bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of protein kinase C to neutrophil membranes in the presence of Ca2+ and its activation by a Ca2+-requiring proteinase.
Authors: Melloni E , Pontremoli S , Michetti M , Sacco O , Sparatore B , Salamino F , Horecker B L
Published: May 2006

In the presence of micromolar concentrations of Ca2+, both protein kinase C and a cytosolic Ca2+-requiring neutral proteinase of human neutrophils become associated with the neutrophil membrane. Binding to the membrane results in activation of the proteinase, which then catalyzes limited proteolysis of the kinase to produce a form that is fully active in the absence of Ca2+ and phospholipid. This irreversibly activated protein kinase is released from the membrane and may thus have access, in the intact cell, to intracellular protein substrates. In the absence of the proteinase, Ca2+ promotes the binding of protein kinase C, but conversion to the Ca2+/phospholipid-independent form does not occur and the kinase remains associated with the membrane fraction.
Gene expression of pigment-binding proteins of the bacterial photosynthetic apparatus: Transcription and assembly in the membrane of
Rhodopseudomonas capsulata
Authors: Klug Gabriele , Kaufmann Norbert , Drews Gerhart
Published: May 2006

Lowering of oxygen partial pressure in chemotrophic cultures or reduction of light intensity in phototrophic cultures of
Rhodopseudomonas capsulata
induced formation of the photosynthetic apparatus. A maximum of mRNA coding for the reaction center (RC) and the light-harvesting 1 B870 antenna complex polypeptides occurred 30 min after induction. Maximal expression of mRNA for B800-B850 antenna proteins appeared with a lag time of about 25 min after RC/B870 mRNA. Pigment-binding polypeptides were inserted into the membrane immediately after mRNA synthesis. It is concluded that the delayed formation of the B800-B850 complex compared to the RC and the B870 complex is caused by sequential expression of the corresponding genes. Biological activity of pigment-protein complexes increased after the incorporation of their polypeptides parallel to the maximum of bacteriochlorophyll synthesis. Studies on mutant strains defective in the formation of pigment-protein complexes suggested that pigment synthesis is of importance for assembly of stable complexes.
Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF.
Authors: Esch F , Baird A , Ling N , Ueno N , Hill F , Denoroy L , Klepper R , Gospodarowicz D , Böhlen P , Guillemin R
Published: May 2006

The two major mitogenic polypeptides for endothelial cells have been purified to homogeneity. The complete primary structure of bovine pituitary basic fibroblast growth factor (FGF) and the amino-terminal amino acid sequence of bovine brain acidic FGF have been established by gas-phase sequence analyses. Homogeneous preparations of these polypeptides are potent mitogens (basic FGF, ED50 approximately equal to 60 pg/ml; acidic FGF ED50 approximately equal to 6000 pg/ml) for many diverse cell types including capillary endothelial cells, vascular smooth muscle cells, and adrenocortical and granulosa cells; in vivo, basic FGF is a powerful angiogenic agent in the chick chorioallantoic membrane assay. The available protein sequence data demonstrate the existence of significant structural homology between the two polypeptides.
The oncofetal domain of fibronectin defined by monoclonal antibody FDC-6: its presence in fibronectins from fetal and tumor tissues and its absence in those from normal adult tissues and plasma.
Authors: Matsuura H , Hakomori S
Published: May 2006

An IgG1 monoclonal antibody, FDC-6, was established, which defines a unique fibronectin (FN) domain, located between the "Hep-2" and the "Fib-2" domains, in the COOH-terminal region of FNs isolated from hepatoma, sarcoma, and fetal fibroblasts. A systematic study with this antibody indicates the presence of two classes of human FNs. (i) FN from fetal connective tissue, placenta, amniotic fluid, hepatoma, and colon carcinoma as well as cell lines from fetal tissues (WI-38), hepatomas (HuH-6 and HuH-7), and sarcoma (VA13) was characterized by the presence of the FDC-6-defined domain and by a high molecular weight (subunit Mr, 310,000-335,000). (ii) In contrast, FN from normal adult tissues and plasma was characterized by a lower molecular weight (subunit Mr, 285,000-295,000) and lack of reactivity with FDC-6 and is therefore devoid of the FDC-6-defined domain. The FDC-6-defined domain is therefore called the "oncofetal" domain, and FN containing this domain is hereby called "oncofetal FN" (onf FN). The onf FN is similar to the previously known "cellular-form" FN. FN from normal adult tissues and plasma, lacking the oncofetal domain, is hereby called "normal FN" (nor FN). The nor FN is similar to the previously known "plasma-form" FN. Development of FN from fetal to adult form is associated with loss of the oncofetal domain defined by the FDC-6 antibody, and oncogenic transformation is associated with activation in synthesis of the oncofetal domain defined by the FDC-6 antibody.
Do agonists promote rapid internalization of beta-adrenergic receptors?
Authors: Mahan L C , Motulsky H J , Insel P A
Published: May 2006

