bluuebunny/crossref_metadata_2025 huggingface dataset:
Antimalarial activities of oligodeoxynucleotide phosphorothioates in chloroquine-resistant Plasmodium falciparum.
Authors: Rapaport E , Misiura K , Agrawal S , Zamecnik P
Published: May 2006

Synthetic oligonucleotides and their chemical modifications have been shown to inhibit viral and cellular gene expression by sequence-specific antisense hybridization to target mRNAs. We now report that oligodeoxynucleotide phosphorothioates and their nuclease-resistant modifications are effective in micromolar and submicromolar concentrations against the growth of both chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum in vitro. Parasitized human erythrocytes were found to be accessible to radioactively labeled oligodeoxynucleotides, whereas the uninfected erythrocytes did not permit any cellular entry of the same compounds. The dihydrofolate reductase-thymidylate synthase gene of P. falciparum was demonstrated to be a good target for sequence-dependent inhibition of plasmodial growth by exogenously administered modified oligonucleotides. The antimalarial activities observed in vitro were identical for chloroquine-sensitive and chloroquine-resistant strains of P. falciparum. The antimalarial activity of oligodeoxynucleotide phosphorothioates is related to sequence complementarity to certain regions of the plasmodial genome as well as to non-sequence-defined activities.
The matrix proteins of neurovirulent subacute sclerosing panencephalitis virus and its acute measles virus progenitor are functionally different.
Authors: Hirano A , Wang A H , Gombart A F , Wong T C
Published: May 2006

Persistence of measles virus in the brains of patients with subacute sclerosing panencephalitis (SSPE) is accompanied by changes in the viral matrix (M) protein. To understand the significance of these changes, cell culture and cell-free assays were developed to compare the functions of the M proteins of an SSPE virus Biken strain and its acute measles virus progenitor Nagahata strain. The Nagahata viral M protein is associated with the intracellular viral nucleocapsids and the plasma membrane, whereas the Biken viral M protein is localized mainly in the cytosol. The lack of M protein in the Biken viral nucleocapsids is due to a failure of the Biken M protein to bind to the viral nucleocapsids. The Biken M protein also fails to bind to the Nagahata viral nucleocapsids. Conversely, the Nagahata M protein can bind to the Biken viral nucleocapsids, although this association is not as stable at physiological salt concentration. These results offer concrete evidence that the M protein of an SSPE virus is functionally different from that of its progenitor acute measles virus.
An inhibitory carboxyl-terminal domain in Ets-1 and Ets-2 mediates differential binding of ETS family factors to promoter sequences of the mb-1 gene.
Authors: Hagman J , Grosschedl R
Published: May 2006

The mb-1 gene is expressed only during the early stages of B-lymphocyte differentiation. Here we show that the mb-1 proximal promoter region contains a functionally important binding site for members of the ETS family of DNA-binding proteins. We found that both the E26 virus-encoded v-ets and the myeloid/B-cell-specific factor PU.1 bind efficiently to this site in vitro. By contrast, Ets-1, the lymphocyte-specific cellular homologue of v-ets, and the related, more ubiquitously expressed Ets-2 protein interacted weakly with this binding site. DNA binding by both Ets-1 and Ets-2, however, could be increased 20- to 50-fold by deleting as few as 16 carboxyl-terminal amino acids. The inhibitory carboxyl-terminal amino acid sequence is highly conserved between Ets-1 and Ets-2 but is not present in either v-ets or PU.1. Replacement of the carboxyl-terminal amino acids of v-ets with those of Ets-1 decreased DNA binding by v-ets drastically. Cotranslation of Ets-1 transcripts encoding proteins of different lengths suggested that Ets-1 binds DNA as a monomer. Therefore, the carboxyl-terminal inhibitory domain appears to interfere directly with DNA binding and not with homodimerization. Finally, the functional relevance of ETS factor binding to the mb-1 promoter site was evidenced by the stimulation of transcription through this site by a v-myb-v-ets fusion protein. Together, these data suggest that one or more ETS family factors are involved in the regulation of mb-1 gene expression.
Cellular and peptide requirements for in vitro clonal deletion of immature thymocytes.
Authors: Iwabuchi K , Nakayama K , McCoy R L , Wang F , Nishimura T , Habu S , Murphy K M , Loh D Y
Published: May 2006

Thymocytes from DO10 T-cell-receptor transgenic mice undergo apoptosis, or programmed cell death, when chicken ovalbumin-(323-339) peptide is administered in vivo. Using DO10 mice thymocytes, we have now developed a simple in vitro model system that recapitulates the in vivo clonal-deletion process. When transgenic thymocytes were cocultured with fibroblasts, B cells, or thymic nurse cell lines (all bearing I-Ad) in the presence of chicken ovalbumin-(323-339), deletion of the transgenic TCR+CD4+CD8+ thymocytes was seen within 8-20 hr. Thymocytes designed to bear I-Ad on their surface could mediate the deletion themselves. Thus, thymocyte clonal deletion entirely depends on the stage at which the thymocytes are vulnerable to the onset of apoptosis, rather than on the nature of the peptide antigen-presenting cells. Furthermore, thymic nurse cell line TNC-R3.1 could cause deletion, strongly suggesting that some thymic epithelial/stromal components are potentially capable of participating in negative selection. In all cases examined, little deletion could be induced at a peptide concentration less than 10 nM, thus defining the minimum amount of peptide antigen required for negative selection. The peptide-dependent in vitro negative-selection system will allow further dissection of the molecular and cellular processes involved in clonal deletion due to apoptosis in the thymus.
Ionizing radiation stimulates unidentified tyrosine-specific protein kinases in human B-lymphocyte precursors, triggering apoptosis and clonogenic cell death.
Authors: Uckun F M , Tuel-Ahlgren L , Song C W , Waddick K , Myers D E , Kirihara J , Ledbetter J A , Schieven G L
Published: May 2006

