bluuebunny/crossref_metadata_2025 huggingface dataset:
Native and mutant 5-lipoxygenase expression in a baculovirus/insect cell system.
Authors: Funk C D , Gunne H , Steiner H , Izumi T , Samuelsson B
Published: May 2006

Human 5-lipoxygenase (EC 1.13.11.34), the key enzyme involved in the transformation of arachidonic acid to the potent biologically active leukotrienes, has been overexpressed in insect cells using a baculovirus expression system. A recombinant baculovirus (3B6) carrying the human 5-lipoxygenase coding sequence downstream of the strong polyhedrin protein promoter was isolated. Approximately 48 hr after infection of Spodoptera frugiperda cells with the recombinant baculovirus, maximal intracellular enzyme activity and protein levels were detected. The recombinant 5-lipoxygenase in 10,000 x g supernatant fractions was able to synthesize large amounts of 5-hydroperoxy-6,8,10,14-icosatetraenoic acid, together with smaller amounts of the nonenzymatic hydrolysis products of leukotriene A4, and exhibited a dependence on Ca2+ and ATP for maximal activity. Immunoblot analysis of supernatant proteins from human leukocytes and recombinant virus-infected cells indicated the presence of indistinguishable approximately 80-kDa bands. However, 5-lipoxygenase protein in recombinant-infected cells was found to be present in amounts 50-200 times that present in leukocytes on a per-cell basis. Histidine-362 and histidine-372, potential iron-atom ligands within a putative iron-binding domain, were changed to serine residues. Recombinant baculoviruses carrying the mutations were isolated and used to infect insect cells. Although infected cells were able to express mutant 5-lipoxygenase protein, enzyme activity was not substantially altered, suggesting the nonessential nature of these histidines in binding iron at the putative ferric catalytic site.
Involvement of vimentin in the reverse transformation reaction.
Authors: Chan D , Goate A , Puck T T
Published: May 2006

An organized cytoskeleton is required for the cAMP-induced reverse transformation reaction in CHO-K1 cells. In the course of the reaction a considerable fraction of the genome changes its nuclease sensitivity. The current paper presents the following evidence that cAMP-induced phosphorylation of vimentin is an early step in this reaction complex. (i) Vimentin is only slightly phosphorylated in transformed CHO-K1 cells but is heavily phosphorylated in normal fibroblasts. (ii) cAMP addition almost triples the vimentin phosphorylation of CHO-K1 cells but does not change that of normal cells. (iii) Vimentin phosphorylation is one of the earliest phenomena to occur after addition of cAMP to CHO-K1 cells, preceding the cell-stretching reaction and other manifestations of reverse transformation. (iv) Indirect immunofluorescence experiments demonstrate that vimentin appears as a condensed mass in transformed CHO-K1 cells but cAMP addition restores the filamentous structure characteristic of the normal fibroblast. (v) Other transformed cells unresponsive to reverse transformation by cAMP failed to demonstrate increased phosphorylation of vimentin on treatment with cAMP. These results support the proposed scheme that phosphorylation of cytoskeletal elements initiates a large-scale genetic regulatory action in which a substantial change in the spectrum of genome exposure and sequestration occurs. A function for intermediate filaments in reverse transformation is implied.
Modulation of 3-methylcholanthrene toxicity in cultured neoplastic keratinocytes by glucocorticoids and retinoids is not accounted for by macromolecular adduct formation.
Authors: Rubin A L , Rice R H
Published: May 2006

3-Methylcholanthrene (3-MC) greatly inhibits the growth of two lines of human squamous carcinoma cells, SCC-9 and SCC-12B2. Exposure of the cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin alone was much less effective and, in the presence of 3-MC, did not alter the sensitivity (EC50 = 0.3 microM) or extent of growth inhibition by the latter. The degree of 3-MC-mediated inhibition, however, was markedly alleviated by inclusion of retinoic acid (EC50 greater than or equal to 0.7 microM) and hydrocortisone (EC50 = 40 nM) or dexamethasone (EC50 = 3 nM) in the culture medium. These physiological effectors, which are known to have opposing actions on keratinocyte character in SCC cells, did not significantly alter either aryl hydrocarbon hydroxylase activity or macromolecular adduct formation. Further analysis of the cellular responses indicated that hydrocortisone and, in some experiments, retinoids increased the growth rate in 3-MC-exposed cultures, while 3-MC increased the saturation density in retinoic acid-exposed cultures, an example of interference with a physiological response of the cells. These results indicate that alteration of the differentiated state, regardless of the direction of the change, can alter the sensitivity of the cells to toxic stimuli. Further investigation of the bases of such toxic responses and their modulation by the microenvironment may enhance our understanding of the target cell specificity of polycyclic aromatic hydrocarbons.
Antibodies specific to the retinoic acid human nuclear receptors alpha and beta.
Authors: Gaub M P , Lutz Y , Ruberte E , Petkovich M , Brand N , Chambon P
Published: May 2006

