bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolution and relatedness in two aminoacyl-tRNA synthetase families.
Authors: Nagel G M , Doolittle R F
Published: May 2006

Sequence segments of about 140 amino acids in length, each containing a selected consensus region, were used in alignments of the aminoacyl-tRNA synthetases with the aim of discerning their evolutionary relationships. In all cases tested, enzymes specific for the same amino acid from a variety of organisms grouped together, reinforcing the supposition that the aminoacyl-tRNA synthetases are very ancient enzymes that evolved to include the full complement of 20 amino acids long before the divergence leading to prokaryotes and eukaryotes. The enzymes are divided into two mutually exclusive groups that appear to have evolved from independent roots. Group I, for which two sequence segments were analyzed, contains the enzymes specific for glutamic acid, glutamine, tryptophan, tyrosine, valine, leucine, isoleucine, methionine, and arginine. Group II enzymes include those activating threonine, proline, serine, lysine, aspartic acid, asparagine, histidine, alanine, glycine, and phenylalanine. Both groups contain a spectrum of amino acid types, suggesting the possibility that each could have once supported an independent system for protein synthesis. Within each group, enzymes specific for chemically similar amino acids tend to cluster together, indicating that a major theme of synthetase evolution involved the adaptation of binding sites to accommodate related amino acids with subsequent specialization to a single amino acid. In a few cases, however, synthetases activating dissimilar amino acids are grouped together.
Localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization.
Authors: Henney A M , Wakeley P R , Davies M J , Foster K , Hembry R , Murphy G , Humphries S
Published: May 2006

The stromelysins are members of a family of extracellular matrix metalloproteinases. These enzymes may erode the connective tissue in atherosclerotic plaques, leading to fissuring and acute thrombotic events. Cell-specific stromelysin expression in human atherosclerotic plaques was studied by in situ hybridization and immunocytochemistry. Sections were taken from nine coronary arteries: eight with well-established plaques and one normal. Unambiguous signals were seen in five plaques, two were inconclusive, and the remaining sample was negative, as was the normal coronary artery. Stromelysin mRNA transcripts were localized to isolated individual cells, some of which were smooth muscle, in the plaque cap, intima, and adventitia, but not the media. Expression was also seen in large clusters of macrophages that contained intracellular lipid deposits. The isolated expression of stromelysin by smooth muscle cells may reflect local connective tissue remodeling associated with growth and the formation of the plaque, whereas the more extensive expression associated with macrophages may be of greater pathological significance, contributing to the destabilization of the extracellular matrix and eventual plaque rupture.
Membrane-bounded nucleoid in the eubacterium Gemmatata obscuriglobus.
Authors: Fuerst J A , Webb R I
Published: May 2006

The freshwater budding eubacterium Gemmata obscuriglobus possesses a DNA-containing nuclear region that is bounded by two nuclear membranes. The membrane-bounded nature of the nucleoid in this bacterium was shown by thin sectioning of chemically fixed cells, thin sectioning of freeze-substituted cells, and freeze-fracture/freeze-etch. The fibrillar nucleoid was surrounded by electron-dense granules that were in turn enveloped by two nuclear membranes separated by an electron-transparent space. Immunogold labeling of thin sections of conventionally fixed cells with anti-double-stranded DNA antibody demonstrated double-stranded DNA associated with fibrillar material within the membrane boundary. The occurrence of a membrane-bounded nucleoid in a eubacterial prokaryote is a significant exception to the evidence supporting the prokaryote/eukaryote dichotomous classification of cell structure.
Expression of biologically active heterodimeric bovine follicle-stimulating hormone in milk of transgenic mice.
Authors: Greenberg N M , Anderson J W , Hsueh A J , Nishimori K , Reeves J J , deAvila D M , Ward D N , Rosen J M
Published: May 2006

Follicle-stimulating hormone (FSH; follitropin) is a pituitary glycoprotein composed of two post-translationally modified subunits, which must properly assemble to be biologically active. FSH has been difficult to purify and to obtain in quantities sufficient for detailed biochemical studies. We have targeted FSH expression to the mammary gland of transgenic mice by using cDNAs encoding the bovine alpha and FSH beta subunits and a modified rat beta-casein gene-based expression system. Lines of bigenic mice expressing both subunits have been generated either by coinjection of the subunit transgenes or by mating mice that acquired and expressed transgenes encoding an individual subunit. Up to 60 international units (15 micrograms) of biologically active FSH per ml was detected in the milk of the bigenic mice. These lines provide a model system for studying the post-transcriptional mechanisms that effect the expression and secretion of this heterodimeric hormone.
Involvement of corticotropin-releasing factor in chronic stress regulation of the brain noradrenergic system.
Authors: Melia K R , Duman R S
Published: May 2006

Corticotropin-releasing factor (CRF) and norepinephrine (NE) mediate many hormonal, autonomic, and behavioral effects of acute stress, and it is possible that an interaction between these neurotransmitters could underlie neuronal adaptations in response to chronic stress. To test this hypothesis, the influence of chronically administered CRF and a specific CRF antagonist, alpha-helical CRF, on the induction of tyrosine hydroxylase, the rate-limiting enzyme in NE biosynthesis, was examined in the rat locus coeruleus (LC). We now report that administration of alpha-helical CRF specifically blocks the induction of tyrosine hydroxylase in response to a repeated intermittent stress paradigm involving foot shock and noise stress but has no effect on steady-state levels of the enzyme in nonstressed animals or on the induction of the enzyme in response to reserpine treatment. In addition, repeated administration of CRF alone for 5 days, like chronic stress, increases levels of tyrosine hydroxylase in LC. The results demonstrate that endogenous CRF is necessary for the induction of tyrosine hydroxylase in response to this stress paradigm and that exogenously administered CRF is sufficient for the regulation of this enzyme in nonstressed rats. These findings may prove important in elucidating mechanisms by which chronic stress triggers and sustains the biochemical alterations associated with some stress-related psychiatric disorders.
Retroviral-like element in a marine invertebrate.
Authors: Springer M S , Davidson E H , Britten R J
Published: May 2006