We used elution of radioligands at low pH to quantitate intracellular beta-adrenergic receptors on intact S49 lymphoma cells. We validated this method with respect to cell viability, beta-adrenergic receptor integrity, and transferrin receptors on these cells. On control cells, about 15% of the radiolabeled beta-adrenergic antagonists [3H]dihydroalprenolol and [125I]iodocyanopindolol specifically bound at 37 degrees C could not be eluted at low pH; these binding sites appear to be intracellular receptors that are inaccessible to the surface-restricted antagonist [3H]CGP-12177 [tritiated (+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride]. Incubation of cells with the agonist isoproterenol at 37 degrees C for 15 min did not change the number of [3H]dihydroalprenolol binding sites but reduced [3H]CGP-12177 binding sites by 50% or more. However, all specifically bound [3H]CGP-12177 and [3H]dihydroalprenolol were eluted by acid. In addition, the number of acid-elution-resistant [125I]iodocyanopindolol binding sites was not increased in cells coincubated with 1 microM isoproterenol and [125I]iodocyanopindolol for 15 min at 37 degrees C, even though those sites show a loss in apparent affinity for isoproterenol of about 2 orders of magnitude, a loss previously attributed to internalization. We conclude that the early phase of agonist-mediated desensitization of beta-adrenergic receptors in S49 cells does not coincide with the movement of receptors to intracellular sites; instead, agonist-modified receptors remain in association with the plasma membrane and are accessible to the extracellular environment. These "redistributed" receptors together with "cell-surface" and "intracellular" receptors represent three classes of beta-adrenergic receptors that can be selectively identified in intact target cells.
Analyzing the components of microtubules: antibodies against chartins, associated proteins from cultured cells.
Authors: Magendantz M , Solomon F
Published: May 2006

In previous work, we have identified cytoplasmic microtubule-associated proteins by isolating the microtubule organelles of several different cultured cells. Among those proteins are the chartins, a family of polypeptides with related sequence but of varying molecular weight and isoelectric point. Biochemical analyses of the distribution of the chartins in the cytoplasm, and among cells with different functions, suggest that they may regulate microtubule structure in vivo. We describe here the preparation and application of antibodies to chartins. These antibodies enable us to demonstrate that the chartins colocalize with assembled tubulin in the cytoplasm, as assessed by immunofluorescence, so that they fulfill a major criterion that has been applied to other putative microtubule components. The results also demonstrate that the tau proteins, which fractionate and copurify with chartins, are in fact clearly distinguishable from them. The implications of these results for evaluating microtubule composition are discussed.
Processing of an anglerfish somatostatin precursor to a hydroxylysine-containing somatostatin 28.
Authors: Spiess J , Noe B D
Published: May 2006

A novel 28-residue somatostatin (SS) has been isolated from anglerfish pancreatic islets and characterized by complete Edman degradation, peptide mapping, and amino acid analysis. The primary structure of this anglerfish SS-28 (aSS-28) containing hydroxylysine (Hyl) was established to be H-Ser-Val-Asp-Ser-Thr-Asn-Asn-Leu-Pro-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys- Lys-Asn-Phe-Tyr-Trp-Hyl-Gly-Phe-Thr-Ser-Cys-OH. This sequence (with the exception of hydroxylysine-23, which is replaced by lysine) is identical to the sequence of the COOH-terminal 28 residues of prepro-SS II predicted on the basis of cDNA analysis [Hobart, P., Crawford, R., Shen, L., Pictet, R. & Rutter, W. J. (1980) Nature (London) 288, 137-141]. This is the first instance in which hydroxylysine (to date characteristically observed in collagen or collagen-like structures) has been found in a potential regulatory peptide. Chromatographic characterization of peptides, radiolabeled in islet culture, revealed that aSS-28 contained 10-12% of the radioactivity incorporated into the 8000- to 1000-dalton SS-like polypeptides, whereas 88-90% of this radioactivity was detected in anglerfish SS-14. It appears probable that aSS-28 represents the predominant primary cleavage product derived from prepro-SS II by cleavage at the COOH-terminal side of a single arginine. Based on knowledge of the collagen biosynthesis, it is speculated that hydroxylation may take place as an early post-translational event.
Covalent enzyme-RNA complex: a tRNA modification that prevents a covalent enzyme interaction also prevents aminoacylation.
Authors: Starzyk R , Schoemaker H , Schimmel P
Published: May 2006

Previous work indicates that aminoacyl-tRNA synthetases make a transient covalent adduct with cognate tRNAs, through Michael addition of an enzyme nucleophile to the carbon-6 position of uridine 8. We report the selective reduction of the 5,6 double bond of 4-thiouridine at position 8 in Escherichia coli tyrosine tRNA, so as to prevent formation of the presumed covalent enzyme-nucleic acid adduct. The completely reduced tRNA molecules are inactivated for aminoacylation. With partial reduction, a mixed pool of active and inactive molecules is created and the degree of inactivation exactly matches the extent of 4-thiouridine reduction. The active molecules recovered from this mixed pool are specifically unaltered at position 8. The results are consistent with the view that the covalent enzyme-RNA adduct is an obligatory intermediate for aminoacylation of this tRNA.
Primary structure of rat thymus prothymosin alpha.
Authors: Haritos A A , Blacher R , Stein S , Caldarella J , Horecker B L
Published: May 2006

