bluuebunny/crossref_metadata_2025 huggingface dataset:
Passenger transgenes reveal intrinsic specificity of the antibody hypermutation mechanism: clustering, polarity, and specific hot spots.
Authors: Betz A G , Rada C , Pannell R , Milstein C , Neuberger M S
Published: May 2006

We have analyzed somatic hypermutation in mice carrying an immunoglobulin kappa transgene in order to discriminate mutations that reflect the intrinsic specificity of the hypermutation mechanism from those highlighted by antigenic selection. We have immunized animals with three different immunogens. With one immunogen, the antigen-specific B cells express a transgenic kappa chain, which does not form part of the antibody; the transgene is a passenger free to accumulate unselected mutations. With the other two immunogens, the transgenic kappa chain constitutes the light chain of the expressed antibody. A comparison of the transgene mutations obtained under these different circumstances allows us to identify common features that we attribute to the intrinsic specificity of the hypermutation process. In particular, it yields only base substitutions and leads to hot spots occurring in individual positions (e.g., the second base of the Ser-31 codon). The mutations preferentially accumulate around the first complementarity-determining region. The process exhibits specific base substitution preferences with transitions being favored over transversions. We propose that these substitution preferences can be used to discriminate intrinsic from antigen-selected hot spots. We also note that hypermutation distinguishes between the coding and noncoding strands since pyrimidines (particularly thymidines) mutate less frequently than purines.
Inhibitors of human immunodeficiency virus integrase.
Authors: Fesen M R , Kohn K W , Leteurtre F , Pommier Y
Published: May 2006

In an effort to further extend the number of targets for development of antiretroviral agents, we have used an in vitro integrase assay to investigate a variety of chemicals, including topoisomerase inhibitors, antimalarial agents, DNA binders, naphthoquinones, the flavone quercetin, and caffeic acid phenethyl ester as potential human immunodeficiency virus type 1 integrase inhibitors. Our results show that although several topoisomerase inhibitors--including doxorubicin, mitoxantrone, ellipticines, and quercetin--are potent integrase inhibitors, other topoisomerase inhibitors--such as amsacrine, etoposide, teniposide, and camptothecin--are inactive. Other intercalators, such as chloroquine and the bifunctional intercalator ditercalinium, are also active. However, DNA binding does not correlate closely with integrase inhibition. The intercalator 9-aminoacridine and the polyamine DNA minor-groove binders spermine, spermidine, and distamycin have no effect, whereas the non-DNA binders primaquine, 5,8-dihydroxy-1,4-naphthoquinone, and caffeic acid phenethyl ester inhibit the integrase. Caffeic acid phenethyl ester was the only compound that inhibited the integration step to a substantially greater degree than the initial cleavage step of the enzyme. A model of 5,8-dihydroxy-1,4-naphthoquinone interaction with the zinc finger region of the retroviral integrase protein is proposed.
Organization of human T-cell receptor beta-chain genes: clusters of V beta genes are present on chromosomes 7 and 9.
Authors: Robinson M A , Mitchell M P , Wei S , Day C E , Zhao T M , Concannon P
Published: May 2006

To ascertain the extent and organization of the germ-line human T-cell receptor (TCR) beta-chain gene repertoire, beta-chain variable region (V beta) genes were mapped by pulsed-field gel electrophoresis, cosmid cloning, and in situ hybridization. Probes derived from the 24 known V beta families were mapped to a total of six Sfi I fragments in DNA samples from multiple individuals representing all possible haplotypes of TCR V- and C (constant)-region insertion/deletion-related polymorphisms. Four of the Sfi I fragments were linked to one another to develop an extended map of the TCR beta-chain gene complex previously localized to chromosome 7q35. The remaining two Sfi I fragments, containing 6 V beta genes, could not be linked to the TCR beta-chain gene complex. Using human-hamster somatic cell hybrids and in situ hybridization, these orphon genes were localized to chromosome 9p. Nucleotide sequences of the orphon V beta genes, derived from cosmid clones, were 93-97% identical to V beta genes in the TCR beta-chain gene complex. Open reading frames in three of the orphon V beta genes were intact as were the recombination signal sequences. As expected, based on their orphon status, none of the V beta genes of chromosome 9 was detected in transcripts containing C beta. These results indicate that the functional germ-line V beta repertoire in humans is substantially (10%) smaller than previously estimated.
Projective resolutions of Weyl modules.
Authors: Buchsbaum D A , Rota G C
Published: May 2006

We present a projective resolution of the two-rowed Weyl module, using techniques of supersymmetric algebra.
Granulocyte-macrophage and macrophage colony-stimulating factors differentially regulate alpha v integrin expression on cultured human macrophages.
Authors: De Nichilo M O , Burns G F
Published: May 2006

The colony-stimulating factors (CSFs) greatly influence mature macrophage function in vitro: macrophage (M)-CSF induces maturation of monocytes and enhances differentiated cell function; granulocyte-macrophage (GM)-CSF stimulates a variety of antimicrobial functions. In vivo M-CSF is thought to promote differentiation, and GM-CSF is thought to potentiate the inflammatory response. One mechanism by which these differential effects may be achieved is through the receptor-mediated interaction of macrophages with their extracellular matrix. Here we show that M-CSF induces specifically the expression of the alpha v beta 5 integrin receptor, whereas GM-CSF rapidly induces mRNA and surface expression of the alpha v beta 3 integrin. The M-CSF-treated cells acquire a flattened epitheloid phenotype, and on vitronectin the alpha v beta 5 is located in adhesion plaques. These cells do not bind collagen or laminin. In contrast, cells treated with GM-CSF adopt an elongated phenotype on a number of substrates, including collagen and laminin, and express alpha v beta 3 at the leading edge of cells on vitronectin. These results suggest that a primary means by which the CSFs exert their individual effects on mature cells may be through regulating integrin expression.
Analysis of homozygous mutant chimeric mice: deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production.
Authors: Jakobovits A , Vergara G J , Kennedy J L , Hales J F , McGuinness R P , Casentini-Borocz D E , Brenner D G , Otten G R
Published: May 2006

