bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence of the Shiga-like toxin genes of Escherichia coli.
Authors: Calderwood S B , Auclair F , Donohue-Rolfe A , Keusch G T , Mekalanos J J
Published: May 2006

We have determined the nucleotide sequence of the sltA and sltB genes that encode the Shiga-like toxin (SLT) produced by Escherichia coli phage H19B. The amino acid composition of the A and B subunits of SLT is very similar to that previously established for Shiga toxin from Shigella dysenteriae 1, and the deduced amino acid sequence of the B subunit of SLT is identical with that reported for the B subunit of Shiga toxin. The genes for the A and B subunits of SLT apparently constitute an operon, with only 12 nucleotides separating the coding regions. There is a 21-base-pair region of dyad symmetry overlapping the proposed promoter of the slt operon that may be involved in regulation of SLT production by iron. The peptide sequence of the A subunit of SLT is homologous to the A subunit of the plant toxin ricin, providing evidence for the hypothesis that certain prokaryotic toxins may be evolutionarily related to eukaryotic enzymes.
Domain structure of the glucocorticoid receptor protein.
Authors: Carlstedt-Duke J , Strömstedt P E , Wrange O , Bergman T , Gustafsson J A , Jörnvall H
Published: May 2006

The purified rat liver glucocorticoid receptor protein was analyzed by limited proteolysis and amino acid sequence determination. The NH2 terminus appears to be blocked. The steroid-binding domain, defined by a unique tryptic cleavage site, corresponds to the COOH-terminal part of the protein with the domain border in the region of residue 518. The DNA-binding domain, defined by a region with chymotryptic cleavage sites, is immediately adjacent to the steroid-binding domain and reflects another domain border in the region of residues 410-414. The results described at the protein level in this report confirm functional data previously obtained by mutations at the genetic level.
Alpha-chain locus of the T-cell antigen receptor is involved in the t(10;14) chromosome translocation of T-cell acute lymphocytic leukemia.
Authors: Kagan J , Finan J , Letofsky J , Besa E C , Nowell P C , Croce C M
Published: May 2006

Human leukemic T cells carrying a t(10;14)(q24;q11) chromosome translocation were fused with mouse leukemic T cells, and the hybrids were examined for genetic markers of human chromosomes 10 and 14. Hybrids containing the human 10q+ chromosome had the human genes for terminal deoxynucleotidyltransferase that has been mapped at 10q23-q25 and for C alpha [the constant region of TCRA (the alpha-chain locus of the T-cell antigen receptor gene)], but not for V alpha (the variable region of TCRA). Hybrids containing the human 14q- chromosome retained the V alpha genes. Thus the 14q11 breakpoint in the t(10;14) chromosome translocation directly involves TCRA, splitting the locus in a region between the V alpha and the C alpha genes. These results suggest that the translocation of the C alpha locus to a putative cellular protooncogene located proximal to the breakpoint at 10q24, for which we propose the name TCL3, results in its deregulation, leading to T-cell leukemia. Since hybrids with the 10q+ chromosome also retained the human terminal deoxynucleotidyltransferase gene, it is further concluded that the terminal deoxynucleotidyltransferase locus is proximal to the TCL3 gene, at band 10q23-q24.
Identification of HLA-DP polymorphism with DP alpha and DP beta probes and monoclonal antibodies: correlation with primed lymphocyte typing.
Authors: Bodmer J , Bodmer W , Heyes J , So A , Tonks S , Trowsdale J , Young J
Published: May 2006

Thirty-four lymphoblastoid cell lines that had been previously typed for HLA-DP antigens by primed lymphocyte typing (PLT) were tested by Southern blotting and by ELISA. Using two DP beta probes and a DP alpha probe with a series of enzymes, it is possible to identify restriction fragment length polymorphism (RFLP) patterns characteristic of DPw1, -2, -3, -4, and possibly -5. ELISA typing results, based on two polymorphic DP antibodies DP11.1 and ILR1, were compared with PLT-defined and RFLP-defined types. Thus, using a range of probes and enzymes it is possible to identify DP polymorphism. The value of monoclonal antibodies for such studies is demonstrated, and the molecular data can, in some cases, pinpoint the amino acids responsible for the specificity of the monoclonal antibodies.
Modulated structures and competing interactions in amphiphilic monolayers
Authors: Andelman David , Brochard Françoise , Joanny Jean-François
Published: May 2006

We investigate the effects of electrostatic interactions on the phase behavior and structure of insoluble Langmuir monolayers at the liquid/air interface. Both for charged and neutral monolayers, the competition between such repulsive long-range and attractive short-range interactions of the monolayer tends to stabilize modulated phases. Phase diagrams are obtained in two limits: (
i
) close to the liquid-gas critical point and (
ii
) at low temperatures.
Fundamental studies of the interactions of adsorbates on organic surfaces
Authors: Dubois Lawrence H. , Zegarski Bernard R. , Nuzzo Ralph G.
Published: May 2006

