000-dalton polypeptide is an evolutionarily conserved antigenic component of Drosophila and HeLa snRNPs.
Complete nucleotide sequence of the neuraminidase gene of influenza B virus.
Authors: Shaw M W , Lamb R A , Erickson B W , Briedis D J , Choppin P W
Published: May 2006

The complete nucleotide sequence of the neuraminidase gene of influenza virus B/Lee/40 was derived from a cloned cDNA copy of virion RNA segment 6 and its corresponding mRNA. The RNA segment contains 1,557 virus-specific nucleotides, and the protein encoded by the longest open reading frame has a total of 466 amino acids with a molecular weight of 51,721. As is the case with the influenza A virus neuraminidases, the deduced amino acid sequence of the influenza B protein includes a single hydrophobic region near the amino terminus which would be capable of spanning the lipid bilayer of the viral or cell membrane. There are four potential glycosylation sites in the protein, two of which are near the amino-terminal hydrophobic region. Comparisons of the nucleotide and amino acid sequences with those of influenza A virus neuraminidases revealed seven regions of extensive homology within the central portion of the molecules, including 12 conserved cysteine residues. Five other cysteine residues in the terminal portions were also conserved.
Vitellogenin genes A1 and B1 are linked in the Xenopus laevis genome.
Authors: Wahli W , Germond J E , ten Heggeler B , May F E
Published: May 2006

Genomic clones containing the Xenopus laevis vitellogenin gene B1 have been isolated from DNA libraries and characterized by heteroduplex mapping in the electron microscope, restriction endonuclease analysis, and in vitro transcription in a HeLa whole-cell extract. Sequences from the 3'-flanking region of the previously isolated A1 vitellogenin gene were found in the 5'-flanking region of this B1 gene. Thus, the two genes are linked, with 15.5 kilobase pairs of DNA between them. Their length is about 22 kilobase pairs (A1 gene) and 16.5 kilobase pairs (B1 gene) and they have the following arrangement: 5'-A1 gene-spacer-B1 gene-3'. The analysis of heteroduplexes formed between the two genes revealed several regions of homology. Both genes are in the same orientation and, therefore, are transcribed from the same DNA strand. The possible events by which the vitellogenin gene family arose in Xenopus laevis are discussed.
Location and nucleotide sequence of the gene for the proton-translocating subunit of wheat chloroplast ATP synthase
Authors: Howe C. J. , Auffret A. D. , Doherty A. , Bowman C. M. , Dyer T. A. , Gray J. C.
Published: May 2006

The proton-translocating subunit of wheat chloroplast ATP synthase is encoded by a chloroplast gene that has been accurately mapped and whose nucleotide sequence has been determined. The predicted sequence of 81 amino acids has been confirmed in part by determination of the sequence of the first 40 amino acids from the NH
2
terminus of the protein, and it shows 100% homology with the known amino acid sequence of the spinach protein but no more than 35% homology with the amino acid sequences of bacterial and mitochondrial proteins. The gene shows no deviation from the “universal” genetic code and is not split. A potential ribosome binding site is located 12 nucleotides upstream from the initiation codon, but sequences homologous to prokaryotic promotors and transcription terminators are not apparent.
Polyamine requirement for efficient translation of amber codons in vivo.
Authors: Tabor H , Tabor C W
Published: May 2006

Multiplication of several amber mutants of bacteriophage T7 was decreased in two polyamine-deficient mutants of Escherichia coli K-12 carrying amber suppressors, relative to the multiplication of wild type bacteriophage T7 in the same hosts. In contrast the same T7 amber bacteriophages multiplied well in these strains when supplemented with polyamines. The requirement for polyamines for optimal translation of amber codons in vivo was confirmed by showing that infection of polyamine-depleted E. coli with bacteriophage T7 carrying an amber mutation in gene 1 resulted in an increased accumulation of the amber fragment of the gene 1 protein and a decreased accumulation of the full-length gene 1 protein compared with infection of an amine-supplemented culture. These results indicate that one important function of polyamines in vivo is concerned with protein translation and the protein-synthesizing ribosomal complex.
In vitro synthesis and integration into mitochondria of porin, a major protein of the outer mitochondrial membrane of Saccharomyces cerevisiae.
Authors: Mihara K , Blobel G , Sato R
Published: May 2006