Very little is known regarding the effects of ionizing radiation on cytoplasmic signal transduction pathways. Here, we show that ionizing radiation induces enhanced tyrosine phosphorylation of multiple substrates in human B-lymphocyte precursors. This response to ionizing radiation was also observed in cells pretreated with vanadate, a potent protein-tyrosine-phosphatase (PTPase) inhibitor, and phosphotyrosyl [Val5]angiotensin II phosphatase assays showed no decreased PTPase activity in irradiated cells. Thus, enhanced tyrosine phosphorylation in irradiated B-lymphocyte precursors is not triggered by inhibition of total cellular PTPase activity. Immune-complex kinase assays using anti-phosphotyrosine antibodies demonstrated enhanced protein-tyrosine kinase (PTK) activity in the immunoprecipitates from irradiated cells, and the PTK inhibitors genistein and herbimycin effectively prevented radiation-induced tyrosine phosphorylation. Immune-complex kinase assays on irradiated and unirradiated B-lymphocyte precursors using antibodies prepared against unique amino acid sequences of p59fyn, p56/p53lyn, p55blk, and p56lck demonstrated that these Src-family tyrosine kinases were not the primary PTKs responsible for enhanced tyrosine kinase activity in the anti-phosphotyrosine antibody immunoprecipitates or for enhanced tyrosine phosphorylation of multiple substrates. Thus, our findings favor the hypothesis that ionizing radiation induces enhanced tyrosine phosphorylation in B-lymphocyte precursors by stimulation of as yet unidentified PTKs. Tyrosine phosphorylation appears to be an important proximal step in radiation-induced apoptosis and clonogenic cell death because inhibition of PTK prevents DNA fragmentation and loss of clonogenicity of irradiated B-lymphocyte precursors. Since PTKs play myriad roles in the regulation of cell function and proliferation, the activation of a PTK cascade, as detailed in this report, may explain some of the pleiotropic effects of ionizing radiation on cellular functions of B-lymphocytes and their precursors.
Cis-acting, orientation-dependent, positive control system activates pheromone-inducible conjugation functions at distances greater than 10 kilobases upstream from its target in Enterococcus faecalis.
Authors: Chung J W , Dunny G M
Published: May 2006

The prgB gene encodes the surface protein, Asc10, which mediates cell aggregation, resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline-resistance plasmid pCF10 in Enterococcus faecalis. Messenger RNA analysis by Northern blot hybridization and primer extension indicates that prgB transcription is pheromone-inducible and monocistronic. Previous transposon mutagenesis and sequencing analysis of a 12-kilobase (kb) region of pCF10 indicated that several genes including prgR and prgS are required to activate expression of prgB. The distance (3-4 kb) between these regulatory genes and prgB suggested that the activation might function in trans. To test this, a promoterless lacZ gene fusion to prgB was constructed and cloned without some or all of the regulatory genes. Several restriction fragments of the regulatory region were cloned in a higher copy-number plasmid, and numerous complementation studies were carried out in E. faecalis. Complementation in trans was not observed in any of these experiments. However, when the regulatory region and target genes were cloned in different sites of the same plasmid, separated by as much as 12 kb, activation of prgB was observed. Interestingly, this activation occurred only when the regions were cloned in the same relative orientation in which they exist on wild-type pCF10. These results suggest that one or more regulatory molecules may bind to an upstream cis-acting site and track along the DNA to reach a target site to activate prgB transcription.
Model studies directed toward the boron neutron-capture therapy of cancer: boron delivery to murine tumors with liposomes.
Authors: Shelly K , Feakes D A , Hawthorne M F , Schmidt P G , Krisch T A , Bauer W F
Published: May 2006

The successful treatment of cancer by boron neutron-capture therapy (BNCT) requires the selective concentration of boron-10 within malignant tumors. The potential of liposomes to deliver boron-rich compounds to tumors has been assessed by the examination of the biodistribution of boron delivered by liposomes in tumor-bearing mice. Small unilamellar vesicles with mean diameters of 70 nm or less, composed of a pure synthetic phospholipid (distearoyl phosphatidylcholine) and cholesterol, have been found to stably encapsulate high concentrations of water-soluble ionic boron compounds. The hydrolytically stable borane anions B10H10(2-), B12H11SH2-, B20H17OH4-, B20H19(3-), and the normal form and photoisomer of B20H18(2-) were encapsulated in liposomes as their soluble sodium salts. The tissue concentration of boron in tumor-bearing mice was measured at several time points over 48 h after i.v. injection of emulsions of liposomes containing the borane anions. Although the boron compounds used do not exhibit an affinity for tumors and are normally rapidly cleared from the body, liposomes were observed to selectively deliver the borane anions to tumors. The highest tumor concentrations achieved reached the therapeutic range (greater than 15 micrograms of boron per g of tumor) while maintaining high tumor-boron/blood-boron ratios (greater than 3). The most favorable results were obtained with the two isomers of B20H18(2-). These boron compounds have the capability to react with intracellular components after they have been deposited within tumor cells by the liposome, thereby preventing the borane ion from being released into blood.
Transcriptional analysis of the mts1 gene with specific reference to 5' flanking sequences.
Authors: Tulchinsky E , Ford H L , Kramerov D , Reshetnyak E , Grigorian M , Zain S , Lukanidin E
Published: May 2006