We have undertaken fundamental studies that characterize the important interactions of adsorbates on organic solids. Model substrates were prepared by the molecular self-assembly of organosulfur compounds of the general structure [S(CH
2
)
n
-X]
2
(
n
= 15-19, X=CH
3
, CONH
2
, CO
2
H, etc.) on a Au(111) single crystal surface. The adsorption of both polar and nonpolar molecules on top of these model surfaces was studied. The bonding of nonpolar molecules is dominated by dispersive forces and is weak in all cases. In contrast to this behavior, polar molecules, which interact weakly with the nonpolar surfaces, bond much more strongly to the polar materials. Our results also suggest an important role for structural transformations in the adsorbed overlayer in these latter cases. The potential relevance of these microscopic observations to important macroscopic properties such as wetting is discussed.
Characterization of a preleukemic state induced by Moloney murine leukemia virus: evidence for two infection events during leukemogenesis.
Authors: Davis B R , Brightman B K , Chandy K G , Fan H
Published: May 2006

A preleukemic state in mice inoculated with Moloney murine leukemia virus (Mo-MuLV) was characterized. Six to 10 weeks after neonatal inoculation, animals developed mild splenomegaly and generalized hematopoietic hyperplasia. The hyperplasia was evident from myeloid and erythroid progenitor assays. A nonleukemogenic variant, Mo+PyF101 Mo-MuLV, did not induce the hyperplasia; this suggests that the hyperplasia is a necessary event in Mo-MuLV leukemogenesis. Another variant, MF-MuLV, which contains the long terminal repeat of Friend MuLV and causes erythroid leukemia instead of T-cell lymphoma, also induced the preleukemic hyperplasia. A model for Mo-MuLV leukemogenesis is presented in which two infection events are necessary: the first leads to generalized hematopoietic hyperplasia, and the second results in site-specific insertion and long terminal repeat activation of cellular protooncogenes.
Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells.
Authors: Berger J , Howard A D , Gerber L , Cullen B R , Udenfriend S
Published: May 2006

Human placental alkaline phosphatase (PALPase) has been transiently expressed in simian (COS) cells by transfection with a eukaryotic expression vector containing the corresponding cDNA. The level of expression of PALPase was high, and it was produced in an enzymatically active form. The bulk of PALPase was associated with the cell membrane as shown by immunocytochemistry and subcellular fractionation studies. The PALPase produced by transfected COS cells, like PALPase in human tissue, was specifically released from the intact cells in a hydrophilic form by phosphatidylinositol-specific phospholipase C and is, therefore, apparently attached to the outer membrane by means of a phosphatidylinositol-glycan. Transfected COS cells appear to be an excellent model for elucidating the mechanism of attachment of this phosphatidylinositol-glycan to a protein moiety.
Rat brain serotonin receptors in Xenopus oocytes are coupled by intracellular calcium to endogenous channels.
Authors: Takahashi T , Neher E , Sakmann B
Published: May 2006

Serotonin activates chloride currents in Xenopus oocytes injected with a subfraction of rat brain poly(A)+ mRNA. Patch-clamp recordings from cell-attached patches showed that serotonin, applied locally outside the patch, caused the opening of channels of approximately equal to 3 pS conductance and an average lifetime of approximately equal to 100 msec. The extrapolated reversal potential indicated that the channels are chloride-selective. Single-channel currents with similar characteristics were observed in inside-out patches from native oocytes in response to elevated calcium concentrations on the cytoplasmic side. Measurements of intracellular calcium concentration ([Ca2+]i) by fura-2 fluorescence showed approximately equal to 10-fold increases in [Ca2+]i in response to serotonin application in both normal and calcium-free Ringer solution in mRNA-injected oocytes. Little or no response to serotonin was observed in native oocytes. These results suggest that serotonin activation of receptors that are inserted into the oocyte membrane following injection of rat brain poly(A)+ mRNA can induce calcium release from intracellular stores. The increase in [Ca2+]i subsequently activates calcium-dependent chloride channels. Because calcium-dependent chloride channels and a receptor-controlled mechanism of internal calcium release have been shown to exist in native oocytes, we conclude that the newly inserted serotonin receptors utilized the endogenous second-messenger-mediated calcium release to activate endogenous calcium-dependent chloride channels.
Localization of beta-adrenergic receptors on differentiated cells of the central nervous system in culture.
Authors: Ventimiglia R , Greene M I , Geller H M
Published: May 2006

Hypothalamic neurons and cortical protoplasmic (type 1) astrocytes in dissociated cultures of rat brain express binding sites for antibodies specific for epitopes related to the beta-adrenergic receptor. Undifferentiated glial progenitor cells of the rat optic nerve do not detectably bind these antibodies, but both of the progeny [oligodendroglia and process-bearing (type 2) astrocytes] express immunologically identified beta-adrenergic receptors. Levels of receptor expression are variable: not all cells of each type express receptors nor is expression uniform on a given cell. The data suggest that cells of the central nervous system express beta-adrenergic receptors but that levels of expression may be determined by the differentiated state of the cells.
Leaf assemblages across the Cretaceous-Tertiary boundary in the Raton Basin, New Mexico and Colorado
Authors: Wolfe Jack A. , Upchurch Garland R.
Published: May 2006

Analyses of leaf megafossil and dispersed leaf cuticle assemblages indicate that major ecologic disruption and high rates of extinction occurred in plant communities at the Cretaceous-Tertiary boundary in the Raton Basin. In diversity increase, the early Paleocene vegetational sequence mimics normal short-term ecologic succession, but on a far longer time scale. No difference can be detected between latest Cretaceous and early Paleocene temperatures, but precipitation markedly increased at the boundary. Higher survival rate of deciduous versus evergreen taxa supports occurrence of a brief cold interval (<1 year), as predicted in models of an “impact winter.”
Formation of the triple-stranded polynucleotide helix, poly(A.A.U).
Authors: Broitman S L , Im D D , Fresco J R
Published: May 2006