Using a recently described method for efficiently deriving homozygous targeted alleles in embryonic stem cells, we produced chimeric mice whose tissues were derived partially from embryonic stem cells bearing homozygous deletion of the mouse immunoglobulin heavy-chain joining (JH) region. Characterization of these chimeric mice indicated that homozygous JH deletion leads to arrest of B-cell development at an early stage, resulting in a total lack of peripheral B cells and serum IgM. These results were confirmed in mice containing the homozygous JH deletion in their germ line. This novel B-cell-deficient mouse strain provides a tool for studying the recombination and expression of exogenous immunoglobulin genes introduced into the mouse germ line.
Hepoxilin A3 is the endogenous lipid mediator opposing hypotonic swelling of intact human platelets.
Authors: Margalit A , Sofer Y , Grossman S , Reynaud D , Pace-Asciak C R , Livne A A
Published: May 2006

When human blood platelets are exposed to hypotonic medium they swell first but, shortly thereafter, revert toward their original volume in a process termed regulatory volume decrease (RVD). RVD is the result of an enhanced efflux of K+ and Cl- ions and associated water. Platelet RVD is controlled by a short-lived lipoxygenase-derived product (LP). By using a combination of high-performance liquid chromatography, gas chromatography-mass spectrometry, and RVD reconstitution bioassay, we show that LP is identical with hepoxilin A3. In addition we demonstrate that authentic hepoxilin A3 possesses the same biological properties on RVD reconstitution as LP and that the activity of both compounds is amplified through epoxide hydrolase inhibition with 3,3,3-trichloropropene-1,2-oxide. Therefore, we report here that volume expansion causes the formation and release of hepoxilin A3 from intact human platelets and that this hepoxilin plays a major role in volume regulation.
p70 lupus autoantigen binds the enhancer of the T-cell receptor beta-chain gene.
Authors: Messier H , Fuller T , Mangal S , Brickner H , Igarashi S , Gaikwad J , Fotedar R , Fotedar A
Published: May 2006

The p70 (Ku) autoantigen has been described as a nonhistone nuclear protein recognized by antibodies from lupus patients. In our studies on the regulation of T-cell receptor (TCR) beta-chain gene expression we have identified the p70 lupus autoantigen as a DNA-binding protein that binds the enhancer of the TCR beta-chain gene. This enhancer is essential for expression of the TCR beta gene. The core TCR beta enhancer contains the E3 motif, which we show here is essential for enhancer activity. The protection of the E3 motif in T cells and the marked reduction in enhancer activity when the E3 motif is mutated underline its physiological importance in regulating beta enhancer activity. The p70 lupus autoantigen gene was identified by screening T-cell lambda gt11 libraries with an E3 probe. The gene encodes a protein which binds the E3 motif in a sequence-specific manner. The identification of a 70-kDa protein as a major E3-binding protein by UV crosslinking is consistent with the conclusion that the p70 lupus autoantigen binds the beta enhancer. Finally, we have shown that T-cell nuclear proteins which bind the E3 motif bear p70 (Ku) lupus autoantigenic determinants. Together these data suggest that the p70 autoantigen binds a critical motif in the beta enhancer and probably regulates TCR beta gene expression.
Kedarcidin chromophore: an enediyne that cleaves DNA in a sequence-specific manner.
Authors: Zein N , Colson K L , Leet J E , Schroeder D R , Solomon W , Doyle T W , Casazza A M
Published: May 2006

Kedarcidin chromophore is a 9-membered enediyne, recently isolated from an actinomycete strain. In vivo studies show this molecule to be extremely active against P388 leukemia and B16 melanoma. Cytotoxicity assays on the HCT116 colon carcinoma cell line result in an IC50 value of 1 nM. In vitro experiments with phi X174, pM2 DNA, and 32P-end-labeled restriction fragments demonstrate that this chromophore binds and cleaves duplex DNA with a remarkable sequence selectivity producing single-strand breaks. The cleavage chemistry requires reducing agents and oxygen similar to the other naturally occurring enediynes. Certain cations (Ca2+ and Mg2+) prevent strand cleavage. High-resolution 1H NMR studies on the chromophore in the presence of calcium chloride implicate the 2-hydroxynaphthoyl moiety in DNA binding. Interestingly, the kedarcidin chromophore appears structurally related to neocarzinostatin yet recognizes specific DNA sequences in a manner similar to calicheamicin gamma 1I, an enediyne with a significantly different structure. Moreover, kedarcidin and calicheamicin share a DNA preferred site, the TCCTN-mer. These observations indicate that the individual structural features of these agents are not solely responsible for their DNA selectivity. Rather, a complementarity between their overall tertiary structure and the local conformation of the DNA at the binding sites must play a significant role in the recognition process.
Mutant human cells defective in induction of major histocompatibility complex class II genes by interferon gamma.
Authors: Mao C , Davies D , Kerr I M , Stark G R
Published: May 2006