We have isolated an outer mitochondrial membrane (OMM) fraction from baker's yeast. Saccharomyces cerevisiae, that possesses porin activity and contains a major polypeptide of 29,000 daltons. By analogy to similar data for an OMM fraction from rat liver and mung bean [Zalman, L. S., Nikaido, N. & Kagawa, Y. (1980) J. Biol. Chem. 255, 1771-1774], the 29,000-dalton polypeptide of the isolated yeast OMM fraction has been tentatively identified as porin. Evidence to substantiate this identification was provided by the finding that both the porin activity and the 29,000-dalton polypeptide were entirely resistant when the OMM fraction was exposed to trypsin digestion, with the 29,000-dalton polypeptide being virtually the only polypeptide in the OMM fraction to be unaffected by trypsin digestion. There was no protection when trypsin digestion was carried out in the presence of detergent. Using monospecific antibodies, we have shown that yeast porin is apparently not synthesized as a larger precursor in a cell-free translation system. In vitro-synthesized porin could not be integrated into dog pancreas microsomal vesicles or into an isolated OMM fraction from yeast, either co- or posttranslationally. In vitro-synthesized porin, however, could be integrated posttranslationally into whole isolated mitochondria. This membrane specificity suggests that integration does not proceed by unassisted partitioning. The integration of porin into whole mitochondria occurred with fidelity by the criterion of its resistance to trypsin. Moreover, integration was not inhibited in the presence of the protonophore carbonyl cyanide m-chlorophenyl-hydrazone whereas translocation into the mitochondrial matrix of the in vitro-synthesized gamma subunit of F1-ATPase was inhibited.
Complete amino acid sequence of pig kidney fructose-1,6-bisphosphatase.
Authors: Marcus F , Edelstein I , Reardon I , Heinrikson R L
Published: May 2006

The covalent structure of the pig kidney fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) subunit has been determined. Placement of the 335 amino acid residues in the polypeptide chain was based largely on automated Edman degradation of eight purified cyanogen bromide fragments generated from the S-carboxymethylated protein. The determination of the amino acid sequence of the largest cyanogen bromide fragment (154 residues) required additional analysis of subfragments obtained by tryptic cleavage at arginyl residues and by mild acid cleavage of an Asp-Pro peptide bond. Alignment of the cyanogen bromide fragments was accomplished by analysis of a product of limited proteolysis by an endogenous protease and by characterization of the tryptic peptides isolated from S-[14C]carboxymethylated fructose-1,6-bisphosphatase. This sequence information has permitted the identification of several reactive sites of functional and structural significance in pig kidney fructose-1,6-bisphosphatase.
Purification and characterization of a highly purified human factor VIII consisting of a single type of polypeptide chain.
Authors: Fay P J , Chavin S I , Schroeder D , Young F E , Marder V J
Published: May 2006

Human factor VIII was purified 350,000-fold (relative to plasma) from a commercial factor VIII concentrate. The procedure used standard protein separation techniques and was performed in the absence of protease inhibitors. The product has a specific activity of 4,900 units/mg, an activity-to-antigen ratio of 75:1 (unit/unit) and no more than 0.1% von Willebrand protein. Electrophoresis of the reduced protein in a denaturing polyacrylamide gel showed a single major band of Mr 100,000. Procoagulant activity was eluted from a nondenaturing gel after electrophoresis in the region of the single major band. Thrombin converted the Mr 100,000 polypeptide to a polypeptide of Mr 75,000. The procoagulant activity was increased 10-fold by thrombin or factor Xa and was completely inhibited by activated protein C or factor VIII inhibitor plasma. This factor VIII preparation consists of a single high molecular weight polypeptide chain and has the highest specific activity thus far reported for human factor VIII.
DNA primase activity associated with DNA polymerase alpha from Xenopus laevis ovaries.
Authors: Shioda M , Nelson E M , Bayne M L , Benbow R M
Published: May 2006