The mts1 gene is specifically expressed in certain metastatic tumors but not in their nonmetastatic counterparts. It is also expressed in several normal cell and tissue types that exhibit the ability to be motile. The gene was cloned from both mouse and human sources and the 5' flanking regions were sequenced. The sequencing data revealed a 135-base-pair region of high homology between the mouse and human mts1 gene. This homology was observed in the vicinity of the TATA box. The 5' region of the mts1 gene was also observed to have a high degree of homology to some known promoter and enhancer sequences. To determine the role this region plays in regulating the transcription of mts1, promoter analysis was performed. Sixteen constructs were prepared in which the chloramphenicol acetyltransferase gene was fused to different regions of the mouse mts1 promoter. These constructs were analyzed in transient transfection assays in two related cell lines derived from mouse mammary adenosarcomas: CSML-0, a nonmetastatic cell line with low levels of mts1 expression, and CSML-100, a metastatic cell line with high levels of mts1 expression. Results of our transient transfection assays in conjunction with results obtained from in vitro and in vivo footprinting of the promoter region show no evidence of cis-acting control elements important for the transcriptional regulation of mts1 in these cell lines. A few nucleotides upstream of the TATA box are sufficient for maximal levels of mts1 transcription. Because no cis-acting control elements were found, restriction of mts1 transcription in CSML-0 cells must exist on some other level. mts1 was found to be hypermethylated in CSML-0 cells but not in CSML-100 cells. The possible role of methylation in progression of the nonmetastatic CSML-0 adenosarcoma cell line toward the metastatic CSML-100 adenosarcoma cell line is discussed.
Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains.
Authors: Wilson K P , Shewchuk L M , Brennan R G , Otsuka A J , Matthews B W
Published: May 2006

The three-dimensional structure of BirA, the repressor of the Escherichia coli biotin biosynthetic operon, has been determined by x-ray crystallography and refined to a crystallographic residual of 19.0% at 2.3-A resolution. BirA is a sequence-specific DNA-binding protein that also catalyzes the formation of biotinyl-5'-adenylate from biotin and ATP and transfers the biotin moiety to other proteins. The level of biotin biosynthetic enzymes in the cell is controlled by the amount of biotinyl-5'-adenylate, which is the BirA corepressor. The structure provides an example of a transcription factor that is also an enzyme. The structure of BirA is highly asymmetric and consists of three domains. The N-terminal domain is mostly alpha-helical, contains a helix-turn-helix DNA-binding motif, and is loosely connected to the remainder of the molecule. The central domain consists of a seven-stranded mixed beta-sheet with alpha-helices covering one face. The other side of the sheet is largely solvent-exposed and contains the active site. The C-terminal domain comprises a six-stranded, antiparallel beta-sheet sandwich. The location of biotin binding is consistent with mutations that affect enzymatic activity. A nearby loop has a sequence that has been associated with phosphate binding in other proteins. It is inferred that ATP binds in this region, adjacent to the biotin. It is proposed that the binding of corepressor to monomeric BirA may promote DNA binding by facilitating the formation of a multimeric BirA-corepressor-DNA complex. The structural details of this complex remain an open question, however.
Emergence of the keratinocyte growth factor multigene family during the great ape radiation.
Authors: Kelley M J , Pech M , Seuanez H N , Rubin J S , O'Brien S J , Aaronson S A
Published: May 2006

The structural gene for human keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, consists of three coding exons and two introns typical of other fibroblast growth factor loci. A portion of the KGF gene, located on chromosome 15, is amplified to approximately 16 copies in the human genome, and these highly related copies (which consist of exon 2, exon 3, the intron between them, and a 3' noncoding segment of the KGF transcript) are dispersed to multiple human chromosomes. The KGF-like sequences are transcriptionally active, differentially regulated in various tissues, and composed of three distinct classes of coding sequences that are 5% divergent from each other and from the authentic KGF sequence. Multiple copies of KGF-like genes were also discovered in the genomic DNAs of chimpanzee and gorilla but were not found in lesser apes (gibbon), Old World monkeys (African green monkey and macaques), mice, or chickens. The pattern of evolutionary occurrence suggests that a primordial KGF gene was amplified and chromosomally dispersed subsequent to the divergence of orangutan from African apes but before the trichotomous divergence of human, chimpanzee, and gorilla 5-8 million years ago. The appearance of a transcriptionally active and chromosomally dispersed multigene KGF family may have implications in the evolution of the great apes and humans.
CD5 acts as a tyrosine kinase substrate within a receptor complex comprising T-cell receptor zeta chain/CD3 and protein-tyrosine kinases p56lck and p59fyn.
Authors: Burgess K E , Yamamoto M , Prasad K V , Rudd C E
Published: May 2006