The primary structure of prothymosin alpha from rat thymus, containing 113 amino acid residues, is reported as follows: (formula; see text) The sequence of the first 28 amino acids at the NH2 terminus is identical to that of calf thymosin alpha 1. The dicarboxylic amino acids, which account for nearly half of the total residues in prothymosin alpha, are largely clustered in the central portion of the polypeptide chain. The polypeptide contains no aromatic or sulfur-containing amino acids. A computer analysis of the three-dimensional structure based on the primary sequence suggests that the molecule is composed of at least five alpha-helical regions interrupted by one short extended chain and three short random coils.
Isolation and analysis of the 21q+ chromosome in the acute myelogenous leukemia 8;21 translocation: evidence that c-mos is not translocated.
Authors: Drabkin H A , Diaz M , Bradley C M , Le Beau M M , Rowley J D , Patterson D
Published: May 2006

Acute myelogenous leukemia (AML), subgroup M2, is associated with a nonrandom chromosomal translocation, t(8;21)(q22,q22). The oncogene c-mos also has been localized to the q22 band on chromosome 8. There is also evidence that genes on chromosome 21 may be important in the development of leukemia. To determine whether the c-mos oncogene has been translocated in AML-M2 with this translocation and to isolate DNA sequences and genes from these two chromosomes that may be important in malignancy, we constructed somatic cell hybrids between a Chinese hamster ovary cell (CHO) mutant defective in glycine metabolism and myeloblasts with an 8;21 translocation from a patient with AML. We isolated the 21q+ chromosome of this translocation in a somatic cell hybrid and showed that the c-mos oncogene had not been translocated to chromosome 21, ruling out the possibility that translocation of c-mos to chromosome 21 is necessary for development of AML-M2. In addition, there was no detectable rearrangement of the c-mos locus within a 12.4-kilobase region surrounding the gene, indicating that rearrangement of the coding region of the gene itself or alteration of proximal 5' or 3' flanking sequences is not involved. We used this hybrid to determine whether specific DNA sequences and biochemical markers from chromosomes 8 and 21 had been translocated in this case.
Chromosomal locations of the human and mouse genes for precursors of epidermal growth factor and the beta subunit of nerve growth factor.
Authors: Zabel B U , Eddy R L , Lalley P A , Scott J , Bell G I , Shows T B
Published: May 2006

DNA probes for pre-pro-epidermal growth factor (EGF) and the precursor of the beta subunit of nerve growth factor (NGF) were used to chromosomally map human and mouse EGF and NGF genes in panels of human-mouse and mouse-Chinese hamster somatic cell hybrids. The EGF and NGF genes were mapped to human chromosomes 4 and 1, respectively, by using human-mouse cell hybrids. A combination of regional mapping using a chromosome 1 translocation and comparative gene mapping suggests that the human NGF gene is in the p21-p22.1 region of chromosome 1. In mouse-Chinese hamster cell hybrids, both genes were assigned to mouse chromosome 3. A knowledge of the chromosomal assignment of these genes should help in our understanding of their regulation and role in development and disease.
Adrenergic regulation of gluconeogenesis: possible involvement of two mechanisms of signal transduction in alpha 1-adrenergic action.
Authors: García-Sáinz J A , Hernández-Sotomayor S M
Published: May 2006

We have previously suggested that the effects of alpha 1-adrenergic agents on hepatocyte metabolism involve two mechanisms: (i) a calcium-independent insulin-sensitive process that is modulated by glucocorticoids and (ii) a calcium-dependent insulin-insensitive process that is modulated by thyroid hormones. We have studied the effect of epinephrine (plus propranolol) on gluconeogenesis from lactate and dihydroxyacetone. It was observed that the adrenergic stimulation of gluconeogenesis from lactate seemed to occur through both mechanisms, whereas when the substrate was dihydroxyacetone the action took place exclusively through the calcium-independent insulin-sensitive process. This effect was absent in hepatocytes from adrenalectomized rats, suggesting that it is modulated by glucocorticoids.
Argininosuccinate synthetase: essential role of cysteine and arginine residues in relation to structure and mechanism of ATP activation.
Authors: Kumar S , Lennane J , Ratner S
Published: May 2006

We have undertaken studies to identify amino acid residues that are involved in the catalytic mechanism of argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5] and have found that a cysteine residue and an arginine residue are required for activity. The reactive cysteine residues are accessible to solvent and available to react with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Four cysteine residues, one per subunit, are shown by enzymatic assay to be required for catalytic activity, suggesting that a reactive cysteine lies within the active site of argininosuccinate synthetase. In the presence of sodium dodecyl sulfate, 12 cysteine residues react with DTNB; consequently, all of the half-cystine residues in the native enzyme are present in the reduced sulfhydryl form. We also present evidence for the participation of arginine groups in the binding of ATP and PPi. Modification of argininosuccinate synthetase with [14C]-phenylglyoxal results in incorporation concomitant with loss of catalytic activity of 4 mol of phenylglyoxal per mol of native enzyme (one arginine per active site). ATP and PPi protect the enzyme from phenylglyoxal incorporation. Based on these results, we propose that the essential arginine in the active site participates in the binding of ATP and PPi. The binding of ATP and PPi at the same site is mutually exclusive; this exclusion is in accord with the finding that argininosuccinate synthetase has one reactive arginine residue per active site per subunit. This is consistent with our previously proposed reaction mechanism.
Cloning and characterization of a nonmuscle myosin heavy chain cDNA.
Authors: DeLozanne A , Lewis M , Spudich J A , Leinwand L A
Published: May 2006