A polynucleotide helical structure containing two strands of poly(A) and one of poly(U) is reported. As shown by spectroscopic observations, the complex only forms when the poly(A) strands are of Mr between 9000 and 50,000 (degree of polymerization congruent to 28-150), whereas the size of the poly(U) strand has no effect. This limitation may explain why poly(A.A.U) was not seen in previous investigations. The potential of the poly(A) tails of mRNA for formation of this triple helix and of A.A.U or/and A.A.T triplet formation to contribute to the binding of specific RNA strands to gene-encoding nucleic acid double helices are noted.
Gene expression from transcriptionally disabled retroviral vectors.
Authors: Yee J K , Moores J C , Jolly D J , Wolff J A , Respess J G , Friedmann T
Published: May 2006

Retroviral vectors are used for the efficient transfer of foreign genes into mammalian cells. We report here the construction of murine retrovirus-based vectors carrying the full-length cDNA for human hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) and from which the enhancer sequences, the "CAAT box," and the "TATA box" in the long terminal repeats (LTRs) have been deleted. After infection of HPRT-deficient rat cells by the vectors, transcriptional activity from the 5' LTR was undetectable and expression of the HPRT cDNA was dependent on an internal promoter. Removal of the LTR regulatory elements increased HPRT gene expression from an internal promoter, indicating interference between the two sets of transcriptional signals. Such disabled vectors may reduce the likelihood of undesirable genetic changes through insertional mutagenesis in cells infected with retroviral vectors.
Plant defense genes are regulated by ethylene
Authors: Ecker Joseph R. , Davis Ronald W.
Published: May 2006

One of the earliest detectable events during plant-pathogen interaction is a rapid increase in ethylene biosynthesis. This gaseous plant stress hormone may be a signal for plants to activate defense mechanisms against invading pathogens such as bacteria, fungi, and viruses. The effect of ethylene on four plant genes involved in three separate plant defense response pathways was examined; these included (
i
and
ii
) genes that encode L-phenylalanine ammonia-lyase (EC 4.3.1.5) and 4-coumarate:CoA ligase [4-coumarate:CoA ligase (AMP-forming), EC 6.2.1.12], enzymes of the phenylpropanoid pathway, (
iii
) the gene encoding chalcone synthase, an enzyme of the flavonoid glycoside pathway, and (
iv
) the genes encoding hydroxyproline-rich glycoprotein, a major protein component(s) of plant cell walls. Blot hybridization analysis of mRNA from ethylene-treated carrot roots reveals marked increases in the levels of phenylalanine ammonia-lyase mRNA, 4-coumarate CoA ligase mRNA, chalcone synthase mRNA, and certain hydroxyproline-rich glycoprotein transcripts. The effect of ethylene on hydroxyproline-rich glycoprotein mRNA accumulation was different from that of wounding. Ethylene induces two hydroxyproline-rich glycoprotein mRNAs (1.8 and 4.0 kilobases), whereas wounding of carrot root leads to accumulation of an additional hydroxyproline-rich mRNA (1.5 kilobases). These results indicate that at least two distinct signals, ethylene and a wound signal, can affect the expression of plant defense-response genes.
Selective transcription and DNase I protection of the rat prolactin gene by GH3 pituitary cell-free extracts.
Authors: Gutierrez-Hartmann A , Siddiqui S , Loukin S
Published: May 2006

Prolactin (PRL) is a member of the growth hormone gene family that is specifically expressed in the lactotroph cells of the anterior pituitary. Whole cell extracts have been prepared from cultured GH3 rat pituitary tumor cells to study lactotroph-specific expression of the rat PRL (rPRL) gene in an in vitro transcription assay. The human alpha 1-globin and Rous sarcoma virus promoters efficiently initiate transcription in both HeLa and GH3 cell extracts, whereas the rPRL promoter containing 425 base pairs of 5' flanking DNA is active only in GH3 pituitary cell-free extracts. Transcription of the rPRL gene was reconstituted in HeLa cell extracts by the addition of GH3 cell extracts. DNase I digestion of the rPRL promoter reveals two protected regions centered at positions -55 (I) and -160 (III) that are GH3 cell-specific and rPRL promoter-selective. These "footprints" overlie a highly conserved 8-base-pair motif, CCTGATAATA. By contrast, footprint II at position -125 is common to both HeLa and GH3 cell extracts and overlies a 15-base-pair sequence found in all members of the growth hormone gene family. Thus, GH3 pituitary cell-free extracts selectively transcribe the rPRL gene and contain cell-specific factors that directly interact with the rPRL promoter. These studies provide useful assays to further identify, purify, and characterize pituitary-specific transcription factors and to address the biochemical mechanisms involved in rPRL gene expression.
High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells.
Authors: Whittaker J , Okamoto A K , Thys R , Bell G I , Steiner D F , Hofmann C A
Published: May 2006

In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10(6) receptors per cell. The cell line with the highest insulin binding (NIH 3T3 HIR3.5) had 6 X 10(6) receptors with a Kd of 10(-9) M. This level was not dependent on exposure to metals but could be increased further to 2 X 10(7) receptors per cell by addition of sodium butyrate to the culture medium. The alpha and beta subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the alpha and beta subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis.
Cofilin is a component of intranuclear and cytoplasmic actin rods induced in cultured cells.
Authors: Nishida E , Iida K , Yonezawa N , Koyasu S , Yahara I , Sakai H
Published: May 2006