Two cDNAs encoding two human receptors for retinoic acid (RA), RAR-alpha and RAR-beta, have been characterized recently. Synthetic peptides corresponding to the cDNA-deduced amino acid sequences unique to RAR-alpha and RAR-beta were used to generate anti-RAR-alpha antiserum (SP171) and anti-RAR-beta antisera (SP172 and SP248). The specificity of these antisera was confirmed both by immunocytochemical detection of these receptors in COS-1 cells transfected with RAR-alpha and RAR-beta expression vectors and by immunoblot analyses performed with whole extracts of these cells. We also demonstrate that these antisera recognize RAR-alpha and RAR-beta endogenously expressed in the RA-responsive human promyelocytic leukemia cell line HL-60.
Changes in stability and allosteric properties of aspartate transcarbamoylase resulting from amino acid substitutions in the zinc-binding domain of the regulatory chains.
Authors: Eisenstein E , Markby D W , Schachman H K
Published: May 2006

Changes in subunit interaction energies linked to the allosteric transition of the regulatory enzyme aspartate transcarbamoylase (ATCase; EC 2.1.3.2) from Escherichia coli are localized in part at interfaces between the six catalytic (c) and six regulatory (r) polypeptide chains. Site-directed mutagenesis has been used to construct enzymes with amino acid substitutions in a limited region of the zinc-binding domain of the r chains. Substitution of Ser or His for r114 Cys, one of four cysteines binding the structural zinc ion in the regulatory chain, leads to incorrectly folded chains as shown by the inability to detect stable assembled holoenzyme in cell extracts. Replacement of r111 Asn by Ala at the interface between an r chain and a c chain in the apposing catalytic trimer causes a complete loss of the homotropic and heterotropic effects characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrated that this mutant enzyme exists in the R ("relaxed") conformation in the absence of active site ligands due to preferential destabilization of the T ("taut") conformation relative to the R state. In contrast, replacement of r113 Asn by Ala at the interface between adjacent r and c chains leads to an increase in the cooperativity of the enzyme. When r139 Lys is replaced by Met, Vmax is reduced by 50% compared to wild-type ATCase, whereas it is increased about 2-fold when r142 Glu is replaced by Asp. Amino acid substitutions in this domain significantly affect subunit interaction energy as measured by rate of subunit exchange when holoenzymes are incubated with isolated catalytic subunits, thus permitting measurements of the effect of the bisubstrate analog N-(phosphonacetyl)-L-asparatate in weakening intersubunit interactions. Subunit exchange increased about 9-fold for the r142 Glu----Asp mutant and almost 20-fold for the r142 Glu----Ala mutant in the presence of the ligand.
Successive steps in the process of immortalization identified by transfer of separate bovine papillomavirus genes into rat fibroblasts.
Authors: Cerni C , Binétruy B , Schiller J T , Lowy D R , Meneguzzi G , Cuzin F
Published: May 2006

Transfer of neor and bovine papillomavirus type 1 (BPV1) DNA into rat embryo fibroblasts led to colony formation in G418-containing medium, with no detectable background in controls with neor DNA alone. More than 50% of the drug-resistant clones could be further propagated in culture. The genetic functions of BPV1 involved in colony formation and in long-term immortalization were investigated by both translation termination mutations in the full-length genome, which inactivate individual open reading frames, and constructs in which these open reading frames were separately expressed under control of long terminal repeat promoter enhancers. Expression of either open reading frame E2 or E5 was sufficient for formation of a drug-resistant colony, but long-term growth in culture required that of E6. No significant cooperative effect was observed upon cotransfection of BPV1 and ras oncogene DNAs. Expression of the early region of the human papillomavirus type 16 also led to immortalization of rat embryo fibroblast cells in the same assay, and, unlike what was previously reported in baby rat kidney cells, it required neither activation by a heterologous promoter, nor a cooperating ras oncogene.
Exonuclease III and the catalase hydroperoxidase II in Escherichia coli are both regulated by the katF gene product.
Authors: Sak B D , Eisenstark A , Touati D
Published: May 2006

The levels of both exonuclease III (exo III, product of xthA) and hydroperoxidase II (HP-II, product of katE) activity in Escherichia coli were influenced by a functional katF gene. The katF gene product is also necessary for synthesis of HP-II. Mutations in either katF or xthA, but not katE, result in sensitivity to H2O2 and near-UV (300-400 nm) radiation. Exo III, encoded by the xthA locus, recognizes and removes nucleoside 5'-monophosphates near apurinic and apyrimidinic sites in damaged DNA. Extracts of katF mutant strains had little detectable exo III activity. When a katF+ plasmid was introduced into the katF mutant, exo III activity exceeded wild-type levels. We propose that the katF gene is a trans-acting positive regulator of exo III and HP-II enzymes, both of which are involved in cellular recovery from oxidative damage.
T-cell mitogenesis stimulates the synthesis of a mRNA species coding for a 43-kDa peptide reactive with CM-H-9, a monoclonal antibody specific for placental isoferritin.
Authors: Moroz C , Shterman N , Kupfer B , Ginzburg I
Published: May 2006