T-cell antigens including CD2, CD4, CD6, CD8, and CD28 serve as coreceptors with the T-cell receptor (TCR)/CD3 complex in control of T-cell growth. The molecular basis by which these antigens fulfill this role has remained a major issue. An initial clue to this question came with our finding that the sensitivity of in vitro kinase labeling (specifically using protein-tyrosine kinase p56lck) allowed detection of a physical association between CD4-p56lck and the TCR/CD3 complexes. Another T-cell antigen, CD5, is structurally related to the macrophage scavenger receptor family and, as such, can directly stimulate and/or potentiate T-cell proliferation. In this study, we reveal that in Brij 96-based cell lysates, anti-CD5 antibodies coprecipitated TCR zeta chain (TCR zeta)/CD3 subunits as well as the protein-tyrosine kinases p56lck and p59fyn. Conversely, anti-CD3 antibody coprecipitated CD5, p56lck, and p59fyn. Indeed, anti-CD5 and anti-CD3 gel patterns were virtually identical, except for a difference in relative intensity of polypeptides. Anti-CD4 coprecipitated p56lck, p32, and CD3/TCR zeta subunits but precipitated less CD5, suggesting the existence of CD4-TCR zeta/CD3 complexes distinct from the CD5-TCR zeta/CD3 complexes. Consistent with the formation of a multimeric CD5-TCR zeta/CD3 complex, anti-CD5 crosslinking induced tyrosine phosphorylation of numerous T-cell substrates, similar to those phosphorylated by TCR zeta/CD3 ligation. Significantly, as for TCR zeta, CD5 was found to act as a tyrosine kinase substrate induced by TCR/CD3 ligation. The kinetics of phosphorylation of CD5 (t1/2 = 20 sec) was among the earliest of activation events, more rapid than seen for TCR zeta (t1/2 = 1 min). CD5 represents a likely TCR/CD3-associated substrate for protein-tyrosine kinases (p56lck or p59fyn) and an alternative signaling pathway within a multimeric TCR complex.
Muscarinic receptor-operated Ca2+ influx in transfected fibroblast cells is independent of inositol phosphates and release of intracellular Ca2+.
Authors: Felder C C , Poulter M O , Wess J
Published: May 2006

Receptor-mediated changes in cytoplasmic calcium concentrations occur either through release from intracellular calcium stores or by the opening of channels in the plasma membrane, allowing influx of calcium from the extracellular fluid. Carbachol, a muscarinic receptor agonist, stimulated both calcium influx and inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular calcium release in A9 fibroblast cells expressing a m3 muscarinic receptor clone. The calcium influx persisted even after pretreatment of cells with phorbol 12-myristate 13-acetate, which completely prevented the rise in inositol phosphates and intracellular calcium levels. The calcium influx was blocked by divalent cations but was not affected by inhibitors of voltage-dependent calcium channels or high potassium depolarization, indicating the presence of a receptor-operated and voltage-insensitive calcium channel in these cells. Calcium influx was not stimulated by the addition of cAMP analogs or arachidonic acid. To examine the possible involvement of G proteins in m3 receptor-activated calcium influx, two chimeric m2 and m3 muscarinic receptors were expressed in A9 cells in which the third cytoplasmic loop (the primary structural determinant in G protein coupling selectivity of muscarinic receptors) had been exchanged between the m2 receptor, which has no effect on calcium influx, and the m3 receptor. Calcium influx was found to be associated with a structural component of the m3 muscarinic receptor other than the third cytoplasmic loop.
The eukaryotic initiation factor 2-associated 67-kDa polypeptide (p67) plays a critical role in regulation of protein synthesis initiation in animal cells.
Authors: Ray M K , Datta B , Chakraborty A , Chattopadhyay A , Meza-Keuthen S , Gupta N K
Published: May 2006

The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa polypeptide (p67) isolated from reticulocyte lysate protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of active eIF-2 kinases. We have now studied the roles of p67 and eIF-2 kinases in regulation of protein synthesis using several animal cell lysates and an animal cell line (KRC-7) in culture under various growth conditions. The results are as follows. (i) Both p67 and eIF-2 kinase(s) are present in active forms in all animal cells under normal growth conditions and p67 protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation, thus promoting protein synthesis in the presence of active eIF-2 kinases. (ii) In heme-deficient reticulocyte lysates and in serum-starved KRC-7 cells in culture, p67 is deglycosylated and subsequently degraded. This leads to eIF-2 kinase-catalyzed eIF-2 alpha-subunit phosphorylation and thus to protein synthesis inhibition. (iii) Addition of a mitogen (namely, phorbol 12-myristate 13-acetate) to serum-starved KRC-7 cells in culture induces an increase of p67 and thus increases protein synthesis. These results suggest the following conclusions. (i) Protein synthesis inhibition in a heme-deficient reticulocyte lysate is not due to the activation of an eIF-2 kinase (heme-regulated inhibitor), as is generally believed, but is due to degradation of p67. The heme-regulated inhibitor is present in an active form and possibly in equal amounts in both heme-deficient and heme-supplemented reticulocyte lysates but cannot phosphorylate eIF-2 alpha subunit because of the presence of p67. (ii) p67 is essential for protein synthesis as it protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of one or more active eIF-2 kinases present in all animal cells. (iii) p67 is both degradable and inducible. Only the p67 level correlates directly with the protein synthesis activity of the cell, indicating that p67 is a critical factor in protein synthesis regulation in animal cells.
Interaction of a synthetic mitochondrial presequence with isolated yeast mitochondria: mechanism of binding and kinetics of import.
Authors: Roise D
Published: May 2006