One of the two forms of DNA polymerase alpha from ovaries of the frog Xenopus laevis catalyzed ribonucleoside triphosphate-dependent DNA synthesis on single-stranded circular fd phage DNA templates. DNA synthesis was dependent on ATP and added template. CTP, GTP, and UTP stimulated DNA synthesis but were not required and could not substitute for ATP. DNA synthesis was not inhibited by alpha-amanitin. Neither poly(dT) nor double-stranded DNA served as template. Analysis of [32P]-dTMP-labeled product by neutral and alkaline agarose gel electrophoresis showed that 0.1- to 1-kilobase DNA fragments (average size of approximately equal to 0.25 kilobase) were synthesized. The fragments were not covalently linked to the template. Either [alpha-32P]NMP, [gamma-32P]ATP, or [gamma-32P]GTP were incorporated also into the product. Analysis of the product after hydrolysis by KOH, alkaline phosphatase, or bacteriophage T4 3' leads to 5' exonuclease showed the presence of a small oligoribonucleotide primer at the 5' end of the newly synthesized DNA. NTP-dependent DNA-synthesizing activity copurified on six columns and cosedimented during glycerol gradient centrifugation with one form of DNA polymerase alpha activity but not with the other form. These results suggest that DNA primase activity is associated with one of the two forms of X. laevis DNA polymerase alpha.
Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.
Authors: Reynafarje B , Alexandre A , Davies P , Lehninger A L
Published: May 2006

The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC 1.9.3.1) of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 in the presence of a protonophore, and (c) a fast-responding membraneless oxygen electrode. The reliability of the rate measurements was first established against the known stoichiometry of the scalar reaction of cytochrome oxidase (2ferrocytochrome c + 2H+ + 1/2O2 leads to 2ferricytochrome c + H2O) in the presence of excess protonophore. With all three methods the directly observed vectorial H+/O ejection ratios in the presence of K+ + valinomycin significantly exceeded 3.0. However, because the rate of backflow of the ejected H+ into the mitoplasts is very high and increases with the increasing delta pH generated across the membrane, there is a very rapid decline in the observed H+/O ratio from the beginning of the reaction. Kinetic analysis of ferrocytochrome c oxidation by the mitoplasts, carried out with a fast-responding membraneless oxygen electrode, showed the reaction to be first order in O2 and allowed accurate extrapolation of the rates of O2 uptake and H+ ejection to zero time. At this point, at which there is zero delta pH across the membrane, the H+/O ejection ratio of the cytochrome oxidase reaction, obtained from the rates at zero time, is close to 4.0.
Initiator proteins for the assembly of the 50S subunit from Escherichia coli ribosomes.
Authors: Nowotny V , Nierhaus K H
Published: May 2006

An rRNA-binding protein that binds to the rRNA independently of other proteins during the course of ribosomal assembly is termed "assembly initiator protein." In spite of the large number of rRNA-binding proteins (more than 17 out of 32 proteins have been identified in the case of the large ribosomal subunit), only a very small number of proteins should actually initiate ribosomal assembly for theoretical reasons. Here we demonstrate that only two of the L proteins derived from the large subunit (50S) function as assembly initiator proteins. Two different techniques are used to identify these initiator proteins: reconstitution experiments with purified proteins and pulse-chase experiments during in vitro assembly. Both methods independently identify L24 and L3 as initiator proteins for the 50S assembly. The existence of two initiator proteins (not just one) resolves an apparent contradiction--namely, that on the one hand, rRNA is synthesized in excess under unfavorable growth conditions, whereas on the other hand, rRNA-binding proteins should be available for translational control.
Evidence for cross-bridge attachment in relaxed muscle at low ionic strength.
Authors: Brenner B , Schoenberg M , Chalovich J M , Greene L E , Eisenberg E
Published: May 2006