In studying the changes that occur in concanavalin A-activated T lymphocytes, an mRNA species was discovered by hybridization of poly(A)+ mRNA with a human ferritin heavy chain cDNA probe. This ferritin mRNA, termed superheavy chain mRNA, differed from the known human ferritin heavy chain mRNAs by its larger size and degree of homology. The superheavy chain mRNA was isolated by sucrose-gradient centrifugation and translated in vitro in a cell-free system. The products obtained included two peptides (superheavy) of 43kDa that reacted with CM-H-9, a monoclonal antibody specific for placental isoferritin. De novo synthesis in intact transformed T cells revealed the synthesis of the superheavy chain peptides that were immunoprecipitated by anti-ferritin monoclonal antibody CM-G-8 and by placental isoferritin specific monoclonal antibody CM-H-9. The above results indicated that blast transformation of human T cells stimulated the appearance of a unique mRNA species that coded for a superheavy chain peptide associated with placental isoferritin, which was not detected in resting T cells.
Antibodies to CD4 in individuals infected with human immunodeficiency virus type 1.
Authors: Kowalski M , Ardman B , Basiripour L , Lu Y C , Blohm D , Haseltine W , Sodroski J
Published: May 2006

The attachment of human immunodeficiency virus type 1 (HIV-1) to target cells is mediated by a specific interaction between the viral envelope glycoprotein (gp120) and the CD4 receptor. Here we report that approximately 10% of HIV-1-infected individuals produce antibodies that recognize the extracellular portion of the CD4 molecule. Carboxyl-terminal deletions of CD4 that do not affect HIV-1 gp120 binding eliminate recognition of CD4 by patient antisera. In contrast, mutations in the amino-terminal domain of CD4 that attenuate HIV-1 gp120 binding do not diminish CD4 recognition by patient antisera. These results suggest that HIV-1 infection can generate antibodies directed against a region of the viral receptor distinct from the virus-binding domain.
Involvement of myosin light-chain kinase in endothelial cell retraction.
Authors: Wysolmerski R B , Lagunoff D
Published: May 2006

Permeabilized bovine pulmonary artery endothelial cell monolayers were used to investigate the mechanism of endothelial cell retraction. Postconfluent endothelial cells permeabilized with saponin retracted upon exposure to ATP and Ca2+. Retraction was accompanied by thiophosphorylation of 19,000-Da myosin light chains when adenosine 5'-[gamma-[35S]thio]triphosphate was included in the medium. Both retraction and thiophosphorylation of myosin light chains exhibited a graded quantitative dependence on Ca2+. When permeabilized monolayers were extracted in buffer D containing 100 mM KCl and 30 mM MgCl2 for 30 min, the cells failed to retract upon exposure to ATP and Ca2+, and no thiophosphorylation of myosin light chains occurred. The ability both to retract and to thiophosphorylate myosin light chains was restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by either alone. These studies indicate that endothelial cell retraction, as does smooth muscle contraction, depends on myosin light-chain kinase phosphorylation of myosin light chains.
Long-distance activation of the Myc protooncogene by provirus insertion in Mlvi-1 or Mlvi-4 in rat T-cell lymphomas.
Authors: Lazo P A , Lee J S , Tsichlis P N
Published: May 2006

T-cell lymphomas induced by Moloney murine leukemia virus frequently have proviruses integrated at the Mlvi-4 and Mlvi-1 loci, which map approximately 30 and 270 kilobases 3' of the promoter region of the Myc protooncogene, respectively. Provirus insertion in these loci is responsible for the activation of adjacent genes. To determine whether Myc expression was also affected by these provirus insertions, we constructed T-cell hybrids between two rat thymic lymphomas containing a provirus in Mlvi-4 or Mlvi-1 and the murine T-cell lymphoma line BW5147. These hybrids segregated the provirus-containing rearranged alleles from the normal nonrearranged alleles of Mlvi-4 and Mlvi-1, and they carried an intact copy of rat Myc. Using an S1 nuclease protection assay, we observed that the expression of the rat Myc cosegregated with the rearranged Mlvi-4 or Mlvi-1 locus. However, provirus insertion in these loci had no effect on promoter utilization or on the expression of the murine Myc locus. We conclude that provirus insertion exerts a long-range cis effect on the expression of Myc. Therefore, provirus integration in a single locus may affect the expression of multiple genes, some of which may be located a long distance from the site of integration.
GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction.
Authors: Baker D , Wuestehube L , Schekman R , Botstein D , Segev N
Published: May 2006

The 21-kDa GTP-binding Ypt1 protein (Ypt1p) is required for protein transport from the endoplasmic reticulum to the Golgi complex in yeast extracts. Ypt1 antibodies block transport; this inhibition is alleviated by competition with excess purified Ypt1p produced in bacteria. Furthermore, extracts of cells carrying the mutation ypt1-1 are defective in transport, but transport is restored if a cytosolic fraction from wild-type cells is provided. The in vitro transport reaction also requires physiological levels of Ca2+. However, Ypt1p functions independently of Ca2+. First, buffering the free Ca2+ at concentrations ranging from 1 nM to 10 microM does not relieve inhibition by Ypt1 antibodies. Second, consumption of a Ca2+-requiring intermediate that accumulates in Ca2+-deficient incubations is not inhibited by anti-Ypt1 antibodies, although completion of transport requires ATP and an N-ethylmaleimide-sensitive factor. Thus, Ypt1p and Ca2+ are required at distinct steps.
Subcellular distribution of an inhalational anesthetic in situ.
Authors: Eckenhoff R G , Shuman H
Published: May 2006