Incubation of cultured cells under specific conditions induces a dramatic change in the actin organization: induction of intranuclear and/or cytoplasmic actin rods (actin paracrystal-like intracellular structures). We have found that cofilin, a 21-kDa actin-binding protein, is a component of these rods. Antibodies directed against cofilin labeled intranuclear actin rods induced in cells treated with dimethyl sulfoxide or exposed to heat shock and also labeled cytoplasmic actin rods induced in cells incubated in specific salt buffers. Moreover, we found that these actin rods are not stained with fluorescent phalloidin derivatives at all and appear to be right-handed helices, different from straight bundles of F-actin such as stress fibers. In vitro experiments revealed that cofilin and phalloidin compete with each other for binding to F-actin. Since cofilin and phalloidin have the ability to stoichiometrically bind actin molecule in the filament in vitro, the above results seem to suggest that cofilin directly binds to actin molecule in nearly an equimolar ratio in these rods. We call these rods "actin/cofilin rods."
Interleukin 1 regulates hematopoietic activity, a role previously ascribed to hemopoietin 1.
Authors: Mochizuki D Y , Eisenman J R , Conlon P J , Larsen A D , Tushinski R J
Published: May 2006

A murine in vitro assay was developed to measure potentiation of a proliferative response to suboptimal concentrations of the hematopoietic regulatory molecule granulocyte/macrophage colony-stimulating factor by an immature bone marrow population. The assay, designated the 5-fluorouracil bone marrow proliferation assay, was used to characterize potentiating activity in serum-free culture supernatants of the human tumor cell line HBT 5637. Molecular and biochemical analyses indicated that the HBT 5637-derived potentiating activity could be attributed to interleukin 1 alpha. Serologic analysis using a monoclonal antibody against purified recombinant interleukin 1 alpha proved conclusively that the potentiating activity in HBT 5637 serum-free supernatants is due to interleukin 1 alpha. From these data, the activity of interleukin 1 alpha seems to be the same synergistic activity formerly ascribed to hemopoietin 1.
Genes encoding alpha and beta subunits of Na,K-ATPase are located on three different chromosomes in the mouse.
Authors: Kent R B , Fallows D A , Geissler E , Glaser T , Emanuel J R , Lalley P A , Levenson R , Housman D E
Published: May 2006

We have made use of a panel of mouse-hamster somatic cell hybrids and restriction fragment length polymorphisms between two mouse species (Mus musculus and Mus spretus) to determine the chromosomal localization of genes encoding the alpha and beta subunits of the Na,K-ATPase (Na+,K+-activated ATP phosphohydrolase, EC 3.6.1.3). DNA probes for three distinct isoforms of the Na,K-ATPase alpha subunit mapped to three different mouse chromosomes: the alpha 1 gene (Atpa-1) cosegregated with the Egf gene on chromosome 3; alpha 2 (Atpa-2) with the cytochrome P-450PB gene family/coumarin hydroxylase locus on chromosome 7; alpha 3 (Atpa-3) with the alpha-spectrin gene on chromosome 1. The Na,K-ATPase beta-subunit gene (Atpb) mapped to the same region of chromosome 1, but it was not tightly linked to the Atpa-3 gene. These results indicate that three isoforms of the Na,K-ATPase alpha subunit are encoded by three distinct genes. The dispersion of Na,K-ATPase genes suggests that their expression is not likely to be controlled by a common cis-acting regulatory element.
Activation of interleukin 2 and interleukin 2 receptor (Tac) promoter expression by the trans-activator (tat) gene product of human T-cell leukemia virus, type I.
Authors: Siekevitz M , Feinberg M B , Holbrook N , Wong-Staal F , Greene W C
Published: May 2006

Cotransfection of cDNA encoding the trans-activator gene product of human T-cell leukemia virus, type I (HTLV-I) (tat-I), which acts in trans to augment viral gene expression, has revealed strong regulatory effects of this viral protein on the inducible cellular promoters governing human interleukin 2 (IL-2) and IL-2 receptor (Tac) gene expression. The tat-I protein stimulates a 3- to 6-fold increase in IL-2 receptor (Tac) promoter activity in transfected Jurkat T cells, but not in the natural killer-like YT cell line, as measured by changes in the expression of the chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) reporter gene linked to this promoter. In contrast, tat-I alone has little or no effect on IL-2 promoter activity in Jurkat T cells but markedly synergizes with other mitogenic stimuli (phytohemagglutinin, phorbol 12-myristate 13-acetate, or the OKT3 monoclonal antibody), which alone are ineffective. The tat-I protein also partially circumvents the pronounced inhibitory effects of cyclosporin A on the IL-2 promoter. Other cellular and viral promoters are unaffected by the tat-I gene product, either alone or in combination with other mitogens. The specific effects of the tat-I gene product on the IL-2 and IL-2 receptor (Tac) promoters suggest the possibility of an autocrine or paracrine mechanism of T-cell growth as an early event in HTLV-I-mediated leukemogenesis.
Expression of an omega-conotoxin-sensitive calcium channel in Xenopus oocytes injected with mRNA from Torpedo electric lobe.
Authors: Umbach J A , Gundersen C B
Published: May 2006