In vitro at low ionic strength (mu = 0.02 M) and 5 degrees C, myosin subfragment-1 shows significant binding to regulated actin in the presence of ATP, independent of the concentration of free Ca2+. Under the same conditions, single skinned rabbit psoas muscle fibers develop force only in the presence of Ca2+ and are relaxed in its absence. However, the stiffness, measured with very rapid stretches (0.5% of muscle length in 0.1 ms), is high even when the fibers are relaxed. This "rapid stiffness" of the resting muscle is sensitive to ionic strength, becoming small at normal ionic strength (mu = 0.17 M). At low ionic strength, the rapid stiffness is approximately proportional to the overlap between the actin and myosin filaments. At zero overlap (sarcomere length = 3.8 microns), the stiffness is less than 20% of the value measured at full overlap. This remaining 20% is relatively insensitive to ionic strength, like the passive resting tension, and it may in fact be due to the structures responsible for the resting tension. Thus, both in vitro binding and the effect of overlap on rapid stiffness measurements in fibers suggest that cross-bridges are attached to actin in relaxed muscle at low ionic strength.
Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog.
Authors: Singer R H , Ward D C
Published: May 2006

The chicken muscle tissue culture system has been used for visualizing actin gene expression after in situ hybridization. Cell differentiation is morphologically distinguishable in this system as the myoblasts fuse into myotubes. This differentiation involves the production of large amounts of actin required for myofibrils. The presence of actin mRNA has been observed in cells preserved with ethanol and paraformaldehyde by hybridizing a recombinant plasmid into which a biotinated analog of dUTP was incorporated by nick-translation. The biotin was then detected by using an anti-biotin antibody and a rhodamine-conjugated second antibody. Alternatively, avidin conjugated to rhodamine or avidin complexed to biotinated peroxidase has been used for mRNA detection. The procedure described preserved morphological detail yet is compatible with hybridization conditions and reveals the disposition of actin mRNA during gene expression.
Differentiation of human T-lymphoid leukemia cells into cells that have a suppressor phenotype is induced by phorbol 12-myristate 13-acetate.
Authors: Ryffel B , Henning C B , Huberman E
Published: May 2006

Treatment of cultured human T-lymphoid (CEM) leukemia cells with nanomolar concentrations of phorbol 12-myristate 13-acetate (PMA) resulted in a reduction in cell growth and in the acquisition of a surface antigenic pattern that is common to both suppressor and cytotoxic T lymphocytes. This antigenic pattern was detected by OKT monoclonal antibodies. PMA treatment did not cause the expression of a cytotoxic function but rather induced the expression of a suppressor cell marker. This marker was characterized by the ability of the treated CEM cells to suppress [3H]thymidine incorporation into phytohemagglutinin-activated peripheral blood lymphocytes. After 4 days of treatment of CEM cells from either cloned or the parental cell population with 16 nM PMA, 71-98% of the cells expressed reactivity with OKT3 and OKT8 antibodies whereas reactivity with OKT4 and OKT6 was detected in less than or equal to 1-8% of the cells. The CEM cells can be divided into five groups based on the antigenic patterns of cells from randomly isolated clones. The cells from four of these groups were characterized by either low or high reactivity with each of the four OKT antibodies. The antigenic pattern of the fifth group resembled that of the parent CEM cells. The acquisition of reactivity with the OKT3 antibody in the CEM cells after PMA treatment was dependent on both time and dose and did not require cell replication. Acquisition of reactivity with OKT3 antibody also occurred after treatment with phorbol 12,13-dibutyrate but not after treatment with phorbol 13-monoacetate, phorbol 12,13-diacetate, or dimethyl sulfoxide. These results indicate that treatment of CEM cells with PMA and related agents can cause the cells to express a phenotype that resembles that of a mature suppressor T lymphocyte.
Highly polymorphic DNA site
D14S1
maps to the region of Burkitt lymphoma translocation and is closely linked to the heavy chain γ1 immunoglobulin locus
Authors: Balazs Ivan , Purrello Michele , Rubinstein Pablo , Alhadeff Becky , Siniscalco Marcello
Published: May 2006