Using immunoselection, we have isolated 11 independent mutant HT1080 fibrosarcoma cell lines defective in the induction by interferon gamma (IFN-gamma) of the expression of the human leukocyte antigen HLA-DRA. The mutations are recessive and fall into five complementation groups. All the mutants are affected mainly in the expression of major histocompatibility complex class II and invariant-chain genes. Type I mutants (three complementation groups) are completely defective in induction of the invariant-chain and class II HLA-DP, -DQ, -DR, and -DM genes, whereas type II mutants (two complementation groups) induce these genes weakly in response to IFN-gamma, in the order DPB > DRA > invariant chain. The induction by IFN-gamma of the mRNAs for class I, TAP1, LMP7, and 9-27 is partially defective and the induction of the proteins IRF-1 and ICAM-1 is normal in both types of mutants. All the mutants respond normally to IFN-alpha. The mutants are stable and thus can be used to clone the affected genes by reversion.
An 83-nucleotide promoter of the acetylcholine receptor epsilon-subunit gene confers preferential synaptic expression in mouse muscle.
Authors: Duclert A , Savatier N , Changeux J P
Published: May 2006

The expression of the acetylcholine receptor epsilon-subunit gene is restricted to the endplate of adult muscle fibers. We have started to study the regulatory elements of the epsilon-subunit gene promoter that are important for its synaptic expression. We used, for this purpose, a rapid method of in vivo expression after DNA injection into the muscle tissue [Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. & Felgner, P. L. (1990) Science 247, 1465-1468]. Our results show that a construction containing 83 nucleotides upstream from the transcription start site is sufficient to obtain preferential endplate expression. Moreover, mutation of a MyoD binding site located around position-70 does not alter this synaptic expression. We also studied the expression of this promoter in vitro in muscle primary cultures and showed the presence of a positive element between positions -122 and -83. Comparison of in vivo and in vitro results reveals that the elements important for in vivo localization at the synapse and in vitro expression in cultured muscle cells may differ.
Affinity of human erythrocyte transglutaminase for a 42-kDa gelatin-binding fragment of human plasma fibronectin.
Authors: Radek J T , Jeong J M , Murthy S N , Ingham K C , Lorand L
Published: May 2006

Complex formation between the human erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) and fibronectin or its fragments was examined by immunoanalytical procedures and by fluorescence polarization. A 42-kDa gelatin-binding structure, obtained from human plasma fibronectin by thermolytic digestion, showed as high an affinity for the cytosolic enzyme as the parent fibronectin chains themselves. A 21-kDa fragment comprising type I modules 8 and 9, the last two modules in the 42-kDa fragment, bound with an affinity 100-fold less than the 42-kDa fragment. Binding was remarkably specific and could be exploited for the affinity purification of transglutaminase directly from the hemoglobin-depleted erythrocyte lysate. In spite of the high affinity, it was possible to elute active enzyme from the 42-kDa fragment column with 0.25% monochloroacetic acid. This solvent might have general applicability in other systems involving separation of tightly bound ligands.
Transgenic mouse model for neurocristopathy: Schwannomas and facial bone tumors.
Authors: Jensen N A , Rodriguez M L , Garvey J S , Miller C A , Hood L
Published: May 2006

We have characterized a strain of double transgenic mice with simian virus 40 large tumor antigen and prokaryotic lacZ under the control of the myelin basic protein promoter that develops spindle-cell sarcomas and osteogenic sarcomas at 5-7 months of age. Although poorly differentiated, the spindle-cell sarcomas were characterized as malignant Schwannomas based on their neural association, the presence of basal lamina, and expression of Schwann cell-specific genes. The osteogenic sarcomas were often multiple and appeared predominantly in the facial bones, less frequently in the ribs and vertebral column, and only rarely in the appendicular skeleton. Benign osteoblastic lesions were often observed adjacent to these sarcomas. Both the osteoblastic cells in the facial skeleton and Schwann cells are regarded as neural crest derivatives. The biological properties and anatomical location of these tumors suggest that they may share a common origin from the neural crest or its derivatives. R.P. Bolande [Hum. Pathol. (1974) 5, 409-429] introduced the term neurocristopathy as a unifying concept to describe such lesions arising from the neural crest or its derivatives. Cell lines established from both bone and Schwann cell tumors arising in these transgenic mice express simian virus 40 large tumor antigen mRNA as well as functional large tumor antigen. Such cell lines are potentially valuable in the search for markers that identify mammalian neural crest derivatives.
Calcium influx mediated by the Escherichia coli heat-stable enterotoxin B (STB).
Authors: Dreyfus L A , Harville B , Howard D E , Shaban R , Beatty D M , Morris S J
Published: May 2006