To better understand the mechanisms and sites of anesthetic action, we determined the subcellular partitioning of halothane in a tissue model. A method was found to fix the in vivo distribution of halothane in rat atrial tissue for subsequent electron microscopy and x-ray microanalysis. Atrial strips were exposed to various concentrations of halothane, rapidly frozen, cryo-sectioned, and cryo-transferred into an electron microscope. Irradiation of the hydrated cryosections with the electron beam caused halothane radiolysis, which allowed retention of the halogen-containing fragments after dehydration of the sections. The bromine from halothane was detected and quantified with x-ray microanalysis in various microregions of atrial myocytes. Halothane (bromine) partitioned largely to mitochondria, with progressively lower concentrations in sarcolemma, nuclear membrane, cytoplasm, sarcomere, and nucleus. Partitioning could not be explained solely by distribution of cellular lipid, suggesting significant and differential physicochemical solubility in protein. However, we found no saturable compartment in atrial myocytes within the clinical concentration range, which implies little specific protein binding.
Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements.
Authors: Band V , Zajchowski D , Kulesa V , Sager R
Published: May 2006

Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing in a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectants tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grow and investigate may also be immortalized by HPV.
Molecular dissection of mutations at the heterozygous thymidine kinase locus in mouse lymphoma cells.
Authors: Applegate M L , Moore M M , Broder C B , Burrell A , Juhn G , Kasweck K L , Lin P F , Wadhams A , Hozier J C
Published: May 2006

The mouse lymphoma L5178Y TK+/- 3.7.2C cell line allows quantitation of induced TK(+/-)----TK-/- mutations at the heterozygous thymidine kinase (Tk) locus. TK-/- mutant colonies show a bimodal size distribution, reflecting a difference in the growth rates of the two size classes that is hypothesized to result from different degrees of genetic damage. The two homologous chromosomes 11 containing the alleles of the Tk gene in L5178Y 3.7.2C TK+/- cells are distinguishable at the cytogenetic level. We find, in addition, that the two alleles are distinguishable at the molecular level because of an Nco I restriction fragment length polymorphism at the 3' end of the gene. In a set of 51 large-colony and 48 small-colony TK-/- mutants induced by ionizing radiation or by chemical mutagens, we find that 78, including all except one of the small-colony mutants, have lost the Tk+ allele and that some of these have two to four copies of the remaining Tk- allele. Nineteen of the large-colony TK-/- mutants that do not show Tk+ allele loss show no other structural changes detectable at the level of Southern blot analysis. One shows a partial deletion. The variety of mutagenic lesions recorded at the heterozygous Tk locus may be representative of events observed in human malignancy and may include a wider range of mutagenic events than can be observed at hemizygous test loci.
Holomorphic curves in surfaces of general type.
Authors: Lu S S , Yau S T
Published: May 2006

This note answers some questions on holomorphic curves and their distribution in an algebraic surface of positive index. More specifically, we exploit the existence of natural negatively curved "pseudo-Finsler" metrics on a surface S of general type whose Chern numbers satisfy c(2)1>2c2 to show that a holomorphic map of a Riemann surface to S whose image is not in any rational or elliptic curve must satisfy a distance decreasing property with respect to these metrics. We show as a consequence that such a map extends over isolated punctures. So assuming that the Riemann surface is obtained from a compact one of genus q by removing a finite number of points, then the map is actually algebraic and defines a compact holomorphic curve in S. Furthermore, the degree of the curve with respect to a fixed polarization is shown to be bounded above by a multiple of q - 1 irrespective of the map.
Changes in the sialylation and sulfation of secreted thyrotropin in congenital hypothyroidism.
Authors: Gyves P W , Gesundheit N , Thotakura N R , Stannard B S , DeCherney G S , Weintraub B D
Published: May 2006

We have examined the oligosaccharide structure of secreted thyrotropin (TSH) in perinatal and mature rats with congenital primary hypothyroidism. Rat pituitaries from euthyroid control animals and those rendered hypothyroid by methimazole treatment were incubated with [3H]glucosamine in vitro. Secreted TSH was purified, and oligosaccharides were enzymatically released and characterized by anion-exchange HPLC. In perinatal hypothyroid animals compared with control animals, oligosaccharides from TSH alpha and beta subunits contained more species with three or more negative charges. Moreover, perinatal hypothyroid animals demonstrated a dramatic increase in the ratio of sialylated to sulfated species within oligosaccharides of the same negative charge (2.9- to 7.4-fold increase for TSH-alpha; 15.1- to 25.5-fold increase for TSH-beta). In mature hypothyroid 9-week-old animals compared with control animals, changes were less pronounced, suggesting that endocrine regulation of oligosaccharide structure is dependent upon the maturational state of the animal. These changes were specific for TSH because glycosylation of free alpha subunit (synthesized by the thyrotroph and gonadotroph) and of total glycoproteins was minimally altered by hypothyroidism. Together, these data provide direct evidence and characterization of specific changes in the structure of a secreted pituitary glycoprotein hormone occurring as a result of in vivo endocrine alterations during early development. Moreover, they provide a potential structural basis to explain the delayed clearance of both TSH and the gonadotropins with end-organ deficiency, which may have important implications for the in vivo biological activities of these hormones. Specifically, such posttranslational changes may be an important adaptive response to prevent the consequences of endocrine deficiency during early development.
Lesion-induced increase in nerve growth factor mRNA is mediated by c-fos.
Authors: Hengerer B , Lindholm D , Heumann R , Rüther U , Wagner E F , Thoenen H
Published: May 2006

