bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and sequence analysis of cDNA for corticotropin-releasing factor precursor from the teleost fish Catostomus commersoni.
Authors: Okawara Y , Morley S D , Burzio L O , Zwiers H , Lederis K , Richter D
Published: May 2006

The sequence of a cDNA encoding the corticotropin-releasing factor precursor has been identified by screening lambda gt11 libraries constructed from poly(A)+ RNA of the hypothalamic region of the white sucker Catostomus commersoni brain with synthetic oligonucleotide probes deduced from the sequence of the rat corticotropin-releasing factor. The amino acid sequence of corticotropin-releasing factor of the sucker is strikingly conserved when compared to its counterpart from rat and differs only in two positions at the carboxyl terminus; in contrast, there is little similarity between their cryptic regions.
At least two non-antigen-binding molecules are required for signal transduction by the T-cell antigen receptor.
Authors: Goldsmith M A , Dazin P F , Weiss A
Published: May 2006

In the T-cell somatic mutant J.CaM1, the T-cell antigen receptor complex is poorly coupled to the inositolphospholipid second messenger system; some antibodies against the invariant CD3 subunit of the receptor retain their agonist function in J.CaM1. Here we show by a combination of complementation assays that the mutation in J.CaM1 affects a molecule other than the antigen-binding Ti subunit, suggesting that Ti is coupled indirectly to the signal transduction apparatus through a pathway involving the CD3 complex. We also describe another mutant, J.CaM2, in which the receptor complex is completely uncoupled from inositolphospholipid hydrolysis. J.CaM2 defines an additional complementation group, suggesting that signal transduction by the antigen receptor depends on at least two molecules distinct from Ti.
Biochemical localization of hepatic surface-membrane Na+,K+-ATPase activity depends on membrane lipid fluidity.
Authors: Sutherland E , Dixon B S , Leffert H L , Skally H , Zaccaro L , Simon F R
Published: May 2006

Membrane proteins of transporting epithelia are often distributed between apical and basolateral surfaces to produce a functionally polarized cell. The distribution of Na+,K+-ATPase [ATP phosphohydrolase (Na+/K+-transporting), EC 3.6.1.37] between apical and basolateral membranes of hepatocytes has been controversial. Because Na+,K+-ATPase activity is fluidity dependent and the physiochemical properties of the apical membrane reduces its fluidity, we investigated whether altering membrane fluidity might uncover cryptic Na+,K+-ATPase in bile canalicular (apical) surface fractions free of detectable Na+,K+-ATPase and glucagon-stimulated adenylate cyclase activities. Apical fractions exhibited higher diphenylhexatriene-fluorescence polarization values when compared with sinusoidal (basolateral) membrane fractions. When 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) was added to each fraction, Na+,K+-ATPase, but not glucagon-stimulated adenylate cyclase activity, was activated in the apical fraction. In contrast, further activation of both enzymes was not seen in sinusoidal fractions. The A2C-induced increase in apical Na+,K+-ATPase approached 75% of the sinusoidal level. Parallel increases in apical Na+,K+-ATPase were produced by benzyl alcohol and Triton WR-1339. All three fluidizing agents decreased the order component of membrane fluidity. Na+,K+-ATPase activity in each subfraction was identically inhibited by the monoclonal antibody 9-A5, a specific inhibitor of this enzyme. These findings suggest that hepatic Na+,K+-ATPase is distributed in both surface membranes but functions more efficiently and, perhaps, specifically in the sinusoidal membranes because of their higher bulk lipid fluidity.
Characterization of the minor groove environment in a drug-DNA complex: bisbenzimide bound to the poly[d(AT)].poly[d(AT)]duplex.
Authors: Jin R , Breslauer K J
Published: May 2006

We compare the fluorescence properties of bisbenzimide (also known as Hoechst 33258) bound to the minor groove of the poly[d(AT)].poly[d(AT)] duplex with the corresponding fluorescence properties of bisbenzimide dissolved in neat organic solvents and mixed organic/aqueous solvents. Based on these comparisons, we conclude that the minor groove of the bisbenzimide-poly[d(AT)].poly[d(AT)] complex is quite nonpolar and exhibits a local dielectric constant of approximately 20 D. We discuss how this insight influences our understanding of the molecular forces that dictate and control the binding affinities and specificities of minor groove-directed DNA binding ligands.
Polyomavirus early-late switch is not regulated at the level of transcription initiation and is associated with changes in RNA processing.
Authors: Hyde-DeRuyscher R , Carmichael G G
Published: May 2006

Polyoma gene expression is temporally regulated during productive infection of mouse cells. Early genes are expressed throughout the viral life cycle, but late mRNAs are not detected until after the onset of DNA replication. At late times, late-strand transcripts represent the great majority of viral-specific RNA in the cell. To learn more about the mechanism by which the early-late switch is regulated, we have carried out a detailed analysis of polyomavirus transcription in mouse NIH 3T6 cells. Nuclei were isolated from cells infected for 6, 12, 18, or 24 hr, and run-on assays were performed. The resulting RNAs were then hybridized to a number of immobilized early- and late-strand-specific probes, which represent the entire polyoma genome. Results indicate that the late promoter is always on, even in the absence of DNA replication. Even though the early-late switch is characterized by a greater than 300-fold difference in the ratio of steady-state early- and late-strand RNAs, there is only a 2-fold effect at the level of transcription initiation. Furthermore, the efficiency of termination for late transcripts is very high at early times during infection (greater than 90%) but drops drastically at late times (less than 40%). In other experiments, we have found an increase in splicing efficiency of late pre-mRNA molecules that parallels the decrease in termination efficiency. These results, taken together with other studies from our laboratory, have led us to propose two possible models for the temporal control of polyomavirus late gene expression.
Selective and indirect modulation of human multipotential and erythroid hematopoietic progenitor cell proliferation by recombinant human activin and inhibin.
Authors: Broxmeyer H E , Lu L , Cooper S , Schwall R H , Mason A J , Nikolics K
Published: May 2006