Despite many biochemical and structural similarities between muscle and nonmuscle myosins, their genes appear to have completely diverged, since muscle myosin molecular clones will not hybridize to RNA from nonmuscle sources. Here we report the isolation and characterization of a partial myosin heavy chain (MHC) cDNA clone from the slime mold Dictyostelium discoideum. We have isolated this clone from a lambda gt11 expression cDNA library by antibody screening. In contrast to the highly conserved sarcomeric muscle MHC multigene families in other organisms, there appears to be only one gene encoding MHC in the Dictyostelium genome. The cloned portion of this gene does not hybridize to the genomic DNAs of other eukaryotic organisms. Analysis of the predicted amino acid sequence of the partial Dictyostelium MHC clone shows that while there is no sequence homology to known striated muscle MHCs, the structure- and coiled-coil-forming capacities have been conserved.
Multiple chemical forms of hepatitis B surface antigen produced in yeast.
Authors: Wampler D E , Lehman E D , Boger J , McAleer W J , Scolnick E M
Published: May 2006

Hepatitis B surface antigen (HBsAg) has been extracted from yeast cells that produce HBsAg. These cells contain the gene for surface antigen carried on a plasmid that replicates in the cells. Analysis of the yeast-derived HBsAg by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis shows that the antigen that is initially released from yeast cells is a high molecular weight aggregate of the fundamental Mr 25,000 subunit. Unlike HBsAg derived from human plasma, the yeast antigen is held together by noncovalent interactions and can be dissociated in 2% NaDodSO4 without the use of reducing agents. During in vitro purification of the yeast antigen, some disulfide bonds form spontaneously between the antigen subunits, resulting in a particle composed of a mixture of monomers and disulfide-bonded dimers. Treatment with 3 M thiocyanate converts the 20-nm particles into a fully disulfide-bonded form that is not disrupted in NaDodSO4 unless a reducing agent is added. This disulfide-bonded particle resembles the naturally occurring, plasma-derived surface antigen particle, and the in vitro formed particle has been used to prepare a vaccine for humans against hepatitis B virus infection.
Interleukin 2-dependent release of interleukin 3 activity by T4+ human T-cell clones.
Authors: Ythier A A , Abbud-Filho M , Williams J M , Loertscher R , Schuster M W , Nowill A , Hansen J A , Maltezos D , Strom T B
Published: May 2006

We have isolated and studied two alloreactive, T4+, human lymphocyte clones that release interleukin 2 (IL-2) and interleukin 3 (IL-3) bioactivities upon stimulation with IL-2, alloantigen, or Sepharose-conjugated antibodies directed against the T3 protein. Anti-IL-2 receptor monoclonal antibodies block IL-2-, alloantigen-, or anti-T3-stimulated IL-3 release. Hence, release of IL-3 activity in each circumstance is rigorously dependent upon activation of the IL-2 receptor. Even low, nonmitogenic concentrations of recombinant IL-2 stimulated IL-3 release.
Differentiating effects of murine nerve growth factor in the peripheral and central nervous systems of Xenopus laevis tadpoles.
Authors: Levi-Montalcini R , Aloe L
Published: May 2006

The present investigation was directed toward exploration of the spectrum of action of murine nerve growth factor (m-NGF) in peripheral cells and neurons in the central nervous system (CNS) of Xenopus laevis tadpoles. It was found that systemic m-NGF injections elicit growth and differentiative effects not only on sensory and sympathetic nerve cells but also on several populations in the CNS. The finding that aminergic and peptidergic neurons in brain centers are highly receptive to m-NGF activity provides evidence for the broad spectrum of action of this molecule in lower vertebrates and calls for a systematic search for these and other putative target cells in the CNS of higher vertebrates.
Identification of Yb-glutathione-S-transferase as a major rat liver protein labeled with dexamethasone 21-methanesulfonate.
Authors: Homma H , Listowsky I
Published: May 2006

Dexamethasone 21-methanesulfonate, an affinity label for glucocorticoid-binding proteins, was incubated with rat liver cytosol preparations. The predominant covalently labeled component was identified as Yb-glutathione-S-transferase on the basis of chromatographic properties, electrophoretic mobility, and specific retention by an anti-Yb-immunoadsorbent. Affinity labeling of this protein was blocked by excess dexamethasone. Preferential reactivity of dexamethasone 21-methanesulfonate with the Yb subclass of glutathione-S-transferase (glutathione transferase, EC 2.5.1.18) was also evident with mixtures containing the multiple forms of the enzyme. Yb-glutathione-S-transferase, the nonsaturable glucocorticoid-binding component of rat liver cytosol should, therefore, be reclassified; because of its high concentration and selective interaction with steroids, this enzyme may be an intracellular glucocorticoid-binding protein and, thereby, influence transport, metabolism, and action of the steroids.
Oxygen binding properties of human mutant hemoglobins synthesized in Escherichia coli.
Authors: Nagai K , Perutz M F , Poyart C
Published: May 2006