Lesion of the sciatic nerve caused a rapid increase in c-fos and c-jun mRNA that was followed about 2 hr later by an increase in nerve growth factor (NGF) mRNA. To evaluate whether the initial increase in c-fos mRNA is causally related to the subsequent increase in NGF mRNA, we performed experiments with fibroblasts of transgenic mice carrying an exogenous c-fos gene under the control of a metallothionein promoter. In primary cultures of these fibroblasts, CdCl2 evoked a rapid increase in exogenous c-fos mRNA, followed immediately by an increase in endogenous c-jun mRNA and with a slight delay by an increase in NGF mRNA. In fibroblasts of C3H control mice, CdCl2 had no effect on the mRNA levels of the protooncogenes c-fos and c-jun or of NGF. Additional evidence for a causal relationship between c-fos induction and the subsequent increase in NGF mRNA was obtained in cotransfection experiments. Fibroblasts of C3H control mice were cotransfected with a metallothionein-promoter-driven c-fos expression vector and a NGF promoter-chloramphenicol acetyltransferase reporter gene construct. Induction of the exogenous c-fos by CdCl2 resulted in increased activity of the NGF promoter. DNase I footprint experiments demonstrated that a binding site for transcription factor AP-1 (Fos/Jun heterodimer) in the first intron of the NGF gene was protected following c-fos induction. That this protected AP-1 site indeed was functional in the regulation of NGF expression was verified by deletion experiments and by a point mutation in the corresponding AP-1 binding region in the NGF promoter-chloramphenicol acetyltransferase reporter construct.
Enzymatic amplification of a Y chromosome repeat in a single blastomere allows identification of the sex of preimplantation mouse embryos.
Authors: Bradbury M W , Isola L M , Gordon J W
Published: May 2006

The polymerase chain reaction (PCR) technique has been adapted to identify the sex of preimplantation mouse embryos rapidly. PCR was used to amplify a specific repeated DNA sequence on the Y chromosome from a single isolated blastomere in under 12 hr. The remainder of the biopsied embryo was then transferred to a pseudopregnant female and carried to term. Using this technique, 72% of embryos can be classed as potentially either male or female. Transfers of such embryos have produced pregnancies with 8/8 fetuses (100%) being of the predicted sex. Variations of the technique have demonstrated certain limitations to the present procedure as well as indicated possible strategies for improvement of the assay. The PCR technique may have wide application in the genetic analysis of preimplantation embryos.
Maize sucrose synthase-1 promoter directs phloem cell-specific expression of Gus gene in transgenic tobacco plants.
Authors: Yang N S , Russell D
Published: May 2006

Tobacco plants were transformed by using a chimeric gene construction, in which a corn sucrose synthase-1 gene (Sh) promoter was used to direct expression of the beta-glucuronidase (Gus) reporter gene. Expression of Sh-Gus activity in these plants was found to be cell type specfifc. GUS activity was detected only in the phloem cells but not in any other cell types of vegetative tissues. In addition, Sh-Gus expression was found to be anaerobically inducible in tobacco roots. Sh-Gus was also expressed at high levels in the endosperm tissue of maturing tobacco seeds. We thus demonstrated that the corn Sh promoter can direct cell-type-specific and inducible expression in a heterologous dicotyledonous plant.
5-Azacytidine-induced reactivation of the human X chromosome-linked PGK1 gene is associated with a large region of cytosine demethylation in the 5' CpG island.
Authors: Hansen R S , Gartler S M
Published: May 2006

Hamster-human cell hybrids containing an inactive human X chromosome were treated with 5-azacytidine and derived clones were examined for phosphoglycerate kinase activity and cytosine methylation in the human PGK1 (X chromosome-linked phosphoglycerate kinase) gene. Comparisons between expressing and nonexpressing clones indicated that demethylation of several methylation-sensitive restriction sites outside of the 5' CpG island were unnecessary for expression. High-resolution polyacrylamide gel analysis of 25 Hpa II, Hha I, and Tha I sites revealed that all clones expressing PGK1 were unmethylated in a large region of the CpG island that includes the transcription start site and 400 base pairs upstream. Many nonexpressing clones had discontinuous patterns of demethylation. Remethylation was often observed in subclones of nonexpressing hybrids. These data suggest that a specific zone of methylation-free DNA within the PGK1 promoter is required for transcription. In addition, the presence of neighboring methylcytosines appears to decrease the heritable stability of unmethylated CpGs in this region.
Role of extracellular calcium in anoxic injury of mammalian central white matter.
Authors: Stys P K , Ransom B R , Waxman S G , Davis P K
Published: May 2006