Activin and inhibin are biomolecules that, respectively, enhance and suppress the release of follicle-stimulating hormone from pituitary cells in vitro. Purified recombinant human (rhu) activin A and inhibin A were assessed for their effects on colony formation in vitro by human multipotential (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells. It was found that (i) rhu-activin A enhances colony formation by normal bone marrow erythroid and multipotential progenitor cells; (ii) purified rhu-inhibin A decreases activin, but not rhu-interleukin 3, rhu-granulocyte-macrophage colony-stimulating factor, or rhu-interleukin 4, enhancement of erythropoietin-stimulated colony formation by erythroid and multipotential progenitor cells; (iii) modulatory actions of rhu-activin and rhu-inhibin are mediated through monocytes and T lymphocytes within the marrow; (iv) actions are apparent in the absence or presence of serum; and (v) rhu-activin and rhu-inhibin have no effect on colony formation by granulocyte-macrophage progenitor cells. This defines an indirect mode of action and a specificity for activin and inhibin on multipotential and erythroid progenitor cells.
Glucocorticoids both stimulate and inhibit production of pulmonary surfactant protein A in fetal human lung.
Authors: Liley H G , White R T , Benson B J , Ballard P L
Published: May 2006

Pulmonary surfactant is a mixture of phospholipids and proteins which stabilizes lung alveoli and prevents respiratory failure. The surfactant-associated protein of Mr = 28,000-36,000 (SP-A) influences the structure, function (film formation), and metabolism of surfactant. We have characterized glucocorticoid regulation of SP-A and SP-A mRNA in explants of fetal human lung. The time course of response to dexamethasone was biphasic, with early stimulation and later inhibition of SP-A accumulation. Maximal induction of SP-A occurred with 3-10 nM dexamethasone and approximately 300 nM cortisol for 72 hr, and stimulation diminished at higher concentrations. SP-A mRNA accumulation was maximally stimulated at 24-48 hr of exposure to dexamethasone (10 nM) and was generally inhibited by 4-6 days. Stimulation was also observed with cortisone and corticosterone but not with sex steroids, suggesting a receptor-mediated process. When explants were exposed to cortisol for only 24 hr, SP-A content was transiently increased above the level in continuously treated tissue and subsequently was similar to control. The content of SP-A and its mRNA was also increased by dibromo-cAMP, terbutaline, and forskolin, and effects were approximately additive with those of dexamethasone. However, elevated in tracellular cAMP did not alter the biphasic time course or dose-response patterns of dexamethasone. We propose that glucocorticoids have both stimulatory and inhibitory effects on SP-A gene expression. This biphasic regulation is not consistent with generalized toxic effects, product-feedback inhibition, or receptor down-regulation, and it appears to be specific for SP-A among the various surfactant components.
Structure and chromosomal localization of the functional intronless human JUN protooncogene.
Authors: Hattori K , Angel P , Le Beau M M , Karin M
Published: May 2006

The JUN protooncogene encodes a protein that is functionally and biochemically identical to the transcription factor AP-1 (activator protein 1). To understand the structure and regulation of this important gene, a genomic clone of human JUN was isolated and its primary structure and transcription pattern were determined. Most surprisingly, the sequence of the genomic clone was found to be contiguous with the sequence of the JUN cDNA, suggesting that it lacks introns. RNase protection experiments confirm that JUN is an intronless gene that yields several transcripts due to 5' and 3' heterogeneities. Transfection experiments show that the cloned gene is functional, as it encodes a trans-acting factor that stimulates transcription of AP-1-dependent reporter gene. In situ hybridization was used to map JUN to chromosomal region 1p31-32. Interestingly, this region is frequently deleted in neuroblastomas, suggesting that elimination of AP-1 may play an important role in the pathogenesis of this disease.
Role of protein N-glycosylation in pathogenesis of human immunodeficiency virus type 1.
Authors: Montefiori D C , Robinson W E , Mitchell W M
Published: May 2006

Human immunodeficiency virus type 1 (HIV-1), the retrovirus responsible for acquired immunodeficiency syndrome (AIDS), contains two heavily glycosylated envelope proteins, gp120 and gp41, which mediate attachment of virions to glycosylated cell surface receptor molecules (CD4 antigens) and appear to be responsible for syncytium formation and associated cytopathic effects of this virus. A comprehensive study of the effects of N-linked glycoprotein processing inhibitors on HIV-1 replication, infectivity, cytopathicity, target-cell infectibility, syncytium formation, and gp120 electrophoretic mobility was conducted to assess the importance of protein glycosylation in the pathogenesis of HIV-1 in vitro. The electrophoretic mobility of gp120 was decreased when gp120 was synthesized in the presence of castanospermine or 1-deoxynojirimycin (inhibitors of glucosidase I), increased when gp120 was synthesized in the presence of 1-deoxymannojirimycin (mannosidase I) or swainsonine (mannosidase II), and unaffected when gp120 was synthesized in the presence of bromoconduritol (glucosidase II). Inhibition by tunicamycin (lipid-linked oligosaccharide precursor synthesis), castanospermine, 1-deoxynojirimycin, and 1-deoxymannojirimycin attenuated HIV-1 infectivity and blocked HIV-1-induced syncytium formation and cytopathicity, whereas bromoconduritol and swainsonine failed to have such effects. None of the inhibitors interfered with virus replication in acutely infected cells or affected the ability of target cells to form syncytia with untreated HIV-1-infected cells. These results demonstrate that protein N-glycosylation is critical to the pathogenesis of HIV-1 at the levels of viral infectivity and cytopathicity but not at the level of virus replication or of host-cell infectibility.
Human granulocyte-macrophage colony-stimulating factor is a growth factor active on a variety of cell types of nonhemopoietic origin.
Authors: Dedhar S , Gaboury L , Galloway P , Eaves C
Published: May 2006