The mechanism of interaction of a presequence with isolated yeast mitochondria was examined. A synthetic peptide corresponding to a matrix-targeting signal was covalently labeled with a fluorescent probe. Binding of the presequence to the surface of the mitochondria and translocation of the presequence into the interior of the mitochondria could then be monitored directly in solution by measuring changes in the steady-state fluorescence of the attached fluorophore. The binding step was rapid and reversible. Quantitation of the binding under equilibrium conditions suggested that the initial association of the presequence with the surface of the mitochondria occurred by partitioning of the presequence directly into the lipid bilayer of the outer membrane. Subsequent translocation of the bound presequence into the mitochondria was monitored by measuring the rate of disappearance of presequences sensitive to digestion by added trypsin. The efficiency of translocation was high, and the rate of the translocation was dependent on the electrical potential across the inner membrane. At physiological concentrations of presequence, the rate displayed first-order kinetics with respect to the concentration of bound presequence and had a rate constant of 0.19 min-1 at 20 degrees C. Several kinetic models for the translocation of the presequence are presented that are consistent with the experimental results.
Treatment of premalignancy: prevention of lymphoma in radiation leukemia virus-inoculated mice by cyclosporin A and immunotoxin.
Authors: Yefenof E , Abboud G , Epszteyn S , Vitetta E S
Published: May 2006

Radiation leukemia virus (RadLV)-induced preleukemic (PL) latency is characterized by the appearance of virus-infected PL cells in the thymus. The survival of these PL cells is dependent upon autostimulation with interleukin 4 (IL-4). We have intervened prophylactically in RadLV-induced preleukemia by using cyclosporin-A (CSA), which inhibits IL-4 production, and an immunotoxin (ITx) that kills PL cells. CSA efficiently inhibited IL-4 secretion from RadLV-induced PL and leukemic cells, and its administration to PL mice caused a significant delay in their death. An ITx consisting of anti-RadLV glycoprotein-70 (gp70) antibody coupled to ricin A chain efficiently inhibited protein synthesis in virus-infected cells in vitro and, when injected into PL mice, also delayed their death. Combined treatment with CSA and ITx prevented 75% of the treated PL mice from developing lymphoma. These results show that the development of malignancy from a premalignant state can be averted by a combination of therapeutic modalities that decrease the size and growth rate of the premalignant cell population.
Mechanisms of the inhibition of reverse transcription by antisense oligonucleotides.
Authors: Boiziau C , Thuong N T , Toulmé J J
Published: May 2006

We have demonstrated that the synthesis of cDNA by avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases can be prevented by oligonucleotides bound to the RNA template approximately 100 nucleotides remote from the 3' end of the primer. The RNA was truncated at the level of the antisense oligonucleotide-RNA duplex during the reverse transcription. The key role played by the reverse transcriptase-associated RNase H activity in the inhibition process was shown by the use of (i) inhibitors of RNase H (NaF or dAMP), (ii) Moloney murine leukemia virus reverse transcriptase devoid of RNase H activity, or (iii) alpha-analogues of oligomers that do not elicit RNase H-catalyzed RNA degradation. In all three cases the inhibitory effect was either reduced (NaF, dAMP) or totally abolished. However, an alpha-oligomer bound to the sequence immediately adjacent to the primer-binding site prevented reverse transcription. Therefore, initiation of polymerization can be blocked by means of an RNase H-independent mechanism, whereas arrest of a growing cDNA strand can be achieved only by an oligonucleotide mediating cleavage of the template RNA.
Immunosuppression in the definitive and intermediate hosts of the human parasite Schistosoma mansoni by release of immunoactive neuropeptides.
Authors: Duvaux-Miret O , Stefano G B , Smith E M , Dissous C , Capron A
Published: May 2006

Evidence supporting the concept that the parasitic trematode Schistosoma mansoni may escape immune reactions from its vertebrate (man) or invertebrate (the freshwater snail Biomphalaria glabrata) hosts by using signal molecules it has in common with these hosts was obtained by the following experiments. The presence of immunoactive proopiomelanocortin (POMC)-derived peptides [corticotropin (ACTH), beta-endorphin] in, and their release from, S. mansoni was demonstrated. Coincubation of adult worms with human polymorphonuclear leukocytes or B. glabrata immunocytes led to the appearance of alpha-melanotropin (MSH) in the medium. The conclusion that this alpha-MSH resulted from conversion of the parasite ACTH by neutral endopeptidase 24.11 (NEP) present on these cells was supported by the fact that the alpha-MSH level in the medium was markedly reduced by addition of the specific NEP inhibitor phosphoramidon. This interpretation is substantiated by the fact that no conversion was observed in comparable tests with human monocytes, which exhibit no NEP activity. alpha-MSH has the capacity to inactivate formerly active immunocytes not only from the definitive host (man, hamster) but also from the intermediate host (B. glabrata), as determined by microscopic computer-assisted examination of conformational changes. POMC-derived peptides have been detected in B. glabrata hemolymph 2, 10, and 24 days after infection by S. mansoni miracidia. Immunocytes from infected snails were found to be inactivated, and this inactivation was prevented by antibodies directed against ACTH and alpha-MSH. The immunoactive beta-endorphin released from S. mansoni does not appear to be subject to enzymatic conversion. Since it is active at lower concentrations, it may be used for distant signaling.
Cloning and chromosomal mapping of a human immunodeficiency virus 1 "TATA" element modulatory factor.
Authors: Garcia J A , Ou S H , Wu F , Lusis A J , Sparkes R S , Gaynor R B
Published: May 2006