White matter (WM) of the mammalian brain is susceptible to anoxic injury, but little is known about the pathophysiology of this process. We studied the mechanisms of anoxic injury in WM using the isolated rat optic nerve, a typical central nervous system WM tract. Optic nerve function, measured as the area under the compound action potential, rapidly failed when exposed to anoxia and recovered to 28.5% of control after a standard 60-min period of anoxia. Irreversible anoxic injury was critically dependent on the molar concentration of extracellular calcium [( Ca2+]o); maintaining the tissue in Ca2(+)-free solution throughout the anoxic period resulted in 100% compound action potential recovery. Increasing perfusion [Ca2+] during anoxia from zero to 4 mM resulted in progressively less recovery. As the introduction of the Ca2(+)-free solution was progressively delayed with respect to the onset of anoxia, a graded decrease in recovery of function was observed, suggesting that in WM the deleterious effects of Ca2+ accumulate slowly during anoxia. At the time of reoxygenation, an additional stepwise increase in injury was observed that was also Ca2(+)-dependent. Mammalian WM, which is relatively resistant to anoxic injury compared with gray matter, is damaged by anoxia in a manner that appears to depend on the gradual accumulation of Ca2+ in a cytoplasmic compartment.
NG-methyl-L-arginine causes endothelium-dependent contraction and inhibition of cyclic GMP formation in artery and vein.
Authors: Gold M E , Wood K S , Byrns R E , Fukuto J , Ignarro L J
Published: May 2006

The objective of this study was to determine whether the vascular smooth muscle contractile effect of NG-methyl-L-arginine (NMA) is endothelium dependent and attributed to a decline in smooth muscle levels of cyclic GMP. Vascular smooth muscle levels of cyclic GMP are severalfold greater in endothelium-intact than in endothelium-denuded preparations because of the continuous formation and release of a lipophilic endothelium-derived chemical factor that diffuses into the underlying smooth muscle and activates cytosolic guanylate cyclase. This chemical substance, believed to be nitric oxide (NO) or a labile nitroso precursor, appears to account for the biological actions of endothelium-derived relaxing factor. NMA inhibits the formation of NO from endogenous L-arginine in endothelial cells. In the present study, NMA caused marked endothelium-dependent contraction of isolated rings of bovine pulmonary artery and vein, and this was similar to the contraction elicited by hemoglobin, an inhibitor of the relaxant action of NO. Both NMA and hemoglobin caused endothelium-dependent potentiation of contractile responses to phenylephrine in artery and vein. NMA caused endothelium-dependent decreases in the resting or basal levels of cyclic GMP in artery and vein to levels that were characteristic of those in endothelium-denuded vessels. Finally, NMA inhibited endothelium-dependent relaxant responses and cyclic GMP formation stimulated by acetylcholine and bradykinin. These observations reveal that interference with the continuous or basal generation of endothelium-derived NO in artery and vein can cause marked increases in vascular smooth muscle tone as a result of inhibition of cyclic GMP formation.
Human genes for the alpha and beta chains of complement C4b-binding protein are closely linked in a head-to-tail arrangement.
Authors: Pardo-Manuel F , Rey-Campos J , Hillarp A , Dahlbäck B , Rodriguez de Cordoba S
Published: May 2006

C4b-binding protein (C4BP) is an important component in the regulation of the complement system and also binds the anticoagulant vitamin K-dependent protein S. These activities are performed by distinct, although structurally related, polypeptides of 70 kDa (alpha chain) and 45 kDa (beta chain), respectively. In this report we have investigated the genetic relationships between these polypeptides. Using pulsed field gel electrophoresis analysis we demonstrate that the genes coding for the alpha (C4BP alpha) and beta (C4BP beta) chains are closely linked within the regulator of complement activation gene cluster. In addition, we have determined that the 3' end of the C4BP beta gene lies 3.5-5 kilobases from the 5' end of the C4BP alpha gene. These findings support the concept that the C4BP alpha and C4BP beta genes are the result of a gene duplication event.
Invariants of sets of linear varieties.
Authors: Huang R Q
Published: May 2006

A minimal set of generators of the ring of invariants for four linear subspaces of dimension n in a vector space of dimension 2n is computed, using the symbolic method introduced by Grosshans et al. [Grosshans, F., Rota, G.-C. & Stein, J. A. (1987) Invariant Theory and Superalgebras (Am. Math. Soc., Providence, RI)].
The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization.
Authors: Bernad A , Lázaro J M , Salas M , Blanco L
Published: May 2006

The alpha-like DNA polymerases from bacteriophage phi 29 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dNTP binding site. We have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved C-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser). Our results indicate that in phi 29 DNA polymerase this consensus sequence, although irrelevant for the 3'----5' exonuclease activity, is essential for initiation and elongation. Based on these results and on its homology with known or putative metal-binding amino acid sequences, we propose that in phi 29 DNA polymerase the Tyr-Gly-Asp-Thr-Asp-Ser consensus motif is part of the dNTP binding site, involved in the synthetic activities of the polymerase (i.e., initiation and polymerization), and that it is involved particularly in the metal binding associated with the dNTP site.
Why do A.T base pairs inhibit Z-DNA formation?
Authors: Dang L X , Pearlman D A , Kollman P A
Published: May 2006