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of a family of glycoprotein hormones that stimulate the proliferation and differentiation of hemopoietic cells in vitro and in vivo. We now report that human GM-CSF can also stimulate the proliferation of two osteogenic sarcoma cell lines, a breast carcinoma cell line, a simian virus 40-transformed marrow stromal cell line, and normal marrow fibroblast precursors. These findings suggest a more general regulatory function of GM-CSF on nonhemopoietic cell types than previously anticipated. They also raise the possibility of adverse side effects of GM-CSF therapy in patients whose malignant cells may be directly stimulated by this molecule and suggest a previously unanticipated role of GM-CSF gene activation in the evolution of solid tumors and in the pathogenesis of myelofibrosis.
Processing of the structural proteins of human immunodeficiency virus type 1 in the presence of monensin and cerulenin.
Authors: Pal R , Gallo R C , Sarngadharan M G
Published: May 2006

The synthesis and processing of structural proteins of human immunodeficiency virus type 1 (HIV-1) were studied in infected cells treated with monensin and cerulenin. In MOLT-3 cells chronically infected with HTLV-IIIB, monensin inhibited the proteolytic cleavage of the env-coded polyprotein gp160 to gp120, leading to the accumulation of the precursor gp160. The formation of syncytia normally observed when CEM cells are cocultivated with HIV-1-infected MOLT-3 cells was significantly inhibited in the presence of monensin. The effect of the ionophore on the culture was reversible, as withdrawal of monensin from the medium restored the ability of the cells to form syncytia with CEM cells and led to the resumption of the processing of gp160 to gp120. Monensin did not affect the synthesis and processing of gag-coded proteins and regulatory proteins. Cerulenin, an inhibitor of de novo fatty acid biosynthesis, inhibited the myristoylation and the proteolytic cleavage of the gag-coded polyprotein Pr53gag to p24 but did not affect the processing of gp160. However, use for monensin and cerulenin as antiviral agents for treatment of HIV-1 infection cannot be foreseen because of the pronounced in vitro toxicity observed.
Vasopressin modulates the spontaneous electrical activity in aortic cells (line A7r5) by acting on three different types of ionic channels.
Authors: Van Renterghem C , Romey G , Lazdunski M
Published: May 2006

A7r5 smooth muscle (aorta) cells have a spontaneous electrical activity. Application of vasopressin produces a hyperpolarization accompanied by an interruption of the spontaneous activity, which is followed by a depolarization associated with a recovery of the spiking activity. Vasopressin action is produced by an action of the peptide on three different types of ionic channels. Vasopressin activates a Ca2+-sensitive K+ conductance, presumably by producing inositol 1,4,5-trisphosphate intracellularly and liberating Ca2+ from internal stores. This activation is transient (0.5-4 min) and is related to the vasopressin-induced hyperpolarization. Intracellular perfusion of inositol trisphosphate triggers by itself a transient K+ current and prevents subsequent activation by vasopressin. Vasopressin inhibits an L-type Ca2+ channel through both protein kinase C activation and a [Ca2+]i-dependent inactivation mechanism triggered by inositol trisphosphate production. The addition of the activation of a Ca2+-sensitive K+ channel and of the inhibition of a voltage-sensitive Ca2+ channel is responsible for the transient blockade of the spontaneous activity. Vasopressin also provokes the activation of an inward current (2-20 min) due to a nonselective channel able to transfer Ca2+, Na+, K+, and Cs+ across the membrane. This effect of the peptide is associated with the depolarization following the hyperpolarization phase.
Translational repression in eukaryotes: partial purification and characterization of a repressor of ferritin mRNA translation.
Authors: Walden W E , Daniels-McQueen S , Brown P H , Gaffield L , Russell D A , Bielser D , Bailey L C , Thach R E
Published: May 2006

Mouse and rabbit ferritin mRNAs translate very poorly in rabbit reticulocyte lysates relative to most other mRNAs. This translational deficiency is not seen in wheat germ lysates, suggesting the presence of an inhibitor in reticulocyte lysate that is specific for ferritin mRNA. A specific repressor of ferritin mRNA translation has been partially purified from rabbit reticulocytes by differential ultracentrifugation, ammonium sulfate fractionation, and chromatography on phosphocellulose, DEAE-cellulose, and Sephacryl S-300. The elution profile from the latter suggests an aggregate molecular mass of approximately 180 kDa for the repressor. The inhibitory activity of this repressor against native ferritin mRNA can be relieved by adding in vitro transcripts of ferritin light-chain RNAs that contain the first 92 nucleotides of the 5' untranslated region. No other sequences appear to be necessary for this effect.
Sustained expression of the human protooncogene MYCN rescues rat embryo cells from senescence.
Authors: Schwab M , Bishop J M
Published: May 2006