A critical regulatory element in many promoters transcribed by RNA polymerase II is the "TATA" box, which is located 25-30 nucleotides upstream of the transcription initiation site. TFIID is a biochemically defined HeLa cell nuclear fraction containing a transcription factor activity that binds specifically to the TATA box and is critical in determining both basal and regulated promoter activity. Recently, the gene for a TATA-binding protein was cloned and found to bind to various TATA elements and to substitute for TFIID in stimulating basal gene expression in in vitro transcription systems. However, it is possible that additional cellular factors can bind to the TATA element and influence the level of gene expression. By using lambda gt11 expression cloning with oligonucleotides corresponding to the human immunodeficiency virus 1 TATA element, we report the identification of a cellular protein with a calculated molecular mass of 123 kDa that we designate TATA element modulatory factor (TMF). TMF binds to the human immunodeficiency virus 1 TATA element in gel-retardation assays and inhibits activation of the viral long terminal repeat by the TATA-binding protein in in vitro transcription assays. TMF contains leucine-zipper amino acid motifs and exhibits homology in its DNA binding domain with the phage-encoded DNA binding protein Ner. Chromosomal mapping localizes the TMF gene to human chromosome 3p12-p21, which is a site of frequent rearrangements in lung and renal carcinomas. Thus, TMF is a transcription factor that likely regulates the expression of both viral and cellular genes.
Retroviral analysis of cardiac morphogenesis: discontinuous formation of coronary vessels.
Authors: Mikawa T , Fischman D A
Published: May 2006

Cellular progenitors of the coronary vasculature are believed to enter the chicken heart during epicardial morphogenesis between stages 17 and 27 (days 3-5) of egg incubation. To trace cells which give rise to the coronary arteries in vivo, we applied retroviral cell tagging procedures and analyzed clonal populations of vascular smooth muscle, endothelium, and connective tissue in the hearts of post-hatch chickens. Our data provide direct proof that (i) vascular smooth muscle progenitors begin to enter the heart at stage 17, substantially after the heart begins propulsive contractions; (ii) cardiac myocytes, vascular smooth muscle, perivascular fibroblasts, and coronary endothelial cells all derive from independent precursors when these cells migrate into the heart; (iii) endothelial cells of the coronary vessels have a different clonal origin than endothelial cells of the endocardium; (iv) coronary arteries form by the coalescence of discontinuous colonies (i.e., in situ vasculogenesis), each derived from a founder cell tagged at the time of retroviral injection (stages 17-18); and (v) coronary arteries contain discrete segments composed of "polyclones." These studies indicate the feasibility of gene targeting to coronary progenitors through the use of recombinant retroviruses.
High-resolution mapping of mammalian genes by in situ hybridization to free chromatin.
Authors: Heng H H , Squire J , Tsui L C
Published: May 2006

Fluorescence in situ hybridization to metaphase chromosomes or chromatin fibers in interphase nuclei is a powerful technique in mapping genes and DNA segments to specific chromosome region. We have been able to release the chromatin fibers from cells arrested at G1 and G2 phases using different drugs and a simple alkaline lysis procedure. We have also demonstrated specific hybridization of fluorescence-labeled probes to single-copy genomic DNA sequences on the free chromatins. Fluorescence in situ hybridization signals have been detected for sequences separated as close as 21 kilobase pairs and as far as 350 kilobase pairs, with excellent correspondence between the observed and expected distances. The resolution of this technique should approach 10 kilobase pairs and its coverage should span millions of base pairs. Therefore, free chromatin mapping can be generally used to study the structure and organization of mammalian genomes.
Birth and migration of neurons in the central posterior/prepacemaker nucleus during adulthood in weakly electric knifefish (Eigenmannia sp.).
Authors: Zupanc G K , Zupanc M M
Published: May 2006

In contrast to mammals, fish maintain their capacity to generate neurons in the central nervous system even during adulthood for prolonged periods of life. By employing immunohistochemical, autoradiographic, and electron microscopic techniques, we studied such a postnatal neurogenesis within the complex of the central posterior/prepacemaker nucleus (CP/PPn) in knifefish (Eigenmannia sp.), a weakly electric teleost. The CP/PPn is a bilateral cluster of neurons in the thalamus. It controls frequency modulations of the electric organ discharge as they are used during social interactions. In the CP/PPn region adjacent to the wall of the third ventricle ("ventricular zone"), cells are born continuously and at high rates. They undergo multiple cell divisions before differentiating into neurons. Concomitant with this development, the newborn neurons migrate toward lateral regions of the CP/PPn. In the course of this lateral migration, they appear to acquire immunological and morphological characteristics that are typical for mature CP/PPn neurons. We hypothesize that at least some of the newly generated cells develop finally into functional CP/PPn neurons.
Farnesylcysteine, a constituent of the alpha and beta subunits of rabbit skeletal muscle phosphorylase kinase: localization by conversion to S-ethylcysteine and by tandem mass spectrometry.
Authors: Heilmeyer L M , Serwe M , Weber C , Metzger J , Hoffmann-Posorske E , Meyer H E
Published: May 2006