The heat-stable enterotoxin B (STB) of Escherichia coli is a 48-amino acid extracellular peptide that induces rapid fluid accumulation in animal intestinal models. Unlike other E. coli enterotoxins that elicit cAMP or cGMP responses in the gut [heat-labile toxin (LT) and heat-stable toxin A (STA), respectively], STB induces fluid loss by an undefined mechanism that is independent of cyclic nucleotide elevation. Here we studied the effects of STB on intracellular calcium concentration ([Ca2+]i), another known mediator of intestinal ion and fluid movement. Ca2+ and pH measurements were performed on different cell types including Madin-Darby canine kidney (MDCK), HT-29/C1 intestinal epithelial cells, and primary rat pituitary cells. Ca2+ and pH determinations were performed by simultaneous real-time fluorescence imaging at four emission wavelengths. This allowed dual imaging of the Ca(2+)- and pH-specific ratio dyes (indo-1 and SNARF-1, respectively). STB treatment induced a dose-dependent increase in [Ca2+]i with virtually no effect on internal pH in all of the cell types tested. STB-mediated [Ca2+]i elevation was not inhibited by drugs that block voltage-gated Ca2+ channels including nitrendipine, verapamil (L-type), omega-conotoxin (N-type), and Ni2+ (T-type). The increase in [Ca2+]i was dependent on a source of extracellular Ca2+ and was not affected by prior treatment of MDCK cells with thapsigargin or cyclopiazonic acid, agents that deplete and block internal Ca2+ stores. In contrast to these results, somatostatin and pertussis toxin pretreatment of MDCK cells completely blocked the STB-induced increase in [Ca2+]i. Taken together, these data suggest that STB opens a GTP-binding regulatory protein-linked receptor-operated Ca2+ channel in the plasma membrane. The nature of the STB-sensitive Ca2+ channel is presently under investigation.
Neuronal synchronization without calcium-dependent synaptic transmission in the hypothalamus.
Authors: Bouskila Y , Dudek F E
Published: May 2006

A critical question in understanding the mammalian brain is how populations of neurons become synchronized. This is particularly important for the neurons and neuroendocrine cells of the hypothalamus, which are activated synchronously to control endocrine glands and the autonomic nervous system. It is widely accepted that communication between neurons of the adult mammalian brain is mediated primarily by Ca(2+)-dependent synaptic transmission. Here we report that synchronous neuronal activity can occur in the hypothalamic suprachiasmatic nucleus without active Ca(2+)-dependent synaptic transmission. Simultaneous extracellular recordings of neuronal activity in the suprachiasmatic nucleus, which contains the mammalian biological clock, confirmed a circadian rhythm of synchronized activity in hypothalamic slices. Ca(2+)-free medium, which blocks chemical synaptic transmission and increases membrane excitability, produced periodic and synchronized bursts of action potentials in a large population of suprachiasmatic nucleus neurons with diverse firing patterns. N-Methyl-D-aspartic acid, non-N-methyl-D-aspartic acid, and gamma-aminobutyric acid type A receptor antagonists had no effect on burst synchrony. Whole-cell patch-clamp recordings confirmed that the Ca(2+)-free solution blocked evoked postsynaptic potentials and that the mixture of antagonists blocked the remaining spontaneous postsynaptic potentials. Therefore, mechanisms other than Ca(2+)-dependent synaptic transmission can synchronize neurons in the mammalian hypothalamus and may be important wherever neuronal networks are synchronized.
Precursor polyprotein for multiple neuropeptides secreted from the suboesophageal ganglion of the silkworm Bombyx mori: characterization of the cDNA encoding the diapause hormone precursor and identification of additional peptides.
Authors: Sato Y , Oguchi M , Menjo N , Imai K , Saito H , Ikeda M , Isobe M , Yamashita O
Published: May 2006

Peptidergic neurons, which serve as source of various endocrine neuropeptides, were identified in the suboesophageal ganglion (SG) and brain of insects. In the silkworm Bombyx mori, SG is known to secrete two neuropeptides, diapause hormone (DH) responsible for induction of embryonic diapause and pheromone biosynthesis-activating neuropeptide, which share a pentapeptide amide, Phe-Xaa-Pro-Arg-Leu-NH2 (Xaa = Gly or Ser), at the C terminus. We have isolated cDNA clones for DH from the cDNA library of SG by using oligonucleotide probes. The molecular characterization of the cDNA reveals that the mRNA encodes an open reading frame consisting of 192 aa residues in which the 24-aa DH peptide is localized at the N-terminal region just after the signal peptide. A homology search proposed that the cDNA encodes pheromone biosynthesis-activating neuropeptide and three other neuropeptides [alpha-, beta-, and gamma-SG neuropeptide (SGNP)] in the region following DH, all of which are flanked by possible tryptic cleavage sites and share the Phe-Xaa-Pro-Arg-Leu-Gly sequence at the C terminus. Northern hybridization analysis clearly showed that the gene expression was limited to SG. We chemically synthesized alpha-, beta-, and gamma-SGNP and used them to identify components in extracts of SG and to examine biological functions, alpha- and gamma-SGNP were identified in extracts of SG, and the synthetic beta- and gamma-SGNP expressed weak DH activity. These results indicate that DH, along with four other neuropeptides, is generated from a common precursor polyprotein that is encoded by a single mRNA transcribed in neurosecretory cells of SG.
Human immunodeficiency virus type 1 coat protein neurotoxicity mediated by nitric oxide in primary cortical cultures.
Authors: Dawson V L , Dawson T M , Uhl G R , Snyder S H
Published: May 2006

The human immunodeficiency virus type 1 coat protein, gp120, kills neurons in primary cortical cultures at low picomolar concentrations. The toxicity requires external glutamate and calcium and is blocked by glutamate receptor antagonists. Nitric oxide (NO) contributes to gp120 toxicity, since nitroarginine, an inhibitor of NO synthase, prevents toxicity as does deletion of arginine from the incubation medium and hemoglobin, which binds NO. Superoxide dismutase also attenuates toxicity, implying a role for superoxide anions.
Identification, by protein sequencing and gene transfection, of sgp-60 as the murine homologue of CD48.
Authors: Cabrero J G , Freeman G J , Lane W S , Reiser H
Published: May 2006