Amplification of the human gene MYCN may play a role in the malignant progression of human neuroblastomas. In pursuit of this possibility, previous studies have shown that the abundant expression of MYCN in cultured cells can elicit several aspects of the transformed phenotype. We now extend those findings by demonstrating that rat embryo cells transfected with MYCN can proliferate for at least 200 generations. Isolation of established cells was dependent on high expression of MYCN and on biological selection to eliminate untransfected cells. The established cells were not tumorigenic in syngeneic rats or athymic mice, failed to grow in soft agar, and required relatively high concentrations of serum for proliferation in culture. Our results show that enhanced expression of MYCN can rescue normal cells from senescence, add to the credentials of MYCN as an authentic protooncogene, and identify an additional biological activity that can be used in the characterization of MYCN.
Genetic diversity in natural populations of a soil bacterium across a landscape gradient
Authors: McArthur J. Vaun , Kovacic David A. , Smith Michael H.
Published: May 2006

Genetic diversity in natural populations of the bacterium
Pseudomonas cepacia
was surveyed in 10 enzymes from 70 clones isolated along a landscape gradient. Estimates of genetic diversity, ranging from 0.54 to 0.70, were higher than any previously reported values of which we are aware and were positively correlated with habitat variability. Patterns of bacterial genetic diversity were correlated with habitat variability. Findings indicate that the source of strains used in genetic engineering will greatly affect the outcome of planned releases in variable environments. Selection of generalist strains may confer a large advantage to engineered populations, while selection of laboratory strains may result in quick elimination of the engineered strains.
Alpha 1(IV) and alpha 2(IV) collagen genes are regulated by a bidirectional promoter and a shared enhancer.
Authors: Burbelo P D , Martin G R , Yamada Y
Published: May 2006

Collagen IV is the major structural component of basement membranes and is a heterotrimer composed of two alpha 1(IV) and one alpha 2(IV) chains. Most collagen genes are dispersed in the human genome, such as the genes for collagen I, which are located on chromosomes 7 [alpha 1(I)] and 17 [alpha 2(I)]. In contrast, we have found that the murine alpha 1(IV) and alpha 2(IV) collagen chain genes exist in a head-to-head arrangement on opposite strands separated by 130 base pairs. By transfecting various portions of these genes into cells, we have found that transcription of the alpha 1(IV) and alpha 2(IV) genes is regulated by a bidirectional promoter located between the two genes working in concert with an enhancer located in the first intron of the alpha 1(IV) chain gene.
Platelet-activating factor acetylhydrolase activity in maternal, fetal, and newborn rabbit plasma during pregnancy and lactation.
Authors: Maki N , Hoffman D R , Johnston J M
Published: May 2006

The inactivation of the biologically active ether-containing glycerophospholipid platelet-activating factor (PAF) is catalyzed by the enzyme PAF acetylhydrolase (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase, EC 3.1.1.48). The specific activity of acetylhydrolase has been assayed in maternal rabbit plasma prior to and throughout pregnancy and after parturition. The specific activity of acetylhydrolase was 131 +/- 8 nmol.min-1.ml-1 of plasma (mean +/- SEM) in the nonpregnant rabbit. Similar specific activities were found throughout the first half of pregnancy; however, on day 15 the acetylhydrolase activity began to decrease and reached a minimum around day 27. Within 24-48 hr following delivery, the specific activity of the enzyme increased to the levels found in the nonpregnant animals. The specific activity of acetylhydrolase in fetuses (gestational ages of 21-30 days) and neonates increased from 22 nmol.min-1.ml-1 of plasma (21-day-old) to 328 nmol.min-1.ml-1 of plasma (35-day-old rabbits). The decrease in enzymatic activity of the pregnant rabbit plasma cannot be accounted for by the presence of an inhibitor as determined by plasma-mixing experiments with nonpregnant and pregnant (27-day) plasma. The properties of this enzyme in the rabbit were consistent with those reported by others, i.e., substrate specificity, inactivation by heat and various inhibitors, and Ca2+ independency. The activity of PAF acetylhydrolase was associated primarily with the high density lipoprotein fraction in rabbit plasma. The decrease of this enzymatic activity during pregnancy may serve to remove the protective effect on myometrium to allow an increased amount of PAF to come in contact with this tissue.
Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro.
Authors: Sampson J R , Uhlenbeck O C
Published: May 2006

A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNA(Phe) is flanked by a T7 promoter and a BstNI restriction site. Runoff transcription of the BstNI-digested plasmid with T7 RNA polymerase gives an unmodified tRNA of the expected sequence having correct 5' and 3' termini. This tRNA(Phe) transcript can be specifically aminoacylated by yeast phenylalanyl-tRNA synthetase and has a Km only 4-fold higher than that of the native yeast tRNA(Phe). The Km is independent of Mg2+ concentration, whereas the Vmax is very dependent on Mg2+ concentration. Comparison of the melting profiles of the native and the unmodified tRNA(Phe) at different Mg2+ concentrations suggests that the unmodified tRNA(Phe) has a less stable tertiary structure. Using one additional DNA oligomer, a mutant plasmid was constructed having a guanosine to thymidine change at position 20 in the tRNA gene. A decrease in Vmax/Km by a factor of 14 for aminoacylation of the mutant tRNA(Phe) transcript is observed.
Characterization of a mammalian smooth muscle myosin heavy chain cDNA clone and its expression in various smooth muscle types.
Authors: Nagai R , Larson D M , Periasamy M
Published: May 2006