Human beta-globin was synthesized in Escherichia coli as a cleavable fusion protein, using the expression vector pLcIIFX beta-globin [Nagai, K. & Thøgersen, H. C. (1984) Nature (London) 309, 810-812]. The fusion protein cIIFX beta-globin was purified to homogeneity and cleaved at the junction by blood coagulation factor Xa; the authentic beta-globin was liberated. Beta-globin was folded in vitro and reconstituted with heme and alpha subunits to form alpha 2 beta 2 tetramers. The oxygen binding properties of reconstituted Hb are essentially the same as those of human native Hb. Two mutant Hbs (Hb Nymphéas [Cys-93 beta----Ser] and Hb Daphne [Cys-93 beta----Ser, His-143 beta----Arg]) were constructed by site-directed mutagenesis using synthetic oligonucleotides. Hb Nymphéas showed a slightly increased oxygen affinity and diminished cooperativity with normal 2,3-diphosphoglyceric acid and slightly reduced alkaline Bohr effects. Hb Daphne showed low cooperativity with high oxygen affinity. The alkaline Bohr effect was slightly reduced but the diphosphoglycerate effect was enhanced by 50% by the His-143 beta----Arg mutation. As arginine is fully charged at physiological pH and has a long flexible side chain, diphosphoglycerate binds more strongly to Hb Daphne.
Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells: fusion-mediated microinjection of ricin A and simian virus 40 DNA.
Authors: Gitman A G , Graessmann A , Loyter A
Published: May 2006

Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA.
Isolation and characterization of nodulation genes from
Bradyrhizobium
sp. (
Vigna
) strain IRc 78
Authors: Noti John D. , Dudas Brigitta , Szalay Aladar A.
Published: May 2006

An 11.76-kilobase-pair (kb) segment of DNA from
Bradyrhizobium
sp. (
Vigna
) strain IRc 78 that hybridizes to nodulation genes of
Rhizobium meliloti
strain 41 was isolated. Hybridization of the 11.76-kb DNA fragment to DNA from other
Bradyrhizobium
species revealed a high degree of sequence conservation in this region. Transfer of the 11.76-kb segment to nodulation-defective (Nod
-
) mutants of
R. meliloti
restored their ability to induce nodules on
Medicago sativa
(alfalfa). Mutants of strain IRc 78 generated by Tn
5
mutagenesis of the 11.76-kb segment fell into three classes according to their symbiotic reaction with
Vigna unguiculata
(cowpea). Class I mutants of strain IRc 78 were unable to induce root-hair curling or to nodulate; class II induced small, ineffective nodules; and class III showed delayed and decreased nodulation with reduction in amount of nitrogen fixed. Furthermore, in contrast to the wild-type strain, class I mutants could not induce nodules on
Glycine max
(soybean),
Cajanus cajan
(pigeon pea), or
Arachis hypogaea
(peanut). This finding suggests a common function of the 11.76-kb region in the infection of host plants by
Bradyrhizobium
either through root hairs or by “crack entry.”
Blockade of beta 1- but not of beta 2-adrenergic receptors replicates propranolol's suppression of the cerebral spread of an engram in mice.
Authors: Flexner J B , Flexner L B , Church A C , Rainbow T C , Brunswick D J
Published: May 2006

Bitemporal injections of puromycin that primarily affect the hippocampal-entorhinal area induce amnesia of aversive maze-learning in mice for 3 days after training but are ineffective 6 or more days after training. At these later times, additional puromycin sites covering widespread forebrain areas are necessary to induce amnesia, a result that we attribute to the cerebral spread of the engram during the 6-day period. We have reported that blockade of about 60% of cerebral beta-adrenergic receptors by a single, subcutaneous injection of (-)-propranolol, a nonselective beta-receptor antagonist, inhibited engram spread for 60-90 days, at which time engram spread spontaneously occurred. In the present experiments using single doses of antagonists that appeared to block 60% of beta 2- or beta 1-adrenergic receptors, it was found that the selective beta 2 antagonist ICI 118,551 was without effect on engram spread, whereas the selective beta 1 antagonist betaxolol inhibited the spread for at least 3 months. Propranolol's effect consequently appears to be accounted for by its blockade of beta 1 receptors.
Isolation and partial characterization of a pair of prolactins released in vitro by the pituitary of a cichlid fish, Oreochromis mossambicus.
Authors: Specker J L , King D S , Nishioka R S , Shirahata K , Yamaguchi K , Bern H A
Published: May 2006

The pituitary of the cichlid fish tilapia secretes two prolactins (PRLs) of molecular masses 20 kDa and 24 kDa. The 20-kDa PRL has an isoelectric point in the range of those of mammalian PRLs (pI 6.7), but the 24-kDa PRL is unusually basic (pI 8.7). Partial sequence information indicates that the PRLs are homologous but distinct proteins, differing by five amino acids within the first 29 NH2-terminal residues. Homology in the known region is higher with chum salmon PRL than with known mammalian PRLs. Reversed-phase HPLC permits isolation of these two PRLs and a single tilapia growth hormone from culture medium or from the pituitary in a single step. HPLC and radio-HPLC analysis of [3H]leucine pulse-chase experiments reveal that each PRL is secreted in vitro at remarkably high rates (21 pmol per gland per hr) and that the two PRLs are released in approximately equimolar amounts, suggesting the coordinate regulation of the secretion. Both PRLs exert characteristic PRL activity in that they prevent the loss of Na+ from the plasma of hypophysectomized tilapia in fresh water.
Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor.
Authors: Hirschman J , Wong P K , Sei K , Keener J , Kustu S
Published: May 2006