Xenopus laevis oocytes were injected with poly(A)+ RNA isolated from the electric lobe of Torpedo californica. Six to nine days after mRNA injection of the oocytes a cadmium-sensitive inward current could be detected in oocytes bathed in a calcium- and chloride-free solution containing 40 mM barium. This inward current could be distinguished from the native barium current of control oocytes by its high sensitivity to blockade by cadmium ions and its inhibition by omega-conotoxin, a peptide neurotoxin from Conus geographicus. Neither the current of control cells nor that of injected cells was detectably affected by nisoldipine (1 microM) or nitrendipine (1 microM). However, the barium current of control oocytes showed appreciably more inactivation (in the barium solution used for recording) than the omega-conotoxin-sensitive current that develops in mRNA-injected oocytes. Culturing of mRNA-injected oocytes in medium containing actinomycin D failed to prevent the appearance of the omega-conotoxin-sensitive current. These results support the conclusion that mRNA from Torpedo electric lobe is translated to produce an additional calcium channel in Xenopus oocytes. The features of this channel suggest that it may be the same type of calcium channel that controls transmitter release at nerve endings in Torpedo electroplax.
Identification of a cell-specific transcriptional enhancer in the first intron of the mouse alpha 2 (type I) collagen gene.
Authors: Rossi P , de Crombrugghe B
Published: May 2006

A transcriptional enhancer has been identified in the first intron of the mouse alpha 2 (type I) collagen gene in a region between +418 and +1524 base pairs from the transcriptional start site. The enhancer functions both when it is located 5' and 3' to the promoter that it activates and is independent of the orientation of the element. The enhancer stimulates both the homologous alpha 2 type I [alpha 2(I)] collagen promoter and the heterologous early simian virus 40 promoter. In transient expression experiments, enhancer-dependent transcription from the alpha 2(I) collagen promoter utilizes the same transcriptional start site as the one used in the endogenous alpha 2(I) collagen gene. The enhancer activates transcription at a distance of at least 3 kilobase pairs from the transcriptional start site. The alpha 2(I) collagen enhancer displays cell specificity, since it is functional in NIH 3T3 fibroblasts but completely inactive in a lymphoid cell line, in contrast to two immunoglobulin gene enhancers that show the opposite behavior. We find several areas of sequence homology with viral enhancers, particularly the enhancer of simian virus 40.
Amino acid sequence of the von Willebrand factor-binding domain of platelet membrane glycoprotein Ib.
Authors: Titani K , Takio K , Handa M , Ruggeri Z M
Published: May 2006

We report the amino acid sequence of a 299-residue segment from the alpha chain of the human platelet membrane glycoprotein Ib. This includes the complete sequence of the amino-terminal tryptic fragment of 290 residues comprising the von Willebrand factor-binding domain. Two primary sets of overlapping fragments were obtained by cleavage of the S-carboxymethylated protein at methionyl and lysyl bonds following treatment with cyanogen bromide and Achromobacter protease I, respectively. Additional fragments were obtained by treatment of native glycocalicin with trypsin, Staphylococcus aureus V8 protease, and Serratia marcescens protease. Analysis of all these fragments provided data that allowed determination of the continuous sequence corresponding to approximately half of the alpha-chain polypeptide. This region of glycoprotein Ib is largely hydrophobic and contains only two N-linked and one O-linked carbohydrate chains. A hydrophilic region exists between residues 215 and 299, which contains a cluster of 10 negatively charged residues at 269-287. This area is likely to attract positively charged molecules. The hydrophilic, highly glycosylated (at serine and threonine residues) region corresponding to the previously described "macroglycopeptide" and representing the carboxyl-terminal half of the alpha chain is likely to begin at residue 292. The determined sequence of the alpha chain of glycoprotein Ib contains a region (residues 29-193) with seven repeats, which is indicative of gene duplication and is highly homologous to human leucine-rich alpha 2-glycoprotein. This protein sequence agrees completely with that deduced from the cDNA sequence reported by Lopez et al. [Lopez, J.A., Chung, D.W., Fujikawa, K., Hagen, F.S., Papayannopoulou, T. & Roth, G.J. (1987) Proc. Natl. Acad. Sci. USA 84, 5615-5619].
Ca2+-mobilizing actions of platelet-derived growth factor differ from those of bombesin and vasopressin in Swiss 3T3 mouse cells.
Authors: Lopez-Rivas A , Mendoza S A , Nånberg E , Sinnett-Smith J , Rozengurt E
Published: May 2006

Addition of the mitogenic peptides bombesin and vasopressin to quiescent Swiss 3T3 mouse cells increased the cytosolic Ca2+ concentration without any measurable delay. In contrast, there was a significant lag period (16 +/- 1.2 s) before platelet-derived growth factor (PDGF) increased cytosolic Ca2+ concentration. This lag was not diminished at high concentrations of either porcine or human PDGF. Similar results were obtained in 3T3 cells loaded with quin-2 or fura-2. The differences in the effects of bombesin, vasopressin, and PDGF on Ca2+ movements were also substantiated by measurements of 45Ca2+ efflux and of cellular 45Ca2+ content. Activation of protein kinase C by phorbol esters inhibited Ca2+ mobilization induced by either bombesin or vasopressin. In contrast, phorbol esters had no effect on PDGF-induced cytosolic Ca2+ concentration increase or acceleration of 45Ca2+ efflux. Finally, bombesin and vasopressin caused a rapid increase in the production of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate, whereas PDGF, even at a saturating concentration, exerted only a small effect. These results indicate that the signal transduction pathways activated by PDGF that lead to Ca2+ mobilization can be distinguished from those utilized by bombesin and vasopressin.
Induction and segregation of glial intermediate filament expression in the RT4 family of peripheral nervous system cell lines.
Authors: Freeman M R , Sueoka N
Published: May 2006