A cDNA clone, SMHC-29, encoding the light meromyosin of smooth muscle myosin heavy chain (MHC), was isolated from a rabbit uterus cDNA library constructed in phage lambda gt11. This smooth muscle MHC cDNA demonstrates significant nucleotide and amino acid sequence homologies with known sarcomeric MHC genes from rabbit, rat skeletal, and nematode body wall myosin, and even with nonmuscle MHC gene from a slime mold (Dictyostelium discoideum), suggesting that smooth muscle, striated muscle, and nonmuscle MHC genes diverged from a common ancestor. The deduced amino acid sequences of the smooth muscle light meromyosin show very similar periodic distributions of hydrophobic and charged residues as found for the light meromyosin of striated muscle MHCs together with a high potential for alpha-helical formation, indicating an alpha-helical coiled-coil structure for the smooth muscle light meromyosin sequences. Furthermore, S1 nuclease mapping has revealed that this smooth muscle MHC gene for SMHC-29 is specifically expressed in smooth muscles of vascular and nonvascular types but not in the striated muscles or nonmuscle cells.
On the role of protein synthesis in the circadian clock of Neurospora crassa.
Authors: Dunlap J C , Feldman J F
Published: May 2006

Inhibitors of protein synthesis reset the biological clocks of many organisms. This has been interpreted to mean either that the synthesis per se of proteins is a step in the oscillatory feedback loop or merely that certain unstable protein(s) are required at certain times of the cycle to complete the feedback loop. We report here that Neurospora strains bearing the clock mutation frq-7 are relatively insensitive to the resetting action of the protein-synthesis-inhibitor cycloheximide. Protein synthesis itself in this mutant is inhibited by the drug to the same extent as in wild type. Since the clock of frq-7 continues to run relatively unimpeded even in the virtual absence of protein synthesis, it is unlikely that synthesis per se can be a part of the feedback cycle. Rather, we suggest that for normal operation of the Neurospora clock, certain protein(s) with a high turnover rate are required daily and, thus, must be resynthesized each day (at least) during discrete times in the cycle. The frq-7 mutation simultaneously alters several distinct clock characteristics--period length, temperature compensation, and resetting by cycloheximide. A model is presented to unify these observations.
Myeloperoxidase: a myeloid cell nuclear antigen with DNA-binding properties.
Authors: Murao S , Stevens F J , Ito A , Huberman E
Published: May 2006

An antigen histochemically localized in the nuclei and cytoplasmic granules of normal and leukemic human myeloid cells has been identified as myeloperoxidase (MPO; EC 1.11.1.7). The localization and amount of the enzyme was determined by using a murine monoclonal antibody designated H-43-5 raised against nuclear proteins derived from human promyelocytic HL-60 leukemia cells. The highest amount of nuclear MPO (3.5 micrograms per 10(6) nuclei) was found in granulocytes; less than half of this amount was detected in nuclei from HL-60 cells. Still lower levels were found in nuclei from monocytes and a series of human monomyelocytic leukemia cells. MPO from HL-60 cells was purified by immunoaffinity chromatography and fractionated into three components (forms I, II, and III) by CM-cellulose chromatography. Chromatography of these MPO forms on DNA-Sepharose columns confirmed that all three forms of MPO were tightly bound to DNA with apparent relative affinities in the order of form III greater than form II greater than form I. The affinity of MPO form III for DNA was sufficient to enable the formation and elution of DNA-MPO complexes during size-exclusion chromatography at high ionic strength and neutral pH. This form of MPO was also able to shield DNA from strand scission induced by active oxygen species generated by xanthine oxidase acting aerobically on xanthine. These data suggest that intranuclear MPO may help to protect DNA against damage resulting from oxygen radicals produced during myeloid cell maturation and function.
Laminin receptors for neurite formation.
Authors: Kleinman H K , Ogle R C , Cannon F B , Little C D , Sweeney T M , Luckenbill-Edds L
Published: May 2006

Laminin, a basement membrane glycoprotein promotes both cell attachment and neurite outgrowth. Separate domains on laminin elicit these responses, suggesting that distinct receptors occur on the surface of cells. NG108-15 neuroblastoma-glioma cells rapidly extend long processes in the presence of laminin. We report here that 125I-labeled laminin specifically binds to these cells and to three membrane proteins of 67, 110, and 180 kDa. These proteins were isolated by affinity chromatography on laminin-Sepharose. The 67-kDa protein reacted with antibody to the previously characterized receptor for cell attachment to laminin. Antibodies to the 110-kDa and 180-kDa bands demonstrated that the 110-kDa protein was found in a variety of epithelial cell lines and in brain, whereas the 180-kDa protein was neural specific. Antibodies prepared against the 110-kDa and 180-kDa proteins inhibited neurite outgrowth induced by the neurite-promoting domain of laminin, whereas antibodies to the 67-kDa laminin receptor had no effect on neurite outgrowth. We conclude that neuronal cells have multiple cell-surface laminin receptors and that the 110-kDa and 180-kDa proteins are involved in neurite formation.
ATP-sensitive K+ channels that are blocked by hypoglycemia-inducing sulfonylureas in insulin-secreting cells are activated by galanin, a hyperglycemia-inducing hormone.
Authors: de Weille J , Schmid-Antomarchi H , Fosset M , Lazdunski M
Published: May 2006