We have recently described a murine lymphocyte protein, provisionally termed sgp-60, which is expressed on virtually all lymphocytes of both T- and B-cell origin. A hamster monoclonal antibody to sgp-60 can inhibit interleukin-2 (IL-2) production, IL-2-receptor expression, and T-cell proliferation, events normally observed after stimulation of T cells with an antibody to the T-cell receptor/CD3 complex or with the lectin concanavalin A. Our previous studies did not reveal the molecular nature of the sgp-60 antigen. Purification of sgp-60 and protein sequencing demonstrate that sgp-60 is identical to the CD48 antigen, a ligand for the CD2 antigen, which is also called Blast-1 in humans, BCM1 in mice, and OX-45 in rats. The identity of sgp-60 and CD48 was independently confirmed in gene transfection experiments. The anti-sgp-60 monoclonal antibody was selectively reactive with COS-7 cells transfected with a BCM1 cDNA clone but not with Kb-transfected controls. The results of the present report, together with our previous functional studies, may have implications for the role of CD2 and CD48 in murine T-cell activation.
Oxygen regulation of nifA transcription in vitro.
Authors: Agron P G , Ditta G S , Helinski D R
Published: May 2006

In Rhizobium meliloti, transcription of the key nitrogen-fixation regulatory genes nifA and fixK is induced in response to microaerobiosis through the action of the FixL and FixJ proteins. These two proteins are sensor and regulator homologues, respectively, of a large family of bacterial two-component systems involved in sensing and responding to environmental changes. A soluble, truncated form of the membrane protein FixL, FixL*, has been shown to be a hemoprotein that phosphorylates and dephosphorylates FixJ in response to oxygen tension. Here we use an in vitro transcription system to prove that FixJ is a transcriptional activator of both nifA and fixK and that phosphorylation of FixJ markedly increases its activity. Phosphorylation was achieved either by preincubating FixJ with FixL* and ATP or by exposing FixJ to the inorganic phospho donor ammonium hydrogen phosphoramidate. Both FixJ and FixJ-phosphate formed heparin-resistant complexes under the assay conditions used. Lastly, we were able to show that anaerobiosis, in the presence of FixL* and ATP, greatly stimulates FixJ activity at the nifA promoter with either Escherichia coli or R. meliloti RNA polymerase. This use of atmospheric oxygen to control nifA transcription in vitro represents a reconstitution of a bacterial two-component signal transduction system in its entirety, from effector to ultimate target, by the use of purified components.
Cdc25M2 activation of cyclin-dependent kinases by dephosphorylation of threonine-14 and tyrosine-15.
Authors: Sebastian B , Kakizuka A , Hunter T
Published: May 2006

Recent evidence has suggested that human cyclin-dependent kinase 2 (CDK2) is an essential regulator of cell cycle progression through S phase. CDK2 is known to complex with at least two distinct human cyclins, E and A. The kinase activity of these complexes peaks in G1 and S phase, respectively. The vertebrate CDC2/cyclin B1 complex is an essential regulator of the onset of mitosis and is inhibited by phosphorylation of CDC2 on Thr-14 and Tyr-15. In vitro, CDC2/cyclin B1 is activated by treatment with the members of the Cdc25 family of phosphatases. We found that, like CDC2, CDK2 is also phosphorylated on Thr-14 and Tyr-15 and that treatment of cyclin A or cyclin E immunoprecipitates with bacterially expressed Cdc25M2 (the mouse homolog of human CDC25B) increased the histone H1 kinase activity of these immune complexes 5- to 10-fold. Tryptic peptide mapping demonstrated that Cdc25M2 treatment of cyclin A or cyclin B1 immune complexes resulted in the specific dephosphorylation of Thr-14 and Tyr-15 on CDK2 or CDC2, respectively. Thus, we have confirmed that Cdc25 family members comprise a class of dual-specificity phosphatases. Furthermore, our data suggest that the phosphorylation and dephosphorylation of CDKs on Thr-14 and Tyr-15 may regulate not only the G2/M transition but also other transitions in the cell cycle and that individual cdc25 family members may regulate distinct cell cycle checkpoints.
Transcription factor E2F binds DNA as a heterodimer.
Authors: Huber H E , Edwards G , Goodhart P J , Patrick D R , Huang P S , Ivey-Hoyle M , Barnett S F , Oliff A , Heimbrook D C
Published: May 2006

E2F is a mammalian transcription factor that appears to play an important role in cell cycle control. DNA affinity column-purified E2F from HeLa cells reproducibly exhibits multiple protein bands when analyzed by SDS/PAGE. After electrophoretic purification, electroelution, and refolding of the individual protein components, the E2F DNA binding activity of the individual proteins was poor. However, upon mixing the individual components together, a dramatic (100- to 1000-fold) increase in specific DNA binding activity was observed. The five protein bands isolated can be separated into two groups based on apparent molecular mass. Optimal reconstitution of activity requires one of the two proteins found in the group of larger molecular mass (approximately 60 kDa) and one of the three proteins in the smaller-sized group (approximately 50 kDa). The reconstituted heterodimer is identical to authentic affinity-purified E2F by three criteria: DNA-binding specificity, DNA pattern, and binding to the retinoblastoma gene product. A recently cloned protein with E2F-like activity, RBP3/E2F-1, is related to the protein components of the group of larger molecular mass, as determined by Western blot analysis and reconstitution experiments. These data suggest that E2F, like many other transcription factors, binds DNA as an oligomeric complex composed of at least two distinct proteins.
Adaptation to alcoholic fermentation in Drosophila: a parallel selection imposed by environmental ethanol and acetic acid.
Authors: Chakir M , Peridy O , Capy P , Pla E , David J R
Published: May 2006