We have found glial fibrillary acidic protein (GFAP), the major component of astrocyte intermediate filaments, to be expressed in cell lines of the RT4 peripheral neurotumor family. The RT4 family is a "stem-cell-like" cell line, RT4-AC, that spontaneously undergoes differentiation in culture to three derivative cell types. This process, termed cell-type conversion, results in a segregation among the derivative cell types of parental cell phenotypes that have been described as neuronal-like or glial-like. We have identified a 50-kDa GFAP-immunoreactive cytoskeletal protein and GFAP mRNA in continuous RT4-AC and RT4-D (glial-type derivative) cell lines, but not in two presumptive neuronal-type cell lines. This result suggests that GFAP gene expression is coordinately coupled with the expression of other glial properties during cell-type conversion. In addition, the RT4-AC and RT4-D sublines were found to significantly express GFAP only at high cell densities and not during logarithmic growth and to express GFAP precociously during morphological differentiation following treatment with 1 mM N6, O2'-dibutyryladenosine 3',5'-cyclic monophosphate. These observations closely reflect reports of glial filament expression in astrocyte cultures, suggesting that a common regulatory mechanism is employed by central and peripheral nervous system glia.
Neuronal loss in the pedunculopontine tegmental nucleus in Parkinson disease and in progressive supranuclear palsy.
Authors: Hirsch E C , Graybiel A M , Duyckaerts C , Javoy-Agid F
Published: May 2006

In the brains of humans and other mammals, there are two principal groups of cholinergic nuclei aside from those forming the cranial motor nuclei. One group lies in the forebrain and includes the nucleus basalis of Meynert. The second group lies in the hindbrain and includes the nucleus tegmenti pedunculopontinus (NPP), identified by Mesulam et al. [Mesulam, M.-M., Mufson, E. J., Wainer, B. H. & Levey, A. I. (1983) Neuroscience 10, 1185-1201] as cholinergic cell group Ch5. The basal forebrain cholinergic cell groups, which innervate widespread areas of the neocortex, undergo degeneration in Alzheimer disease and also in parkinsonism associated with dementia. We here report that the hindbrain NPP Ch5 cell group, thought to innervate many nuclei of the extrapyramidal motor system, the superior colliculus, and the substantia innominata, undergoes degeneration in idiopathic Parkinson disease and in the parkinsonian syndrome of progressive supranuclear palsy. These findings strongly suggest that degeneration in the brainstem in Parkinson disease is not confined to catecholamine-containing neurons, but that cholinergic neurons of the NPP are also vulnerable. The findings further raise the possibility that certain symptoms of Parkinson disease and progressive supranuclear palsy have their genesis in pathology of these cholinergic neurons.
Characterization of the phosphatidylinositol-glycan membrane anchor of human placental alkaline phosphatase.
Authors: Howard A D , Berger J , Gerber L , Familletti P , Udenfriend S
Published: May 2006

Placental alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] is a member of a diverse group of membrane proteins whose attachment to the lipid bilayer is mediated by a phosphatidylinositol-glycan. To investigate structural aspects of the glycolipid anchor, cultured WISH cells were used because we found that they produce the enzyme in abundant quantities. When cell suspensions were incubated with purified phosphatidylinositol-specific phospholipase C, most of the placental alkaline phosphatase was released from membranes in a hydrophilic form. On incubation of the cells with [14C]ethanolamine, [14C]myristic acid, or myo-[3H]inositol, each was incorporated into the phosphatase near the carboxyl terminus, showing that these components, which are found in other phosphatidylinositol membrane-linked proteins, are also present in placental alkaline phosphatase.
Two promoter-specific host factors interact with adjacent sequences in an EIA-inducible adenovirus promoter.
Authors: SivaRaman L , Thimmappaya B
Published: May 2006

We previously reported the identification of a host factor (EIIA-EF) specific for an upstream transcriptional control sequence (-82 to -66) of the EIA-inducible adenovirus EIIA early promoter. The levels of this factor remained unchanged after virus infection of human cells. Another study also identified a factor (EIIF) specific for this same promoter, but the activity of this second factor was shown to increase severalfold after virus infection. We now show that these dramatically different results, both based on gel shift assays on the same promoter, may be explained by variations in protocol details and actually identify two distinct factors. When synthetic DNA copolymers [poly(dI).poly(dC) or poly(dI-dC).poly(dI-dC)] are used as competitors in gel shift assays, a factor specific for DNA sequences between -82 and -66 can be identified, whereas when natural eukaryotic DNAs (salmon sperm or calf thymus) are used as competitors a different factor specific for DNA sequences between -69 and -33 can be identified. We have mapped the DNA-protein contact residues for the EIIF by analyzing a series of linker scan mutants in gel shift assays and methylation interference experiments. The EIIA-EF and EIIF bind to two distinct but adjacent sequences. Competition experiments indicate that these two activities are due to two different factors. Consistent with the earlier reports, the levels of one (EIIA-EF) do not change after virus infection of human cells, whereas the levels of the other (EIIF) are increased severalfold.
Pairings and polarities of the 14 strands in sickle cell hemoglobin fibers.
Authors: Rodgers D W , Crepeau R H , Edelstein S J
Published: May 2006