The action of the hyperglycemia-inducing hormone galanin, a 29-amino acid peptide named from its N-terminal glycine and C-terminal amidated alanine, was studied in rat insulinoma (RINm5F) cells using electrophysiological and 86Rb+ flux techniques. Galanin hyperpolarizes and reduces spontaneous electrical activity by activating a population of ATP-sensitive K+ channels with a single-channel conductance of 30 pS (at -60 mV). Galanin-induced hyperpolarization and reduction of spike activity are reversed by the hypoglycemia-inducing sulfonylurea glibenclamide. Glibenclamide blocks the galanin-activated ATP-sensitive K+ channel. 86Rb+ efflux from insulinoma cells is stimulated by galanin in a dose-dependent manner. The half-maximum value of activation is found at 1.6 nM. Galanin-induced 86Rb+ efflux is abolished by glibenclamide. The half-maximum value of inhibition is found at 0.3 nM, which is close to the half-maximum value of inhibition of the ATP-dependent K+ channel reported earlier. 86Rb+ efflux studies confirm the electrophysiological demonstration that galanin activates an ATP-dependent K+ channel.
Spatial pattern representation and transformation in monkey somatosensory cortex.
Authors: Phillips J R , Johnson K O , Hsiao S S
Published: May 2006

Embossed letters, used previously in pattern recognition experiments in humans, were used to study the spatial patterns of neural activity evoked in peripheral fibers and cortical neurons in areas 3b and 1 of the primary somatosensory cortex of alert rhesus (Macaca mulatta) monkeys. The object was to investigate the representation and transformation of spatial information during the early stages of peripheral and cortical neural processing. Our method consisted of sweeping each letter of the alphabet across the skin repeatedly and constructing a two-dimensional plot (called a spatial event plot) of the action potentials evoked in afferent fibers and cortical neurons. By using this method, slowly and rapidly adapting primary afferents were shown to transmit isomorphic neural images of the letters. Although the slowly adapting images were more spatially acute, both populations conveyed images of sufficient quality to account for human psychophysical performance. In the cortical areas studied, the slowly adapting neurons of area 3b stood out for the acuity, complexity, and variety of their responses. Some of the spatial event plots for these neurons were isomorphic and at least as acute as those obtained from any primary afferent. Others were highly structured but nonisomorphic. The quality and variety of responses in area 3b slowly adapting neurons suggest that they play an important role in the processing of information underlying tactual pattern recognition. The rapidly adapting neurons of area 3b and all types of neurons in area 1 yield much less structured and differentiated responses.
A phospholipase D specific for the phosphatidylinositol anchor of cell-surface proteins is abundant in plasma.
Authors: Low M G , Prasad A R
Published: May 2006

An enzyme activity capable of degrading the glycosyl-phosphatidylinositol membrane anchor of cell-surface proteins has previously been reported in a number of mammalian tissues. The experiments reported here demonstrate that this anchor-degrading activity is also abundant in mammalian plasma. The activity was inhibited by EGTA or 1,10-phenanthroline. It was capable of removing the anchor from alkaline phosphatase, 5'-nucleotidase, and variant surface glycoprotein but had little or not activity toward phosphatidylinositol or phosphatidylcholine. Phosphatidic acid was the only 3H-labeled product when this enzyme hydrolyzed [3H]myristate-labeled variant surface glycoprotein. It could be distinguished from the Ca2+-dependent inositol phospholipid-specific phospholipase C activity in several rat tissues on the basis of its molecular size and its sensitivity to 1,10-phenanthroline. The data therefore suggest that this activity is due to a phospholipase D with specificity for glycosyl-phosphatidylinositol structures. Although the precise physiological function of this anchor-specific phospholipase D remains to be determined, these findings indicate that it could play an important role in regulating the expression and release of cell-surface proteins in vivo.
Solid-phase peptide synthesis and solid-state NMR spectroscopy of [Ala3-15N][Val1]gramicidin A.
Authors: Fields G B , Fields C G , Petefish J , Van Wart H E , Cross T A
Published: May 2006

[Ala3-15N][Val1]Gramicidin A has been prepared by solid-phase peptide synthesis and studied by solid-state 15N nuclear magnetic resonance spectroscopy. The synthesis of desformyl[Ala3-15N][Val1]gramicidin A employed N-hydroxysuccinimide esters of 9-fluorenylmethoxycarbonyl-N alpha-amino acids and completely avoided the use of acid. Since deblocking was done with piperidine and the peptide was removed from the resin by treatment with ethanolamine, this synthetic protocol prevented oxidation of the indole rings of this tryptophan-rich peptide and reduced truncations produced by acid hydrolysis. After formylation and purification by anion-exchange and high-pressure liquid chromatography, the peptide was obtained in an overall yield of 30%. Solid-state 15N nuclear magnetic resonance spectra of this peptide and uniformly labeled [15N]gramicidin A' oriented in hydrated lipid bilayers have been obtained, allowing unambiguous assignment of the [15N]Ala3 resonance in the latter. The solid-state 15N nuclear magnetic resonance experiments provide evidence that [Val1]gramicidin A is rotating about an axis that is perpendicular to the plane of the lipid bilayer and that the N--H axis is nearly parallel with the rotational axis. This study demonstrates that site-specifically labeled [15N]gramicidin A analogs prepared by solid-phase peptide synthesis are valuable tools in the study of the solid-state nuclear magnetic resonance spectra of samples in oriented lipid bilayers.
Functional role of cysteine-146 in Escherichia coli thymidylate synthase.
Authors: Dev I K , Yates B B , Leong J , Dallas W S
Published: May 2006