Besides ethanol, acetic acid is produced in naturally fermenting sweet resources and is a significant environmental stress for fruit-breeding Drosophila populations and species. Although not related to the presence of an active alcohol dehydrogenase, adult acetic acid tolerance was found to correlate with ethanol tolerance when sensitive (Afrotropical) and resistant (European) natural populations of Drosophila melanogaster were compared. The same correlation was found when comparing various Drosophila species. Tolerance to acetic acid also correlated with the tolerance to longer aliphatic acids of three, four, or five carbons but did not correlate with the tolerance to inorganic acids (i.e., hydrochloric and sulfuric acids). These observations suggest that acetic acid is detoxified by the conversion of acetate into acetyl-CoA, a metabolic step also involved in ethanol detoxification. Future investigations on the adaptation of Drosophila to fermenting resources should consider selective effects of both ethanol and acetic acid.
Identification of HS1 protein as a major substrate of protein-tyrosine kinase(s) upon B-cell antigen receptor-mediated signaling.
Authors: Yamanashi Y , Okada M , Semba T , Yamori T , Umemori H , Tsunasawa S , Toyoshima K , Kitamura D , Watanabe T , Yamamoto T
Published: May 2006

Crosslinking of membrane-bound immunoglobulins, which are B-cell antigen receptors, causes proliferation and differentiation of B cells or inhibition of their growth. The receptor-mediated signaling involves tyrosine phosphorylation of cellular proteins and rapid activation of Src-like kinases. The amino acid sequences of five proteolytic peptides of p75, a major substrate of protein-tyrosine(s) in the signaling, showed that p75 is the human HS1 gene product. The HS1 gene is expressed specifically in hematopoietic cells and encodes p75HS1, which carries both helix-turn-helix and Src homology 3 motifs. p75HS1 showed rapid tyrosine phosphorylation and association with a Src-like kinase, Lyn, after crosslinking of membrane-bound IgM. Thus, p75HS1 may be an important substrate of Lyn and possibly other protein-tyrosine kinases upon B-cell antigen receptor-mediated signaling.
Immunoglobulin heavy chain enhancer is located near or in an initiation zone of chromosomal DNA replication.
Authors: Ariizumi K , Wang Z , Tucker P W
Published: May 2006

In several animal viruses, enhancers have been implicated in both DNA replication and transcriptional activation. The linkage of the two mechanisms appears intimate, in that common DNA binding factors can be shared. The immunoglobulin heavy chain (Igh) intronic [heavy chain joining region (JH)-mu chain constant region (C mu)] enhancer (E mu) is required for tissue-specific transcription of Igh genes and is essential for somatic recombination of diversity (D) and J segments. We show here that E mu is located at or near an origin of chromosomal DNA replication, which is more active in B lymphocytes than fibroblasts. E mu does not fulfill two criteria demonstrated for some cellular origins. E mu can initiate but not maintain autonomous replicating activity in B cells. E mu is unable to impart early replication timing to a transfected VDJ-C mu Igh locus in B cells. Instead we propose that E mu-associated ori activity contributes to tissue-specific Igh expression through local effects on chromatin structure leading to subsequent accessibility of transcription and/or recombination factors for the enhancer.
Initial localization of regulatory regions of the cardiac sarcolemmal Na(+)-Ca2+ exchanger.
Authors: Matsuoka S , Nicoll D A , Reilly R F , Hilgemann D W , Philipson K D
Published: May 2006

We have analyzed the regulatory properties of the wild-type cardiac Na(+)-Ca2+ exchanger expressed in Xenopus laevis oocytes using the giant excised patch technique. The exchanger is activated by cytoplasmic application of chymotrypsin and exhibits a number of properties that can be changed or abolished by chymotrypsin treatment, including cytoplasmic Na(+)-dependent inactivation, secondary regulation by free cytoplasmic Ca2+, and inhibition by exchanger inhibitory peptide. Thus, the cloned exchanger expressed in oocytes exhibits regulatory properties similar to those of the native sarcolemmal exchanger. The exchanger protein contains a large (520 amino acids) hydrophilic domain modeled to be intracellular. The role of this region in exchanger function and regulation was examined by deletion mutagenesis. Mutants with residues 240-679 and 562-685 deleted exhibited exchange activity, indicating that this extensive region is not essential for transport function. Both mutants were stimulated by chymotrypsin treatment. Neither mutant demonstrated regulation by free cytoplasmic Ca2+ (Ca2+i) or inhibition by exchanger inhibitory peptide (XIP). However, mutant delta 562-685 but not delta 240-679 displayed Na(+)-dependent inactivation. The data suggest that the binding sites for XIP and regulatory Ca2+ may reside in the region encompassed by residues 562-685. A chimera made from renal and cardiac exchangers has normal regulatory characteristics and helps to further define these sites.
Vascular cell adhesion molecule 1: contrasting transcriptional control mechanisms in muscle and endothelium.
Authors: Iademarco M F , McQuillan J J , Dean D C
Published: May 2006