Using a phage λ Charon 4A recombinant DNA clone (λCH4A-rHs18) from a human genomic library, Wyman and White detected a multiallelic common polymorphism at an
Eco
RI site (
D14S1
) flanking the DNA region homologous to the probe [Wyman, A. R. & White, R. (1980)
Proc. Natl. Acad. Sci. USA
77, 6754-6758]. Subsequent studies, carried out with the cell hybrid approach and the use of a subclonal derivative (pAW101) from λCH4A-rHs18 have assigned this locus to autosome 14 between 14q21 and 14qter [De Martinville, B., Wyman, A. R., White, R. & Franke, U. (1982)
Am. J. Hum. Gen.
34, 216-226]. The data presented here permit the precise mapping of this locus to the subtelomeric region of autosome 14, below band 14q32, in close proximity to the heavy chain γ1 immunoglobulin locus. These conclusions are supported by three independent lines of evidence, including studies on gene dosage, somatic cell hybrids, and pedigree analysis. Our results are in agreement with the recent assignment of the heavy chain γ1 immunoglobulin locus to band 14q32 [Kirsch, I. R., Morton, C. C., Nakahara, K. & Leder, P. (1982)
Science
216, 301-303] and are consistent with the generally held contention that one unit of meiotic recombination corresponds approximately to one million base pairs. It is to be expected that the location of the highly polymorphic
D14S1
site at a measurable distance from the cluster of the heavy chain genes will provide new opportunities for a genetic approach to the question of the specific gene order within the cluster, its possible individual variation, and its biological significance in normal development and disease. It is worthwhile to point out that such a highly polymorphic DNA sequence is located in the same chromosomal region where so much somatic rearrangement goes on normally (i.e., switch region between classes of heavy chain constant region genes) and which is involved with
de novo
translocations associated with malignancies.
Molecular and cellular pathogenesis of hemoglobin SC disease.
Authors: Bunn H F , Noguchi C T , Hofrichter J , Schechter G P , Schechter A N , Eaton W A
Published: May 2006

Solution and cell studies were performed to ascertain why individuals with hemoglobin (Hb) SC have disease whereas those with Hb AS do not. The polymerization of deoxygenated mixtures containing sickle cell Hb (Hb S; alpha 2 beta 2(6)Glu leads to Val) and Hb C (alpha 2 beta 2(6)Glu leads to Lys) was investigated by measurements of delay times and solubilities. In mixtures containing more than 40% Hb S, polymerization takes place by the same mechanism as in solutions of Hb S alone, with no evidence for independent crystallization of Hb C. A detailed comparison of Hb S/Hb C and Hb S/Hb A mixtures with identical concentrations and proportions of Hb S show that there is no significant difference in the tendency of Hb C and Hb A to copolymerize with Hb S. In 50:50 Hb S/Hb C mixtures, polymerization is about 15 times more rapid than in 40:60 Hb S/Hb A mixtures at the same total Hb concentration. Measurements on density-fractionated erythrocytes show that SC cells contain a higher total Hb concentration and a more uniform distribution of reticulocytes compared to normal (AA) or sickle trait (AS) cells. The concentration distribution for C trait (AC) cells is much closer to that of SC cells than to AS or AA cells. It appears, therefore, that the presence of Hb C results in the SC cell beginning its life with an abnormally high Hb concentration. From these findings we conclude that both the larger proportion of Hb S and the higher intracellular Hb concentration contribute to the pathogenesis of Hb SC disease.
Thyroid hormone regulation of heme oxidation in the liver.
Authors: Smith T J , Drummond G S , Kourides I A , Kappas A
Published: May 2006