In enteric bacteria the products of two nitrogen regulatory genes, ntrA and ntrC, activate transcription of glnA, the structural gene encoding glutamine synthetase, both in vivo and in vitro. The ntrC product (gpntrC) is a DNA-binding protein, which binds to five sites in the glnA promoter-regulatory region and appears to activate transcription initiation. Using as an assay the stimulation of glnA transcription in a coupled in vitro transcription-translation system, we have partially purified the ntrA gene product (gpntrA). The following evidence is consistent with the view that gpntrA is a sigma subunit for RNA polymerase: (i) The gpntrA activity copurifies with the sigma 70 holoenzyme (E sigma 70) and core (E) forms of RNA polymerase through several steps but can be separated from them by chromatography on heparin agarose. (ii) After further purification by molecular sieve chromatography, the partially purified gpntrA fraction allows transcription of glnA from the same startpoint used in vivo; transcription is dependent on gpntrC and on added E. The gpntrA fraction does not allow transcription from promoters that we have used as controls, including lacUV5. E sigma 70 has the reverse specificity.
Expression in Escherichia coli of a cloned DNA sequence encoding the pre-S2 region of hepatitis B virus.
Authors: Offensperger W , Wahl S , Neurath A R , Price P , Strick N , Kent S B , Christman J K , Acs G
Published: May 2006

A DNA sequence encoding the entire pre-S2 region (amino acids 120-174; serotype ayw) of human hepatitis B virus envelope protein has been inserted into the lacZ gene of the plasmid pSKS105 yielding a recombinant, pWS3. Lac+ colonies of the Escherichia coli M182 delta (lacIOPZYA), isolated after transformation with pWS3, produced a pre-S2 peptide-beta-galactosidase fusion protein. This fusion protein, which comprised as much as 3% of the total bacterial protein, was purified to greater than 90% homogeneity by affinity chromatography on p-aminophenyl-beta-D-thiogalactoside-Sepharose. It is immunoprecipitable with rabbit antibodies to a synthetic peptide corresponding to amino acids 120-145 of the pre-S2 region of serotype adw [pre-S(120-145)] or with antibodies to hepatitis B virus. pre-S(120-145) completely blocked the binding of either antibody to the pre-S2 peptide-beta-galactosidase fusion protein. These results indicate that there are antigenic determinants on the fusion protein that are closely related to, if not identical to, determinants on synthetic pre-S(120-145) and on pre-S2 sequences of native hepatitis B virus particles. Thus, bacteria transformed with pWS3 can provide an abundant source of pre-S2-beta-galactosidase fusion protein, which may prove useful either as a diagnostic reagent possessing marker enzyme activity suitable for ELISA tests or as an immunogen with potential to contribute to active prophylaxis of hepatitis B.
Studies on the mechanism of oxidative phosphorylation: effects of specific F0 modifiers on ligand-induced conformation changes of F1.
Authors: Matsuno-Yagi A , Yagi T , Hatefi Y
Published: May 2006

Aurovertin is a fluorescent antibiotic that binds to the catalytic beta subunits of the mitochondrial F1-ATPase and inhibits ATP synthesis and hydrolysis. ATP, ADP, and membrane energization in submitochondrial particles (SMP) alter the fluorescence of F1-bound aurovertin. These fluorescence changes are considered to be in response to the conformation changes of F1-ATPase. This paper shows that the ATP-induced fluorescence change of aurovertin bound to SMP or complex V (purified ATP synthase complex F0-F1) is inhibited when these preparations are pretreated with oligomycin or N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition is not seen with isolated F1-ATPase. These and other results have suggested that modifications of the DCCD-binding protein in the membrane sector (F0) of the ATP synthase complex are communicated to F1, thereby altering the binding characteristics of ATP to the beta subunits. By analogy, it is proposed that modifications (e.g., protonation/deprotonation) of the DCCD-binding protein effected by protonic energy alter the conformation of F1 and bring about the substrate/product binding changes that appear to be essential features of the mechanism and regulation of oxidative phosphorylation.
A rapid and sensitive method for detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin.
Authors: Billingsley M L , Pennypacker K R , Hoover C G , Brigati D J , Kincaid R L
Published: May 2006

Purified bovine brain calmodulin was biotinylated with biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide. Biotinylated calmodulin was used to detect and quantify calmodulin-binding proteins following both protein blotting and slot-blot procedures by using alkaline phosphatase or peroxidase coupled to avidin. When purified bovine brain calcineurin, a calmodulin-dependent protein phosphatase, was immobilized on nitrocellulose slot blots, biotinylated calmodulin bound in a calcium-dependent saturable manner; these blots were then quantified by densitometry. Biotinylated calmodulin was able to detect as little as 10 ng of calcineurin, and the binding was competitively inhibited by addition of either native calmodulin or trifluoperazine. When biotinylated calmodulin was used to probe protein blots of crude brain cytosol and membrane preparations after gel electrophoresis, only protein bands characteristic of known calmodulin-binding proteins (i.e., calmodulin-dependent protein kinase, calcineurin, spectrin) were detected with avidin-peroxidase or avidin-alkaline phosphatase procedures. Purified calcineurin was subjected to one- and two-dimensional gel electrophoresis and protein blotting; as expected, only the 61-kDa calmodulin-binding subunit was detected. When the two-dimensional protein blot was incubated with biotinylated calmodulin and detected with avidin-alkaline phosphatase, several apparent forms of the 61-kDa catalytic subunit were detected, consistent with isozymic species of the enzyme. The results of these studies suggest that biotinylated calmodulin can be used as a simple, sensitive, and quantifiable probe for the study of calmodulin-binding proteins.
Chromosomal location of the gene encoding the neural cell adhesion molecule (N-CAM) in the mouse.
Authors: D'Eustachio P , Owens G C , Edelman G M , Cunningham B A
Published: May 2006