Interaction between vascular cell adhesion molecule 1 (VCAM-1), which appears on the surface of endothelial cells in response to inflammation, and its integrin counter receptor, alpha 4 beta 1, on immune cells is responsible for targeting these immune cells to cytokine-stimulated endothelium. In addition to its role in the immune system, VCAM-1 is also expressed in a developmentally specific pattern on differentiating skeletal muscle, where it mediates cell-cell interactions important for myogenesis through interaction with alpha 4 beta 1. In contrast to endothelium, there is high basal expression of VCAM-1 in skeletal muscle cells and the expression is not cytokine-responsive. Here, we examine the molecular basis for these contrasting patterns of expression in muscle and endothelium, using VCAM-1 promoter constructs in a series of transfection assays. In endothelial cells, octamer binding sites act as silencers that prevent VCAM-1 expression in unstimulated cells. Tumor necrosis factor alpha overcomes the negative effects of these octamers and activates the promoter through two adjacent NF-kappa B binding sites. In muscle cells, a position-specific enhancer located between bp -21 and -5 overrides the effect of other promoter elements, resulting in constitutive VCAM-1 expression. A nuclear protein binds the position-specific enhancer in muscle but not endothelial cells; thus the pattern of expression of this protein could control enhancer activity.
Identification of nine tissue-specific transcription factors of the hepatocyte nuclear factor 3/forkhead DNA-binding-domain family.
Authors: Clevidence D E , Overdier D G , Tao W , Qian X , Pani L , Lai E , Costa R H
Published: May 2006

Hepatocyte nuclear factor (HNF)-3 alpha, -3 beta, and -3 gamma are liver transcription factors that mediate the coordinate expression of a number of hepatocyte-specific genes. The HNF-3 proteins share DNA-binding-domain homology among themselves and with the Drosophila homeotic protein forkhead (fkh). The HNF-3/fkh DNA-binding domain constitutes an uncharacterized protein motif that recognizes its cognate DNA binding site as a monomer. Additional HNF-3/fkh-related proteins are known to be required for determination events during embryogenesis in Drosophila and Xenopus. In this report, we describe the isolation of nine additional HNF-3/fkh homologue (HFH) clones from rodent tissue cDNAs by using both low-stringency hybridization and a polymerase chain reaction protocol. Many of the HFH genes exhibit a tissue-restricted expression pattern and are transcribed in tissues other than liver, including brain, kidney, lung, and intestine. The HNF-3/fkh motif therefore comprises a large gene family of transcription factors that play a role in tissue-specific gene regulation and development.
Oltipraz, an inhibitor of human immunodeficiency virus type 1 replication.
Authors: Prochaska H J , Yeh Y , Baron P , Polsky B
Published: May 2006

Glutathione depletion may play a pivotal role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1) infection. Since certain compounds prevent experimental carcinogenesis by elevating the levels of glutathione and phase II detoxication enzymes, we compared the potencies of several inducers with their ability to inhibit basal levels of HIV-1 replication in H9 cutaneous T-cell lymphoma cells. All monofunctional inducers tested elevated the levels of glutathione and quinone reductase, a marker for phase II enzyme induction. However, only oltipraz [4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione] was effective at inhibiting HIV-1 replication (IC50 = 14.8 +/- 3.1 microM). The antiviral effect of oltipraz was potentiated by 3'-azido-3'-deoxythymidine. Thus, 1,2-dithiole-3-thiones represent a hitherto unrecognized class of anti-HIV-1 agents. Oltipraz behaves kinetically as an irreversible inhibitor of HIV-1 reverse transcriptase in the template-primer binding domain. Oltipraz has been used to treat schistosomiasis in humans and is undergoing clinical evaluation as an anticarcinogen. Thus, oltipraz (and other 1,2-dithiole-3-thiones) may have therapeutic utility in HIV-1-infected individuals, not only because of their antiretroviral activity, but also by preventing the development of HIV-1-associated neoplasms.
Evidence from in vitro replication that O6-methylguanine can adopt multiple conformations.
Authors: Dosanjh M K , Loechler E L , Singer B
Published: May 2006

The effect of O6-methylguanine (m6G) on replication, in a partially double-stranded defined 25-base oligonucleotide, has been studied under nonlimiting conditions of unmodified dNTPs and over an extended time period, using the Klenow fragment of Escherichia coli DNA polymerase I. The sequence surrounding m6G has flanking cytosines (C-m6G-C), and the initial steady-state kinetics have been reported. When the primer was annealed so that the first base to be replicated was m6G, replication was virtually complete in approximately 5 min, although the reaction appears biphasic. When annealed with a primer where thymine or cytosine is paired opposite template m6G, about half the molecules were replicated in the first 15 sec, and no significant further replication was seen over a 1-hr period. When m6G was dealkylated by DNA-O6-methylguanine-methyltransferase, replication was rapid with no blockage. These data suggest that there can be two (or more) conformations of m6G. In these studies the term syn refers to conformers interfering with base-pairing, whereas anti refers to those allowing such base-pairing. Previous physical studies by others indicate that syn- and anti-conformers of the methyl group relative to the N1 of guanine are possible. Here molecular modeling/computational studies are described, suggesting that syn- and anti-m6G can be of similar energy in DNA, and, therefore, these two conformers may explain the two types of species observed during in vitro replication. An alternative explanation could be the possibility that the different species may manifest differential interactions of m6G with Klenow fragment. These results may provide a rationale for why m6G lesions in vivo have been reported to be lethal as well as mutagenic.
A protein phosphatase related to the vaccinia virus VH1 is encoded in the genomes of several orthopoxviruses and a baculovirus.
Authors: Hakes D J , Martell K J , Zhao W G , Massung R F , Esposito J J , Dixon J E
Published: May 2006