The effects of 3,5,3'-triiodothyronine (T3) on heme oxygenase (EC 1.14.99.3) activity and cytochrome P-450 content in liver were examined in thyroidectomized rats. T3, when administered for 5 days at a dose of 6 micrograms/100 g of body weight, stimulated basal heme oxygenase activity approximately equal to 2-fold compared to diluent-treated animals. The induction of heme oxygenase by cobalt heme also was enhanced approximately equal to 3-fold in T3-treated animals. T3 treatment lowered cytochrome P-450 content by approximately equal to 50% and potentiated the depletion of this heme protein after cobalt heme administration. Reverse T3 had no effect either on cytochrome P-450 content or on heme oxygenase activity in liver. The time course of response to a single dose of T3 (50 micrograms/100 g of body weight) revealed that both basal and cobalt heme-induced heme oxygenase activity peaked at 48 hr and that cytochrome P-450 content declined to approximately equal to 40% of controls at 96 hr. Examination of microsomal proteins by polyacrylamide gel electrophoresis after T3 treatment disclosed that major bands in the Mr approximately equal to 50,000-55,000 region were diminished. The administration of T3 together with SKF-525A, a compound known to complex with the heme prosthetic group of cytochrome P-450, resulted in partial preservation of these proteins. These data indicate that thyroid hormone can regulate heme oxygenase activity and concomitantly can lower cytochrome P-450 content in liver. The hormone also can act in a synergistic fashion to enhance the response of hepatic heme oxygenase to a chemical inducer of the enzyme. Thyroid status thus may be a potentially significant determinant of the rate of heme oxidation in the liver.
Combination of rat lutropin subunits occurs early in the secretory pathway.
Authors: Hoshina H , Boime I
Published: May 2006

The combination of lutropin (LH; luteinizing hormone) alpha and beta subunits was examined in rat pituitaries incubated with [35S]methionine or [35S]sulfate. Combination was assessed by using antiserum directed against the beta subunit. The data show that combination of most of the subunits proceeds rapidly, well before the addition of sulfate and prior to the processing of asparagine-linked oligosaccharides to the complex form. Thus, combination appears to initiate in the endoplasmic reticulum and does not require those post-translational modifications. We observed that two forms of the LH-alpha subunit were processed--one that is secreted into the medium not associated with the LH beta subunit and another secreted as part of the alpha-beta dimer. Both forms of the alpha subunit are sulfated, and the data suggest that subsequent to sulfate addition, secretion of free alpha subunit and the dimer occur independently by separate pathways.
Insertion of a repetitive element at the same position in the 5'-flanking regions of two dissimilar yeast tRNA genes.
Authors: Sandmeyer S B , Olson M V
Published: May 2006

The regions 5' proximal to many yeast tRNA genes exhibit a high frequency of DNA sequence polymorphisms. DNA sequence analysis of polymorphic variants of SUQ5, a tRNA Ser UCA gene, and SUP2, a tRNA Tyr gene, shows that in each case one sequence variant of the tRNA gene is 346 base pairs longer than the other. The longer variants appear to have arisen from the shorter ones by the insertion of nearly identical copies of a 341-base pair sigma element into a site 16 base pairs upstream from the 5' ends of the tRNA-coding regions. The sequences of the two copies of the sigma element differ at only five positions. The element has a number of properties that are typical of many transposable elements: (i) there is a perfect eight-base-pair inverted repeat at its ends, (ii) these ends are flanked by a five-base-pair direct repeat of a sequence that occurs only once in the target DNA, (iii) there are approximately 20 copies of the element in the yeast genome, and (iv) there is considerable strain-to-strain variation in the sizes of the restriction fragments on which these copies lie. The presence of the sigma element has no gross effect on the phenotype of a SUP2 ochre suppressor. Analysis of the SUQ5 and SUP2 sequences favors the hypothesis that sigma is a transposable element with a novel type of insertion specificity, which is primarily based on the presence of a tRNA-coding region a fixed distance from the insertion site, rather than on the immediate target sequences.
Effects of small doses of cytochalasins on fibroblasts: preferential changes of active edges and focal contacts.
Authors: Domnina L V , Gelfand V I , Ivanova O Y , Leonova E V , Pletjushkina O Y , Vasiliev J M , Gelfand I M
Published: May 2006