Analysis of mutant Escherichia coli thymidylate synthases (EC 2.1.1.45) with various amino acids substituted for cysteine at position 146 revealed the cysteine to be involved in the binding of 2'-deoxyuridylate as well as initiating the catalytic process. The substitution of a serine or alanine residue at position 146 did not appreciably alter the binding affinity for 2'-deoxyuridylate but the serine mutant enzyme was less active by a factor of 5000, whereas the alanine mutant enzyme was catalytically inactive. In contrast, the substitution of a glycine or threonine at position 146 created inactive enzymes with higher 2'-deoxyuridylate dissociation constants. The dissociation constant values for 2'-deoxyuridylate were used to estimate the overall contribution of the side chain of the amino acid at position 146 to substrate binding. The results suggested that the side chains of cysteine, alanine, and serine make nonspecific but effective van der Waals contacts with 2'-deoxyuridylate, thereby contributing about 0.82 kcal.mol-1 (1 cal = 4.184 J) to the apparent binding energy of the substrate.
Oligonucleotide that binds nuclear factor NF-kappa B acts as a lymphoid-specific and inducible enhancer element.
Authors: Pierce J W , Lenardo M , Baltimore D
Published: May 2006

The immunoglobulin kappa light chain gene contains a lymphoid-specific enhancer that includes several short protein-binding sequences. The sequence that binds the nuclear factor NF-kappa B was tested for its ability to act independently as an enhancer element by inserting it into test plasmids containing the chloramphenicol acetyltransferase gene. When analyzed for activity by transient transfection into lymphoid and nonlymphoid cells, a single copy of the NF-kappa B binding site could act as a tissue-specific upstream activating element. Two copies (dimer) showed 10-fold higher activity than did one copy and could act as an enhancer element 2.5 kilobases downstream of the transcriptional start site. The enhancer activity of this sequence was correlated with the presence of the cognate binding protein, NF-kappa B. This sequence acted as an inducible enhancer under conditions that induce NF-kappa B binding activity. Thus, the NF-kappa B binding site acts by itself as a tissue-specific and inducible enhancer element, and two copies show cooperative interaction.
Diffusion models of ion-channel gating and the origin of power-law distributions from single-channel recording.
Authors: Millhauser G L , Salpeter E E , Oswald R E
Published: May 2006

The lifetimes of the unitary currents from ion channels, as revealed from single-channel recording, are traditionally thought to follow exponential or multiexponential distributions. The interpretation of these event-time distributions is that the gating process follows Markov kinetics among a small number of states. There is recent evidence, however, that certain systems exhibit distributions that follow power laws or functions related to power laws. Likewise, it has been suggested that data sets that appear to be multiexponential may be fit to simple power laws as well. In this paper we propose a different view of ion-channel-gating kinetics that is consistent with these recent experimental observations. We retain the Markovian nature of the kinetics, but, in contrast to the traditional models, we suggest that ion-channel proteins have a very large number of states all of similar energy. Gating, therefore, resembles a diffusion process. We show that our simplest one-dimensional model exhibits single-channel distributions that follow power laws of the form t-a, where 1/2 less than or equal to a less than or equal to 3/2. Exponents determined from recent experiments approximately fall within this range. We believe that this model is consistent with modern views of protein dynamics and, thus, may provide a key to the molecular details of the gating process.
An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins.
Authors: Strauch K L , Beckwith J
Published: May 2006

A fusion between tsr (encoding the inner membrane protein Tsr) and phoA (encoding the periplasmic protein alkaline phosphatase, AP) generates a membrane-bound hybrid protein (Tsr-AP 2) with AP enzymatic activity. The hybrid protein is proteolytically unstable and is broken down to yield a smaller, soluble species with AP activity. We devised a genetic screen to distinguish between cells containing only membrane-bound AP and those containing soluble AP. The screen depends on diffusion of soluble AP away from cells with a leaky outer membrane to produce a halo of AP activity around colonies on solid growth medium. Several mutants lacking this halo show reduced degradation of Tsr-AP 2. One mutant is also defective in breakdown of five other abnormal periplasmic proteins but not of two cytoplasmic proteins. The mutation in this strain, degP4::Tn5, defines a locus distinct from previously identified loci that affect protein stability or protease activities. This strain may be useful for preventing the breakdown of unstable foreign proteins in Escherichia coli.
Sodium-independent in vitro induction of Na+,K+-ATPase by aldosterone in renal target cells: permissive effect of triiodothyronine.
Authors: Barlet-Bas C , Khadouri C , Marsy S , Doucet A
Published: May 2006

The aim of this study was to develop an in vitro system in which we could study the causal relationship between short-term stimulation of Na+,K+-ATPase in the collecting tubule by aldosterone on the one hand and protein synthesis and changes in intracellular Na+ concentration on the other hand. Previous in vivo studies suggested that triiodothyronine might facilitate aldosterone-induced stimulation of Na+,K+-ATPase. Results show that when segments of cortical collecting tubules microdissected from collagenase-treated kidneys of adrenalectomized rats were incubated for 3 hr in the presence of either 10(-8) M aldosterone or 10(-8) M triiodothyronine alone Na+,K+-ATPase activity was not altered, whereas the addition of both hormones markedly stimulated the activity and the number of catalytic sites of Na+,K+-ATPase. This stimulation was abolished by actinomycin D and cycloheximide, whereas it was not altered in the absence of extracellular sodium or in the presence of the luminal Na+-channel blocker amiloride. Thus, triiodothyronine facilitates the in vitro induction of Na+,K+-ATPase synthesis by aldosterone. Aldosterone action on Na+,K+-ATPase is independent of Na+ availability.
UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA.
Authors: Burckhardt S E , Woodgate R , Scheuermann R H , Echols H
Published: May 2006