We have carried out free energy perturbation calculations on DNA double-stranded hexanucleotides. The sequence d(CGCGCG)2 has been "mutated" into d(CGTGCG).d(CGCACG) with the oligonucleotide in the A, B, and Z structural forms, both in vacuo and in aqueous solution. In addition, model free energy calculations have been carried out in which the electrostatic charges of the H-bonding groups of the bases in the major and minor grooves of the DNA are reduced to zero as a way of assessing the relative solvation effects of these groups in the different structural forms of DNA. Finally, energy component analyses have been carried out to assess the relative roles of different intranucleotide interactions on the B----Z equilibrium as a function of base sequence. In vacuo, the free energy for changing a G.C to an A.T base pair is largest in the Z conformation; in the A and B conformations, the free energy cost is approximately 2 kcal/mol lower (1 cal = 4.184 J). The results are similar when the simulations are run in explicit solvent: the change costs 3 kcal/mol more in the Z conformation than in the B form. These results are consistent with experimental data, where it is clear that A.T sequences are significantly more "Z-phobic" than G.C sequences. The calculations indicate that both intranucleotide and solvation interactions contribute to this Z-phobicity.
Preincubation in acid medium increases Na/H antiporter activity in cultured renal proximal tubule cells.
Authors: Horie S , Moe O , Tejedor A , Alpern R J
Published: May 2006

Chronic acidosis in vivo leads to an increase in proximal tubule Na/H antiporter activity that persists when the transporter is studied out of the acidotic environment. It is presently not clear whether a decrease in extracellular fluid pH alone is sufficient to elicit this adaptation. The present studies examined the effect of acid preincubation on Na/H antiporter activity in cultured proximal tubule cells. Antiporter activity was examined after a 2-day preincubation in control or acid medium, 1 hr after removal from the preincubation fluid. Na/H antiporter activity was assayed as the initial rate of Na-dependent alkalinization after cell acidification in the absence of CO2/HCO3. Preincubation in low [HCO3] acid medium or in high PCO2 acid medium led to increases in amiloride-sensitive Na/H antiporter activity. This adaptation was inhibited by addition of cycloheximide to the preincubation medium. Preincubation of fibroblasts in low [HCO3] acid medium did not lead to increased Na/H antiporter activity but rather caused a small inhibition. These studies demonstrate an adaptation in Na/H antiporter activity elicited by a low pH of the extracellular fluid, which is dependent on protein synthesis, and may be unique to certain H/HCO3-transporting epithelia.
Retinoic acid, dibutyryl-cAMP, and differentiation affect the expression of retinoic acid receptors in F9 cells.
Authors: Martin C A , Ziegler L M , Napoli J L
Published: May 2006

Expression of the retinoic acid receptors alpha and beta (RAR-alpha and RAR-beta) was examined in F9 cells, an embryonal carcinoma cell model established for the study of retinoid metabolism and function. Addition of retinoic acid to F9 cell medium caused a dose-dependent increase in RAR-beta mRNA within 3 hr that reached 5- to 30-fold greater than the constitutively expressed mRNA by 24 hr. The elevation in mRNA resulted from increased transcription, as demonstrated by nuclear run-on transcription, did not require protein synthesis, and required the constant presence of retinoic acid. N6,O2'-Dibutyryl-cAMP attenuated the retinoic acid-induced increase in RAR-beta mRNA by a post-transcriptional mechanism. In contrast, RAR-alpha mRNA in F9 stem cells was affected less (1.2- to 1.4-fold increase) by retinoic acid and decreased 3-fold transiently when fresh serum was added to the medium. Differentiation of F9 cells resulted in increased steady-state levels of RAR-beta mRNA in primitive (4-fold), parietal (3-fold), and visceral (8-fold) endoderm but decreased steady-state levels of RAR-alpha mRNA in primitive (2-fold), parietal (3-fold), and visceral (1.4-fold) endoderm. These data demonstrate that RAR-beta is a primary target gene for retinoic acid in a characterized model of retinoid function, indicate that constitutive expression of both RAR-beta and RAR-alpha is dependent upon the differentiation state, and suggest hormonal modulation of RAR-beta by cAMP and modulation of RAR-alpha by serum factors. These results distinguish the effects of serum, cAMP, and retinoic acid on the expression of RAR from the effects mediated by differentiation.
Haplotyping the human T-cell receptor beta-chain gene complex by use of restriction fragment length polymorphisms.
Authors: Charmley P , Chao A , Concannon P , Hood L , Gatti R A
Published: May 2006