The effects of low doses of cytochalasin B (2 micrograms/ml) and cytochalasin D (0.2 microgram/ml) on the spreading of normal mouse fibroblasts in culture were investigated to find out which components of cell-substrate interactions are most sensitive to alterations of the state of actin cytoskeleton. Cytochalasin B disorganized the cortical layer of actin microfilaments and caused partial or complete disappearance of microfilament bundles; focal contacts with the substrate seen by interference-reflection microscopy also disappeared. Diffuse close contacts were apparently insensitive to cytochalasin B. Low doses of cytochalasin B did not inhibit the outgrowth and maintenance of lamellas at the cell periphery. However, in contrast to controls, these lamellas had no distal zones with convex outer edges and ruffles at the upper surfaces. The disappearance of these ruffling active edges was accompanied by loss of the ability to clear the surface of the lamellas from the concanavalin A receptors crosslinked by the corresponding ligand. The effects of cytochalasin D were similar to those of cytochalasin B. Thus, ruffling active edges and focal contacts can be regarded as specialized parts of lamellas with increased sensitivity to cytochalasins; the presence of ruffling active edges is essential for the initiation of centripetal movement of the patches of crosslinked surface receptors.
Selective release of glutamate from cerebellar granule cells differentiating in culture
Authors: Gallo V. , Ciotti M. T. , Coletti A. , Aloisi F. , Levi G.
Published: May 2006

The aim of the present study was to assess whether endogenous and newly synthesized glutamate can be released from differentiating cultured cerebellar granule cells in a way compatible with a neurotransmitter role. Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calf serum for 2-12 days
in vitro
(DIV), then washed with Krebs-Ringer medium, and labeled for 45 min with tracer amounts of radioactive glutamine. Subsequently, the release of endogenous glutamate and of newly formed radioactive glutamate was measured in basal conditions and upon depolarization with elevated K
+
concentration or veratridine. At 2 DIV, the release of endogenous and newly synthesized glutamate evoked by high K
+
concentration was small and Ca
2+
independent, but it progressively and steadily increased (up to 8- to 10-fold) and became Ca
2+
dependent (up to 80-85%) at later stages (4, 8, and 12 DIV). Veratridine was almost ineffective with cells at 2 DIV but greatly increased glutamate release (endogenous and neosynthesized) at 8 DIV, and its action was totally antagonized by tetrodotoxin. The level and synthesis of glutamate remained fairly constant in cells from 2 to 12 DIV. γ-Aminobutyric acid synthesis from radioactive glutamine was about 3% of that of glutamate, and γ-aminobutyric acid release (endogenous and neosynthesized) was not measurable. Aspartate synthesis was about 10% of that of glutamate, and the high K
+
concentration-evoked release of this amino acid was modest and scarcely affected by Ca
2+
. Neither high K
+
concentration nor veratridine was able to induce glutamate release from confluent cerebellar astrocyte cultures at 14 DIV, although the level and synthesis of the amino acid were comparable to those in granule cells. In conclusion, the data show that a stimulus-coupled release of endogenous and neosynthesized glutamate is progressively expressed by cerebellar granule cells differentiating in culture, and this strongly supports the concept that glutamate is the neurotransmitter of these cells.