The gene encoding the neural cell adhesion molecule, N-CAM, has been localized on mouse chromosome 9. A BALB/cJ mouse genomic library prepared in lambda bacteriophage EMBL4 was screened by using a cDNA probe, pEC204, that corresponds to the coding region of the chicken N-CAM gene. Four weakly reactive and one strongly reactive recombinant phage were isolated. A region of the latter that was strongly homologous to pEC204 was subcloned to yield a new probe, pEC501. RNA transfer blots and nucleotide sequencing indicated that pEC501 encoded part of the mouse N-CAM gene. This probe defined a unique genetic locus, Ncam, associated with a restriction fragment length polymorphism that allowed the definition of two alleles. The locus could be provisionally assigned either to chromosome 9 or to chromosome 10 by correlating the presence or absence of mouse-specific DNA fragments reactive with the probe in a panel of somatic hybrid cell lines with the presence or absence of the various mouse chromosomes. Analysis of the inheritance of the Ncam-associated DNA polymorphism in recombinant inbred strains of mice revealed close linkage between Ncam and the Lap-1, Sep-1, and Thy-1 loci on chromosome 9. This result suggests an additional linkage between Ncam and the locus for the cerebellar mutation staggerer (sg). The Ncam locus provides an important reference point for mapping the genes for additional cell adhesion molecules as well as genes for other molecules involved in neural development and function.
Properties of memory T lymphocytes isolated from the mixed leukocyte reaction.
Authors: Inaba K , Koide S , Steinman R M
Published: May 2006

During the primary mixed leukocyte reaction, T lymphocytes of the lyt-2- helper subclass proliferate in response to transplantation antigens on allogeneic dendritic cells. We have isolated populations of antigen-specific proliferating lymphoblasts and recultured them in fresh medium. Within 2 days, the blasts become smaller in size, lose responsiveness to T-cell growth factor or interleukin 2, but retain vigorous reactivity to the original alloantigen. Two new biologic properties of these "memory" lymphocytes have been noted. First, they primarily respond to alloantigen on dendritic cells, whereas freshly sensitized lymphoblasts react to allogeneic dendritic cells, macrophages, and B lymphocytes. Second, the memory lymphocytes quickly aggregate with dendritic cells that are either syngeneic or allogeneic, but not with B cells. The aggregates that form with syngeneic dendritic cells disassemble within hours and do not release interleukin 2 or proliferate. The aggregates that form with allogeneic dendritic cells remain intact, release large amounts of interleukin 2 on the first day of culture, and synthesize DNA on the second day. Therefore, dendritic cells actively cluster memory lymphocytes by an antigen-independent mechanism, and this may underlie the heightened functional activity of each cell type.
Cloning of the human estrogen receptor cDNA.
Authors: Walter P , Green S , Greene G , Krust A , Bornert J M , Jeltsch J M , Staub A , Jensen E , Scrace G , Waterfield M
Published: May 2006

Poly(A)+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and fractions enriched in estrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt10 and lambda gt11 vectors. Clones corresponding to ER sequence were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five were isolated after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kilobase pairs) and lambda OR8 (2.1 kilobase pairs), isolated by using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains the DNA sequence expected from the two ER peptides and crosshybridizes with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Use of lambda OR8 as a hybridization probe revealed a single poly(A)+ RNA band of approximately equal to 6.2 kilobase pairs in the ER-containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-negative cell line HeLa. The same probe hybridizes to a chicken gene that is expressed in oviduct tissue as a 7.5-kilobase-pair poly(A)+ RNA.
Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon.
Authors: Becerra S P , Rose J A , Hardy M , Baroudy B M , Anderson C W
Published: May 2006

The three major capsid proteins of adeno-associated virus type 2 (AAV2) virions are designated A, B, and C and have molecular sizes of 90, 72, and 60 kDa, respectively. These proteins are related, and genetic studies have shown they are encoded by a long open reading frame located in the right half of the genome. The coding capacity distal to the first ATG in this reading frame is only 503 amino acids (i.e., a protein about the size of protein C), but an open frame sequence devoid of ATG codons extends upstream for an additional 184 codons. Although the amino terminus of the C capsid protein is blocked, partial amino acid sequence analyses of peptides from C have confirmed that it is encoded within the portion of the reading frame distal to the first ATG at nucleotide (nt) location 2810. The amino terminus of the B capsid protein is not blocked, and its sequence begins with alanine. The triplet encoding this alanine lies 64 codons upstream from the initiation site for protein C and is immediately preceded by the threonine codon, ACG, at nt 2615. This ACG codon lies in the most favorable sequence context for protein synthesis initiation. All three AAV2 capsid proteins are labeled in vitro with formyl[35S]methionyl-tRNAf, indicating that synthesis of each protein is initiated independently. Our data suggest that the nt 2615 ACG codon directs the methionyl-tRNA-dependent initiation of the AAV2 B capsid protein. Proteins B and C may be synthesized from the same mRNA species and their relative abundance could be determined by the efficiencies of their respective initiation codons.
Binding of pea cytochrome f to the inner membrane of Escherichia coli requires the bacterial secA gene product.
Authors: Rothstein S J , Gatenby A A , Willey D L , Gray J C
Published: May 2006