Sickle cell anemia results from the formation of hemoglobin S fibers in erythrocytes, and a greater understanding of the structure of these fibers should provide insights into the basis of the disease and aid in the development of effective antisickling agents. Improved reconstructions from electron micrographs of negatively stained single hemoglobin S fibers or embedded fiber bundles reveal that the 14 strands of the fiber are organized into pairs. The strands in each of the seven pairs are half-staggered, and from longitudinal views the polarity of each pair can be determined. The positions of the pairs and their polarities (three in one orientation; four in the opposite orientation) suggest a close relationship with the crystals of deoxyhemoglobin S composed of antiparallel pairs of half-staggered strands.
Iron distances in hemoglobin: comparison of x-ray crystallographic and extended x-ray absorption fine structure studies.
Authors: Fermi G , Perutz M F , Shulman R G
Published: May 2006

A comparison is presented of the structures obtained around the iron atom in deoxyhemoglobin (Hb). The data come from extended x-ray absorption fine structure (EXAFS) studies of the iron, which gave Fe-porphyrin nitrogen distances of 2.06 +/- 0.01 A, and from the most recent high-resolution x-ray crystallographic study, which gave exactly the same distance--2.06 +/- 0.02 A. The distance of Fe above the plane of the porphyrin nitrogens was 0.38 +/- 0.04 A from the crystallographic study; this value is not far from the upper limit of the distances 0.20 +/- (0.10)0.20 A calculated from the EXAFS experiment by triangulation. These distances above the nitrogen plane are shorter than those estimated in the earliest x-ray structures.
Coupling of replication type histone mRNA levels to DNA synthesis requires the stem-loop sequence at the 3' end of the mRNA.
Authors: Levine B J , Chodchoy N , Marzluff W F , Skoultchi A I
Published: May 2006

The role of the 3' end of mRNA in coupling between the level of histone mRNAs and DNA synthesis was examined. We introduced modified mouse histone H3 genes into mouse fibroblasts and studied the regulation of several different H3 mRNAs that are not terminated with a normal histone 3' end. In two cases, the stem-loop sequences were deleted from the mRNAs and replaced either by 3' sequences flanking the H3 gene or by globin 3' untranslated region sequences including the polyadenylylation signal. In the former case, approximately equal to 50% of the modified mRNA was polyadenylylated, whereas in the latter case all of the mRNA had a polyadenylylated terminus. In contrast to the normal histone mRNAs, these mRNAs, including the nonadenylylated form, were stable when DNA synthesis was inhibited with several drugs. The levels of two other histone mRNAs, each containing the stem-loop sequences as an internal part of the mRNA, also were stable when DNA synthesis was inhibited. These results indicate that the posttranscriptional coupling of histone mRNA levels to DNA synthesis requires the presence of the stem-loop sequences at the 3' end of the mRNA.
Spectra of spontaneous mutations in Escherichia coli strains defective in mismatch correction: the nature of in vivo DNA replication errors.
Authors: Schaaper R M , Dunn R L
Published: May 2006

We have determined the DNA sequence changes in 487 spontaneous mutations in the N-terminal part of the lacI gene in mutH, mutL, and mutS strains of Escherichia coli. These strains display elevated spontaneous mutation rates because of a deficiency in the process of postreplicative mismatch correction. As a consequence the mutational spectra reveal the nature of spontaneous DNA replication errors. The spectra consist of base substitutions (75%) and single-base deletions (25%). Among the base substitutions, transitions (both A.T----G.C and G.C----A.T) are strongly favored over transversions (96% versus 4%). Large site-to-site differences are observed among identical base substitutions, presumably reflecting the modulating effects of neighboring bases. The single-base-deletion spectrum is dominated by a large hotspot at a run of adjacent identical base pairs, implying a Streisinger-slippage mechanism. The data, when compared to a previously determined wild-type spectrum, also provide information on the specificity of the mismatch repair system.
In situ detection of enteroviral genomes in myocardial cells by nucleic acid hybridization: an approach to the diagnosis of viral heart disease.
Authors: Kandolf R , Ameis D , Kirschner P , Canu A , Hofschneider P H
Published: May 2006

We have developed an in situ hybridization assay capable of detecting enteroviral RNA in myocardial cells, using molecularly cloned coxsackievirus B3 cDNA as a diagnostic probe. Because of the high degree of nucleic acid sequence homology among the numerous enteroviral serotypes, including the group A and B coxsackieviruses and the echoviruses, detection of these various agents commonly implicated in human viral heart disease is possible in a single hybridization assay. We demonstrate the considerable potential of this method for an unequivocal diagnosis of enteroviral heart disease as well as for pathogenicity studies. Using athymic mice persistently infected with coxsackievirus B3 as a model system, we show that the myocardium is affected in a disseminated, multifocal manner.
mRNA for low density lipoprotein receptor in brain and spinal cord of immature and mature rabbits.
Authors: Hofmann S L , Russell D W , Goldstein J L , Brown M S
Published: May 2006