The primary structure of the alpha and beta subunits of phosphorylase kinase reveals that both proteins contain a carboxyl-terminal CA1A2X motif (where C is cysteine, A1 and A2 are aliphatic amino acids, and X is an uncharged amino acid), the recognition signal for a protein polyisoprenyltransferase. The product, a polyisoprenylated cysteine, can be detected by phenylthiocarbamoylamino acid analysis and by microsequencing following conversion to S-ethylcysteine. Mass spectrometry confirms a covalently linked farnesyl residue in both subunits. Tandem mass spectrometry localizes these modifications at the cysteine residues present in the carboxyl-terminal CAMQ and CLVS sequences of the alpha and beta subunits, respectively. Membrane association of phosphorylase kinase, probably mediated by these farnesyl residues, is discussed.
Sphingosine 1-phosphate, a specific endogenous signaling molecule controlling cell motility and tumor cell invasiveness.
Authors: Sadahira Y , Ruan F , Hakomori S , Igarashi Y
Published: May 2006

Sphingosine 1-phosphate (Sph-1-P), the initial product of Sph degradation by Sph kinase, was shown to be a strong inhibitor of cell motility and phagokinesis of B16 melanoma and other types of cells at 10-100 nM concentration. It also inhibited "chemoinvasion" of tumor cells through a thick layer of Matrigel on a filter membrane. Such inhibitory effects were produced minimally or not at all by Sph, N-methyl derivatives of Sph, or other related sphingolipids and phospholipids. Sph-1-P did not inhibit cell proliferation or protein kinase C (PKC) activity, in contrast to Sph and N-methyl-Sph, which inhibit PKC activity and cell growth in general. Radiolabeled [3H]Sph and [14C]N-methyl-Sph were rapidly incorporated into B16 melanoma cells. However, [14C]N-methyl-Sph was not metabolically converted into other compounds, whereas [3H]Sph was efficiently converted within 10 min to Sph-1-P, followed by conversion to other sphingolipids and phospholipids. The inhibitory effect of Sph-1-P on cell motility and tumor cell invasiveness could be a specific phenomenon independent of PKC and other known transmembrane signaling mechanisms, based on an unknown mechanism. It may directly affect organizational assembly of actin filaments. Since exogenous Sph is rapidly converted into Sph-1-P, some reported effects of Sph may be ascribable to such conversion.
Regulation of the reverse transcriptase of human immunodeficiency virus type 1 by dNTPs.
Authors: West A B , Roberts T M , Kolodner R D
Published: May 2006

Reverse transcriptase (RNA-directed DNA polymerase, EC 2.7.7.49) of human immunodeficiency virus type 1 has been examined with respect to the steady-state kinetics of polymerization of dNTPs into product DNA. With dNTPs as variable substrate, the kinetics of polymerization deviated from standard Michaelis-Menten kinetics. Substrate inhibition was observed at high substrate concentrations and negative cooperativity was seen at lower substrate concentrations. Examination of incorporation of substrate dNMPs in the presence of nucleotides not complementing the template demonstrated that dNTPs may act as noncompetitive inhibitors, as well as substrate. The Ki of the enzyme for dNTPs was 104 microM. A working model is presented that accounts for the substrate inhibition. In this model, the reverse transcriptase is a multisubunit holoenzyme, where noncompetitive inhibition is mediated by one subunit binding nucleotide and down-regulating the enzymatically active 64-kDa subunit. With additional assumptions, this model can accommodate the negative cooperativity observed.
Homology of a vesicular amine transporter to a gene conferring resistance to 1-methyl-4-phenylpyridinium.
Authors: Stern-Bach Y , Keen J N , Bejerano M , Steiner-Mordoch S , Wallach M , Findlay J B , Schuldiner S
Published: May 2006

The vesicular amine transporter (VAT) catalyzes transport and storage of catechol and indolamines into subcellular organelles in a wide variety of cells. It plays a central role in neurotransmission and is the primary target for several pharmacological agents. One of the drugs, reserpine, binds very tightly to the transporter and remains bound even after solubilization, a finding that has proven useful for purification of the transporter from bovine adrenal medulla in a fully functional state. The sequences of 26 N-terminal amino acids and of an additional 7-amino acid internal peptide are presented. Antibodies against a synthetic peptide based on the above sequences immunoprecipitate the transporter, confirming the conclusion that the peptide sequence is derived from bovine VAT. To our knowledge, documentation of sequences of vesicular neurotransmitter transporters has not been presented previously. In addition, the sequences obtained are highly homologous to the predicted sequence of a protein from PC12 cells that confers to Chinese hamster ovary cells resistance to 1-methyl-4-phenylpyridinium (MPP+), an agent that causes parkinsonism in model systems, confirming the hypothesis that the protein conferring resistance to MPP+ is a VAT.
Establishment of a highly sensitive and specific exon-trapping system.
Authors: Hamaguchi M , Sakamoto H , Tsuruta H , Sasaki H , Muto T , Sugimura T , Terada M
Published: May 2006