The mutation rate of Escherichia coli increases approximately 100-fold after treatment with replication-inhibiting agents such as UV light. This enhanced mutation rate requires the action of the UmuD and UmuC proteins, which are induced as part of the SOS response to DNA damage. To initiate a biochemical characterization of the role of these proteins, we have developed a plasmid system that gives efficient expression of the umuD and umuC genes. The umuD and umuC genes were placed under the control of a regulated phage lambda PL promoter and a synthetic ribosome-binding site, and the distance to the UmuD start was adjusted to maximize gene expression. Starting from this overproduction system, we have purified the UmuD protein and studied its interaction with RecA. The SOS response is turned on by the capacity of RecA protein to mediate cleavage of the LexA repressor for SOS-controlled operons. Others have shown that UmuD exhibits sequence homology to LexA around the cleavage site, suggesting a possible cleavage reaction for UmuD. We show that RecA mediates cleavage of UmuD, probably at this site. As with LexA, UmuD also undergoes a self-cleavage reaction. We infer that RecA-mediated cleavage of UmuD is another role for RecA in SOS mutagenesis, probably activating UmuD for its mutagenic function.
Imaging asynchronous changes in intracellular Ca2+ in individual stimulated tumor mast cells.
Authors: Millard P J , Gross D , Webb W W , Fewtrell C
Published: May 2006

Changes in the level of intracellular free calcium ([Ca2+]i) are associated with the secretion of mediators of immediate hypersensitivity by rat basophilic leukemia (RBL) cells, a mast cell line. Digital fluorescence ratio imaging of fura-2 was used to measure [Ca2+]i in individual RBL cells. Changes in [Ca2+]i that occurred in response to crosslinking of IgE receptors on the cell surface or to application of the Ca2+ ionophore ionomycin were studied. Stimulation of RBL cells with antigen resulted in rapid increases in [Ca2+]i following lag times that varied widely from cell to cell. Simple averaging of the Ca2+ responses of many cells yielded a gradual response profile that closely resembled that of suspensions of cells measured in the fluorometer. The results show that single cells can respond much more rapidly to antigen than has previously been suggested by studies on populations of cells. The lag time between addition of antigen and initiation of the increase in [Ca2+]i varied considerably between cells in the same field of view. Both the rise time and the variability and average duration of the lag time increased with decreasing antigen concentration.
Characterization of 1-methyladenine binding in starfish oocyte cortices.
Authors: Yoshikuni M , Ishikawa K , Isobe M , Goto T , Nagahama Y
Published: May 2006

1-Methyladenine (1MeAde) is the naturally occurring maturation-inducing hormone of starfish oocytes. We have prepared a biologically active [3H]1MeAde of high purity and relatively high specific radioactivity. This ligand binds to cortices isolated from full-grown prophase-arrested oocytes of the starfish Asterina pectinifera. The binding of [3H]1MeAde to cortices was rapid and reached equilibrium in approximately 15 min. This is in excellent agreement with the hormone-dependent period required for the induction of oocyte maturation. Binding was maximal between pH 6.4 and 8.0 and diminished sharply above and below this range. Analysis of Scatchard plots of the equilibrium binding of [3H]1MeAde to cortices indicates the existence of a single class of binding site with a dissociation constant of 0.3 microM and a binding capacity of 0.02 fmol per cortex. Whereas biologically active analogs (1-benzyladenine, 1-ethyladenine) inhibited the specific binding of [3H]1MeAde by cortices, biologically inactive analogs (2-methyladenine, 3-methyladenine, 1,9-dimethyladenine, and 1-methylhypoxanthine) did not. These results suggest that the 1MeAde binding characterized herein is necessary for the maturational action of 1MeAde on starfish oocytes.
Isolation and characterization of a Drosophila gene that encodes multiple neuropeptides related to Phe-Met-Arg-Phe-NH2 (FMRFamide).
Authors: Schneider L E , Taghert P H
Published: May 2006

A Drosophila gene that encodes neuropeptides related to molluscan Phe-Met-Arg-Phe-NH2 (FMRFamide) was isolated by screening a genomic library with a fragment of an Aplysia Phe-Met-Arg-Phe-NH2 cDNA and with synthetic oligonucleotides. This gene was used to isolate a cDNA from a Drosophila adult head cDNA library. The cDNA was defined by sequence analysis to encode 13 peptides that have Phe-Met-Arg-Phe-NH2 or related sequences at their carboxyl termini. Other putative neuropeptides, including one that has homology to mammalian corticotropin-releasing factor, are present in the deduced approximately equal to 39-kDa precursor. Southern blot analysis suggested the presence of a single Phe-Met-Arg-Phe-NH2-like gene within the haploid genome. RNA blot analysis indicated the expression of at least two transcripts of approximately equal to 1.7 and approximately equal to 0.7 kilobases. Both transcripts are evident throughout larval, pupal, and adult developmental stages. In situ hybridization was used to localize this neuropeptide gene to band 46C on the right arm of the 2nd chromosome. These data provide the basis for utilizing the advanced genetics and molecular techniques of Drosophila to address complex aspects of neuropeptide expression and function.