Hybridization studies with [32P]cDNA probes revealed detectable amounts of mRNA for the low density lipoprotein (LDL) receptor in the central nervous system (CNS) of rabbits. mRNA levels were highest in the medulla/pons and spinal cord, which were the most heavily myelinated regions that were studied. Lower, but detectable levels were present in cerebral cortex, hypothalamus, thalamus, midbrain, and cerebellum. In the medulla/pons and spinal cord, the levels of receptor mRNA were in a range comparable to that detected in the liver. The levels of receptor mRNA in whole brain were constant from 3 days of age to adulthood and, thus, did not vary in proportion to the rate of myelin synthesis. LDL receptor mRNA in the CNS was produced by the same gene that produced the liver and adrenal mRNA as revealed by the demonstration of a deletion in the neural mRNA of Watanabe-heritable hyperlipidemic (WHHL) rabbits identical to the deletion in the LDL receptor gene of these mutant animals. Using antibodies directed against the bovine LDL receptor, we showed that LDL receptor protein is present in the medulla/pons of adult cows. The cell types that express LDL receptors in the CNS and the functions of these receptors are unknown.
Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events.
Authors: Hauber J , Perkins A , Heimer E P , Cullen B R
Published: May 2006

Human immunodeficiency virus encodes a gene product termed tat that is able to activate viral gene expression when present in trans. The mechanism of action of the tat gene product appears to be bimodal, resulting in both an increase in the steady-state level of viral mRNA and the enhanced translation of that RNA. In this report we have examined the mechanism by which tat elevates viral mRNA levels. Data are presented demonstrating that tat acts by increasing the rate of viral transcription, rather than by modulating the stability of viral mRNA. Indirect immunofluorescence was used to show that tat is predominantly localized in the nucleus of expressing cells, a location consistent with a role in the regulation of viral transcription. These results suggest that tat could play a role in human immunodeficiency virus replication essentially similar to that proposed for the trans-acting nuclear gene products described for several other virus species.
cAMP-mediated protein phosphorylation of microsomal membranes increases mannosylphosphodolichol synthase activity.
Authors: Banerjee D K , Kousvelari E E , Baum B J
Published: May 2006

We have investigated the possible role of a cAMP-mediated protein-phosphorylation event(s) as the key regulatory mechanism in beta-adrenoreceptor-stimulated activation of mannosylphosphodolichol (Man-P-Dol) synthase (GDP-mannose:dolichyl-phosphate O-beta-D-mannosyltransferase, EC 2.4.1.83) in rat parotid acinar cells. Microsomal membranes isolated from these cells pretreated with 10 microM isoproterenol for 60 min showed approximately 40-80% enhanced Man-P-Dol synthase activity compared to the untreated controls. This change in enzyme activity was not associated with a significant alteration in apparent Km for GDP-mannose, but the Vmax was enhanced 2-fold. When microsomal membranes isolated from control cells were phosphorylated in vitro by a cAMP-dependent protein kinase, an increase in Man-P-Dol synthase activity, similar to that with membranes from isoproterenol-treated cells, was observed (i.e., a moderate change in Km for GDP-mannose but a 2-fold higher Vmax). Furthermore, treatment of in vitro phosphorylated microsomal membranes by alkaline phosphatase led to a substantial reduction in Man-P-Dol synthase activity. Increased Man-P-Dol synthesis (approximately 30-40%) was also observed in bovine brain and hen oviduct microsomal membranes after in vitro protein phosphorylation. In aggregate, these results strongly suggest that agents that increase cAMP in cells may modulate protein N-glycosylation in those cells by activating this key glycosyltransferase of the dolichol cascade by a cAMP-dependent protein kinase-mediated protein phosphorylation/dephosphorylation cycle.
Structure of the chicken link protein gene: exons correlate with the protein domains.
Authors: Kiss I , Deák F , Mestrić S , Delius H , Soos J , Dékány K , Argraves W S , Sparks K J , Goetinck P F
Published: May 2006

The structure of the chicken link protein gene has been determined from a series of genomic clones that cover the entire coding region as well as the complete 3'-untranslated region and a small portion of the 5'-untranslated region. The gene is greater than 80 kilobase pairs long and is present in a single copy in the chicken genome. The link protein gene contains at least five exons with four encoding the entire protein. The domain of link protein that has homologies with immunoglobulin-like proteins and the tandemly repeated hyaluronic acid binding domains are each encoded by separate exons. The exon-intron structure indicates that the link protein gene may have arisen by exon duplication and exon shuffling.
Three-dimensional structure of a genetically engineered variant of porcine growth hormone.
Authors: Abdel-Meguid S S , Shieh H S , Smith W W , Dayringer H E , Violand B N , Bentle L A
Published: May 2006

The three-dimensional structure of a genetically engineered variant of porcine growth hormone, methionyl porcine somatotropin (MPS), has been determined at 2.8-A resolution, using single crystal x-ray diffraction techniques. Phases were obtained by use of a single isomorphous K2OsCl6 derivative and were improved by use of the density modification procedure. The MPS structure is predominantly helical. It consists mainly of four antiparallel alpha-helices arranged in a left twisted helical bundle, a structural motif observed in a number of other unrelated proteins. However, the way the four helices are connected in the bundle is unusual and, to our knowledge, has never been reported before. Alignment of the amino acid sequence of MPS with that of other growth hormones reveals that residues within the alpha-helices are predominantly invariant and thus these invariant residues are necessary to maintain the structural integrity of these proteins.