Various sequences from the 5' end of the pea chloroplast gene for cytochrome f have been fused in the correct reading frame with lacZ, and the cellular location of the hybrid polypeptides in Escherichia coli has been examined. Hybrid polypeptides containing N-terminal parts of cytochrome f are located in the cytoplasmic membrane of E. coli. Membrane localization is most efficient when the intact signal sequence of cytochrome f is present at the N-terminal end of the fusion proteins. Fusion within the signal sequence, so that the processing site is absent, reduces the efficiency of membrane binding. Membrane insertion of fusion proteins containing signal sequences is prevented in a temperature-sensitive secA strain at the nonpermissive temperature and the hybrid proteins accumulate in the cytoplasm. This indicates that specific recognition of the chloroplast signal sequence occurs in the bacterial secretory pathway.
Characterization of antigens recognized by monoclonal and polyclonal antibodies directed against uvomorulin.
Authors: Peyriéras N , Louvard D , Jacob F
Published: May 2006

Uvomorulin is a cell surface glycoprotein involved in compaction of early mouse embryo. Antibodies, either monoclonal or polyclonal, raised against a purified tryptic fragment of uvomorulin recognize, in a detergent lysate of embryonal carcinoma cells metabolically labeled with 35S, three molecules (120, 100, and 88 kDa) that are not related, as judged by peptide mapping. Only the 120-kDa form is related to the tryptic fragment of uvomorulin and, thus, is considered as the native form of uvomorulin. Although all three products are apparently detectable at the cell surface, only the 120-kDa form is glycosylated. Coimmunoprecipitation of the three different polypeptides is probably due to shared epitopes rather than to their presence in a multimeric complex.
Interleukin 1 can act as a B-cell growth and differentiation factor.
Authors: Pike B L , Nossal G J
Published: May 2006

Splenic B lymphocytes specifically reactive to the hapten fluorescein (FLU) were prepared from nonimmune adult mice by affinity fractionation on hapten-gelatin. These FLU-specific B cells were cultured as single cells or in small numbers in 10-microliter wells either in the absence of any feeder, filler, or accessory cell or in the presence of 3T3 fibroblasts acting as filler cells. A selected batch of a "T-cell-independent" antigen, FLU-Ficoll, which induces growth and differentiation only in the presence of lymphokines or cytokines acting as B-cell growth and differentiation factors (BGDF), was used as the antigenic stimulus. It was found that murine interleukin 1 prepared by recombinant DNA technology was an effective, although weak, BGDF when acting with antigen on B cells cultured either under filler cell-free conditions or in the presence of 3T3 cells. When the murine interleukin 1 was used in combination with recombinant human interleukin 2, itself a weak but effective BGDF in the system, an additive effect was observed. The results challenge the notion that interleukin 1 is exclusively or even primarily an activating cytokine. This system, in which pure factors are able to act with specific antigen on single hapten-specific B cells, will prove helpful for the further dissection of the respective roles of the various factors that can act on B cells.
Influence of the human T-lymphotropic virus/lymphadenopathy-associated virus on functions of human lymphocytes: evidence for immunosuppressive effects and polyclonal B-cell activation by banded viral preparations.
Authors: Pahwa S , Pahwa R , Saxinger C , Gallo R C , Good R A
Published: May 2006

The etiologic agent for the acquired immunodeficiency syndrome (AIDS) is now firmly established as the retrovirus termed the human T-lymphotropic virus type III (HTLV-III) or the lymphadenopathy-associated virus, LAV. The disease is characterized by profound and progressive loss of immunity, but molecular evidence indicates that only a few cells in peripheral blood are being productively infected with this virus. In the present study we have investigated a disrupted HTLV-III viral preparation for biologic effects on normal lymphoid cells. Relatively dilute concentrations of this preparation were found to stimulate immunoglobulin secretion by peripheral blood lymphocytes; at the same dosages, the preparation was inhibitory for the B-cell differentiation responses that are induced with other known polyclonal B-cell activators, pokeweed mitogen, Staphylococcus aureus, and Epstein-Barr virus. This preparation was also inhibitory at high concentrations for T-lymphocyte proliferative responses to phytomitogens and antigens and resulted in a reduced expression of Tac antigen on phytohemagglutinin-activated lymphocytes. Paradoxically, incubation of lymphocytes of certain healthy donors with the HTLV-III preparation alone resulted in increased expression of Tac and Leu-12 antigens. These findings show that a disrupted preparation of HTLV-III virus can mimic many of the immunologic abnormalities present in patients with HTLV-III infection. Nonviable viral proteins may be responsible for some of the immunologic perturbations that occur in HTLV-III-infected states.