We have studied the genetic segregation of human T-cell receptor beta-chain (TCR beta) genes on chromosome 7q in 40 CEPH (Centre d'Etude du Polymorphisme Humain) families by using restriction fragment length polymorphisms (RFLPs). We constructed haplotypes from eight RFLPs by using variable- and constant-region cDNA probes, which detect polymorphisms that span more than 600 kilobases of the TCR beta gene complex. Analysis of allele distributions between TCR beta genes revealed significant linkage disequilibrium between only 6 of the 28 different pairs of RFLPs. This linkage disequillibrium strongly influences the most efficient order to proceed for typing of these RFLPs in order to achieve maximum genetic informativeness, which in this study revealed a 97.3% level of heterozygosity within the TCR beta gene complex. Our results should provide new insight into recent reports of disease associations with the TCR beta gene complex and should assist in designing future experiments to detect or confirm the existence of disease-susceptibility loci in this region of the human genome.
Spantide II, an effective tachykinin antagonist having high potency and negligible neurotoxicity.
Authors: Folkers K , Feng D M , Asano N , Håkanson R , Weisenfeld-Hallin Z , Leander S
Published: May 2006

Spantide (D-Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-D-Trp7-Phe8-D-Trp9-++ +Leu10-Leu11-NH2) was introduced as a tachykinin antagonist in 1984 and has served as a starting point in the design of new antagonists that have proven to be more effective and have exhibited no neurological side effects. The most remarkable and unpredictable structural change that significantly increased potency was deletion of a methylene group by changing Gln6 to Asn6. On the basis that D-Arg1 and Lys3 of spantide contribute to neurological side effects, many new designs led to D-Lys(Nic)1-Pro2-Pal(3)3-Pro4-D-Phe(Cl2)5-Asn6-D-Trp7-Phe8-D-Trp9- Leu10-Nle11- NH2 [spantide II, where D-Lys(Nic) is N epsilon-nicotinoyllysine, Pal(3) is 3-(3-pyridyl)alanine, D-Phe(Cl2) is 3,4-dichloro-D-phenylalanine, and Nle is norleucine], which is a potent antagonist without neurotoxicity. Spantide II, an undecapeptide, has a total of seven substitutions in the sequence of substance P, consisting of two natural L amino acids, and one unnatural L amino acid, and four unnatural D amino acids. The pi- and sigma-bond amino acid substituents of substance P and spantide II are compared toward a future understanding of the essential substituents for mechanism and inhibition binding. Spantide II has five pi-bond and six sigma-bond amino acid moieties, and substance P has two pi-bond and nine sigma-bond moieties.
Evidence that a guanine nucleotide-binding protein linked to a muscarinic receptor inhibits directly phospholipase C.
Authors: Bizzarri C , Di Girolamo M , D'Orazio M C , Corda D
Published: May 2006

The mechanism of phospholipase C regulation by inhibitory receptors was analyzed both in intact and in permeabilized rat thyroid cells (FRTL5). In this system, the muscarinic agonist carbachol inhibited phospholipase C, as indicated by the decrease in the basal levels of inositol 1,4,5-trisphosphate as well as by the reduced adrenergic stimulation of phosphoinositol accumulation, which was paralleled by a fall in the cytosolic Ca2+ levels. This inhibition involved an M2 muscarinic receptor because it was abolished by atropine but not by the M1 antagonist pirenzepine. Cells pretreated with pertussis toxin were not responsive to carbachol, indicating the involvement of a guanine nucleotide-binding protein in this inhibitory process. This possibility was further evaluated in permeabilized cells, where the carbachol inhibition was shown to be completely dependent on GTP. Known second messengers were not involved in this inhibitory process since Ca2+, cAMP, and activators of protein kinases were not able to mimic or prevent the carbachol effect either in intact or in permeabilized FRTL5 cells. In this system, the phospholipases C and A2 are coupled to two classes of muscarinic receptors that display a different sensitivity to pertussis toxin. The carbachol inhibitory effect occurred under conditions that prevented activation of phospholipase A2, excluding a role of the arachidonic acid metabolism in this process. Taken together these data provide the strongest support to date that an inhibitory guanine nucleotide-binding protein sensitive to pertussis toxin can directly mediate receptor-induced inhibition of phospholipase C.
Molecular cloning and expression of an additional epidermal growth factor receptor-related gene.
Authors: Plowman G D , Whitney G S , Neubauer M G , Green J M , McDonald V L , Todaro G J , Shoyab M
Published: May 2006

Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-alpha in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-beta. Amphiregulin also appears to abrogate the stimulatory effect of TGF-alpha on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth.
Endogenous retroviral long terminal repeats within the HLA-DQ locus.
Authors: Kambhu S , Falldorf P , Lee J S
Published: May 2006

Two endogenous retroviral long terminal repeats (LTRs) were found in the human major histocompatibility complex locus HLA-DQ. The solo LTRs, unlinked to retrovirus structural genes, are located approximately 5 kilobases apart from each other and in the same transcriptional orientation, which is opposite to that for the HLA-DQB1 gene. These elements exhibit greater than 90% homology to the LTRs of the human endogenous retrovirus HERV-K10. The conservation of putative regulatory elements found within the LTRs and their position relative to the HLA-DQB1 gene suggest that these elements may confer distinct regulatory properties on genes in the HLA-DQ region. Polymorphic variation between different HLA haplotypes for the presence of the LTRs at this location and of the molecular architecture within this subregion is supported by polymerase chain reaction and Southern blot analysis. Comparisons of chromosomes with and without the LTRs in this region will provide a unique opportunity in the human genome to analyze transposition or integration of retroviral sequences.