We have established a highly sensitive and specific exon-trapping system (SETS) with a specific plasmid vector in which an exon in a given DNA segment is identified by its ability to remain as a mature mRNA after splicing. The SETS provides us with the isolation of possible exons rapidly and easily from DNA fragments in chromosomal regions of more than 300 kilobase pairs. Genomic DNA fragments were partially digested and subsequently cloned into plasmid pMHC2, an exon-trapping vector we have constructed. These constructs were transfected into COS-7 cells, and consequent RNA transcripts were spliced in the cells. The resulting mature mRNA was harvested and amplified by using reverse transcription-PCR. Possible exons can be recognized by the sizes of PCR products and cloned into a plasmid vector. The SETS provides a direct means of cloning exons from genomic DNA of more than 300 kilobase pairs within a short period of time. Using this system, we have screened 300-kilobase-pair genomic DNA segments derived from human chromosome 11q13. Human chromosome 11q13 may contain genes responsible for human cancers, because DNA amplification is observed in several malignant tumors. We have successfully identified exon 2 of the HST1 gene and additional transcribed sequences.
Second-site revertants of an arginine-210 to lysine mutation in the a subunit of the F0F1-ATPase from Escherichia coli: implications for structure.
Authors: Howitt S M , Cox G B
Published: May 2006

Arg-210 of the a subunit of the Escherichia coli F0F1-ATPase has been proposed previously as a component of the proton pore. A mutant in which lysine was substituted for Arg-210 was generated and was found to be unable to translocate protons. A plasmid carrying this mutation, along with wild-type genes encoding the c and b subunits, was unusual in that it failed to complement a chromosomal c-subunit mutation on succinate minimal medium. Three revertants on succinate minimal medium contained plasmids that showed complementation with chromosomal c-subunit but not with a-subunit mutations. One of these had a deletion in the a subunit. The other two were point mutations, resulting in the substitution of aspartic acid by Gly-53 and of arginine for Leu-211. The Gly-53 to aspartic acid change implied that Gly-53 and Arg-210 are normally in close proximity. To test this idea further, a series of mutants in which aspartic acid was placed in helix I at positions ranging from 42 to 57 was generated. Full complementation was regained only when the aspartic acid residue was present on the same side of a putative helix as Gly-53 over a span of three turns of the alpha-helix. These results and others suggest modifications of a previously proposed model for the transmembrane helices of the F0 portion of the F0F1-ATPase. The implications of these modifications for the mechanism of proton translocation are discussed.
Antirepression function in Escherichia coli for the cAMP-cAMP receptor protein transcriptional activator.
Authors: Forsman K , Sondén B , Göransson M , Uhlin B E
Published: May 2006

The cAMP receptor protein (CRP) complex (cAMP-CRP) is a global regulator of gene expression. It influences transcription from a number of promoters in Escherichia coli, including two divergently oriented promoters in the pap pili-adhesin gene system. To further define the role of cAMP-CRP in pap regulation we monitored protein-DNA interactions in vitro and levels of pap transcription in vivo in wild-type and mutant pap-containing clones. The results showed that activation was mediated by a single cAMP-CRP-binding site centered at nucleotide positions -215.5 and -115.5 relative to the transcriptional start points. A target for the pap-specific regulatory protein PapB was localized adjacent to the cAMP-CRP-binding site. The long-range effects exerted from the protein-binding sites were consistent with the idea that cAMP-CRP caused a change in the local DNA conformation and that a nucleoprotein complex (involving cAMP-CRP and PapB) was formed in the region between the pap promoters. Moreover, transcription became independent of activation of cAMP-CRP and the PapB protein in a mutant lacking the nucleoid-associated protein H-NS. Our findings suggest that the cAMP-CRP complex mediates its positive regulatory function by alleviating transcriptional silencing and, as such, plays a role as antirepressor.
A protein methyltransferase specific for altered aspartyl residues is important in Escherichia coli stationary-phase survival and heat-shock resistance.
Authors: Li C , Clarke S
Published: May 2006

Proteins are subject to spontaneous degradation reactions including the deamidation, isomerization, and racemization of asparaginyl and aspartyl residues. A major product of these reactions, the L-isoaspartyl residue, is recognized with high affinity by the protein-L-isoaspartate(D-aspartate) O-methyltransferase (EC 2.1.1.77). This enzyme catalyzes the methyl esterification of the L-isoaspartyl residue in a reaction that can initiate its conversion to the normal aspartyl configuration. To directly study the physiological role of this methyltransferase, especially with respect to the potential repair of isomerized aspartyl residues in aging proteins, we examined the ability of the bacterium Escherichia coli to survive in the absence of its activity. We utilized gene disruption techniques to replace the chromosomal copy of the pcm gene that encodes the methyltransferase with a kanamycin-resistance cassette to produce mutants that have no detectable L-isoaspartyl methyltransferase activity. Although no changes in exponential-phase growth were observed, pcm- mutants did not survive well upon extended culture into stationary phase or upon heat challenge at 55 degrees C. These results provide genetic evidence for a role of the L-isoaspartyl methyltransferase in the metabolism of altered proteins that can accumulate in aging cells and limit their viability.