The vaccinia virus VH1 gene product is a dual specificity protein phosphatase with activity against both phosphoserine- and phosphotyrosine-containing substrates. We investigated the potential presence of VH1 analogs in other viruses. Hybridization and sequence data indicated that a phosphatase related to the VH1 phosphatase is highly conserved in the genomes of smallpox variola virus and other orthopoxviruses. The open reading frames from the raccoonpox virus and the smallpox variola virus Bangladesh major strain genomes encoding the VH1 analogs were sequenced and found to be highly conserved with the vaccinia virus VH1. An open reading frame from the baculovirus Autographa californica has sequence similarity to the VH1 phosphatase. The viral proteins appear to be structurally related to the cell cycle control protein p80cdc25. A recombinant phosphatase expressed from the baculovirus gene was found to share with the VH1 phosphatase the ability to hydrolyze substrates that contained both phosphoserine and phosphotyrosine.
Expression of functional hepatitis B virus polymerase in yeast reveals it to be the sole viral protein required for correct initiation of reverse transcription.
Authors: Tavis J E , Ganem D
Published: May 2006

Replication of hepatitis B viruses proceeds by reverse transcription of an RNA intermediate, a reaction catalyzed by the virus-encoded polymerase (P protein). The reaction product is a partially duplex DNA whose (-)-strand is covalently linked to the P protein. Efforts to understand the mechanism of the reaction have been severely retarded by an inability to express functional polymerase outside of viral particles. Here we report the successful expression of enzymatically active polymerase in yeast cells, by fusing the P gene to coding sequences of the retrotransposon Ty1. The enzyme initiates correctly on viral RNA in yeast cells in vivo, producing nascent DNA chains covalently linked to protein, exactly as found in virus-infected cells. Replication complexes isolated from these yeast are enzymatically active in vitro, synthesizing DNA in a reaction that is actinomycin D-resistant but sensitive to RNase pretreatment. These results indicate that P protein is the sole viral protein required for the correct priming of reverse transcription and establish a tractable system for the biochemical dissection of the reaction.
Reconstitution of functional interleukin 2 receptor complexes on fibroblastoid cells: involvement of the cytoplasmic domain of the gamma chain in two distinct signaling pathways.
Authors: Asao H , Takeshita T , Ishii N , Kumaki S , Nakamura M , Sugamura K
Published: May 2006

We have previously shown that the interleukin 2 (IL-2) receptor gamma chain is a member of the cytokine receptor superfamily and is indispensable for the formation of receptor complexes with high and intermediate affinities for IL-2. The present study demonstrates that the alpha beta gamma heterotrimer and beta gamma heterodimer complexes of IL-2 receptor reconstituted on murine fibroblast L929 cells can transduce IL-2-mediated signals for activation of tyrosine kinase and for induction of c-myc, c-fos, and c-jun expression. A mutant of the gamma chain lacking the C-terminal 68 amino acids in its cytoplasmic region showed a loss of such signal-transducing ability when incorporated into the IL-2 receptor complexes but brought no effect on IL-2 binding and IL-2 internalization. Another mutant, with a C-terminal deletion of 30 amino acids, retained the ability to activate a tyrosine kinase and to induce c-myc expression but lost the ability to induce c-fos and c-jun expression. These results suggest that at least two distinct signals, one for c-myc induction, which parallels tyrosine kinase activation, and the other for c-fos and c-jun induction, can be transduced from the IL-2 receptor complexes reconstituted on fibroblastoid cells.
Altered processing of Alzheimer amyloid precursor protein in response to neuronal degeneration.
Authors: Iverfeldt K , Walaas S I , Greengard P
Published: May 2006

In the brains of individuals with Alzheimer disease, senile plaques containing aggregates of beta-amyloid peptide, derived from the beta-amyloid precursor protein (APP), are seen in association with degenerating nerve terminals. It is not known whether the degenerating nerve terminals cause the formation of these aggregates or whether beta-amyloid peptide in the aggregates causes nerve-terminal degeneration. In the present study of rat brain, degeneration either of local neurons or of nerve terminals caused decreased levels of a neuron-enriched isoform of APP, increased levels of a glia-enriched isoform of APP, and increased levels of potentially amyloidogenic, as well as nonamyloidogenic, COOH-terminal fragments of APP. Our results demonstrate that neuronal degeneration affects APP processing and suggest that it may contribute to amyloid formation in mammalian brain.
Description of the leukocyte function-associated antigen 1 (LFA-1 or CD11a) promoter.
Authors: Cornwell R D , Gollahon K A , Hickstein D D
Published: May 2006

The CD11a/CD18 (leukocyte function-associated antigen 1 or LFA-1) leukocyte integrin is expressed at high levels on the cell surface of T lymphocytes and macrophages, where it mediates homotypic and heterotypic adherence between leukocytes and other cell types by binding to intracellular adhesion molecules 1 and 2 on the conjugate cell. To initiate studies of the molecular regulation of expression of the CD11a molecule, we isolated genomic clones corresponding to the 5'-flanking region of CD11a, identified the transcriptional start sites for CD11a, and characterized the CD11a promoter sequence in transient expression assays. The CD11a promoter (1.7 kb) directed functional activity of a heterologous reporter gene in the T-lymphocyte cell line Jurkat and the myeloid cell line HL-60 but did not direct functional activity in three different nonleukocyte cell lines. Deletional analysis of the CD11a promoter sequence indicated the presence of distinct, cell-type-specific regulatory sequences with the region from -40 to -17 relative to the transcription start sites responsible for most of the in vitro activity of the CD11a promoter in the Jurkat T-cell line, and the promoter sequence located within the first 17 bp relative to the transcription start sites responsible for CD11a promoter activity in the HL-60 cell line. Identification of the CD11a promoter provides the opportunity to identify unique cis-acting elements and trans-acting factors responsible for the cell-type-specific expression of CD11a in human leukocytes. Further, the CD11a promoter may be useful in transgenic constructs and in retroviral vectors to direct expression of heterologous genes selectively in leukocytes.