Retroviral-like elements (RL elements) include retroviruses and long terminal repeat (LTR)-containing retrotransposons. We report the presence of sea urchin RL elements (termed SURL) in eight species of sea urchins and find that these RL elements belong to several subfamilies. The complete DNA sequence of one SURL element in Tripneustes gratilla is 5266 base pairs long, including 254-nucleotide-long identical long terminal repeats (LTRs). It contains a single open reading frame nearly 4 kilobases long including the gag and pol genes. Comparison of conserved DNA sequences of RL elements from different sea urchin species indicates that active elements have been inserting copies into echinoid genomes for at least 200 million years.
Identification and partial characterization of six members of the kinesin superfamily in Drosophila.
Authors: Stewart R J , Pesavento P A , Woerpel D N , Goldstein L S
Published: May 2006

Recent evidence has suggested that the principal polypeptide component of the microtubule motor protein kinesin may be a member of an extended superfamily of related motor proteins. To gain insight into how large the kinesin superfamily might be and to begin determining the potential functions in which various superfamily members might participate, we identified and partially characterized six additional members of the Drosophila kinesin superfamily. Genes encoding these proteins were identified by using the polymerase chain reaction with degenerate primers corresponding to highly conserved regions of the kinesin heavy-chain motor domain. Partial sequencing of the six genes revealed that they encode proteins that are 40-60% identical to the motor domain of the kinesin heavy-chain sequence. The cytogenetic locations as well as the developmental and tissue-specific expression patterns have been determined. The data suggest that each of these six kinesin-like proteins may have functions in a wide variety of cell types and tissues.
Immunoregulatory activity of the T-cell receptor alpha chain demonstrated by retroviral gene transfer.
Authors: Green D R , Bissonnette R , Zheng H G , Onda T , Echeverri F , Mogil R J , Steele J K , Voralia M , Fotedar A
Published: May 2006

We have previously described an antigen-specific I-Ad-restricted T-cell hybridoma, A1.1, that constitutively releases an antigen-specific immunoregulatory activity into supernatants. Using retrovirally mediated gene transfer, we have found that transfer of the T-cell receptor alpha chain (TCR alpha) gene from A1.1 to a number of other T-cell hybridomas effectively transferred the ability to produce the activity. Gene transfer of the TCR beta chain (TCR beta), however, did not transfer this ability. The regulatory activity from cells expressing the A1.1 TCR alpha bound to and was eluted from an anti-TCR alpha monoclonal antibody and displayed fine antigenic specificity identical to that of supernatants from A1.1. The possibility that this activity represents a secreted form of the TCR alpha (as opposed to shed cell-surface TCR) was examined in BW1100 cells, lacking TCR alpha and TCR beta, which produced the antigen-specific activity after gene transfer of the A1.1 TCR alpha gene. The expression of the immunoregulatory activity in supernatants correlated with a direct antigen-binding activity as detected by ELISA, thus raising the possibility that antigen binding is relevant to the mechanism of action of the soluble TCR alpha. We discuss these observations and our earlier studies suggesting an immunoregulatory role for soluble TCR alpha.
Dual activation of a sex pheromone-dependent ion channel from insect olfactory dendrites by protein kinase C activators and cyclic GMP.
Authors: Zufall F , Hatt H
Published: May 2006

Olfactory transduction is thought to take place in the outer dendritic membrane of insect olfactory receptor neurons. Here we show that the outer dendritic plasma membrane of silkmoth olfactory receptor neurons seems to be exclusively equipped with a specific ion channel activated by low concentrations of the species-specific sex pheromone component. This so-called AC1 channel has a conductance of 56 pS and is nonselectively permeable to cations. The AC1 channel can be activated from the intracellular side by protein kinase C activators such as diacylglycerol and phorbolester and by cGMP but not by Ca2+, inositol 1,4,5-triphosphate, or cAMP. Our results imply that phosphorylation of this ion channel by protein kinase C could be the crucial step in channel opening by sex pheromones.
Protein-DNA interactions in vivo of an erythroid-specific, human beta-globin locus enhancer.
Authors: Reddy P M , Shen C K
Published: May 2006

The 5' DNase I-hypersensitive site 2 (5' HS-2) is an erythroid-specific enhancer located 11 kilobases (kb) upstream of the human beta-globin gene cluster. Presence in cis of 5' HS-2 confers a high level of erythroid cell-specific and developmentally regulated promoter activities of human globin genes in transfected cell cultures and in transgenic mice. Combining the use of the methylation protection assay and polymerase chain reaction, we have studied nuclear factor-DNA interactions of the 5' HS-2 enhancer in vitro and in vivo. The data from analyses of three different sequence motifs within 5' HS-2 represent three different modes of protein-DNA interaction with respect to cell-type specificities and in vivo vs. in vitro differences. First, a GATA-1 motif was found to bind nuclear factor(s), presumably the GATA-1 factor, present in K-562 cell extracts and in living K-562 cells. No such binding was seen in nonerythroid HeLa cells or extract. A second motif, NF-E2/AP1 (nuclear factor-erythroid 2/activator protein 1), consists of tandemly arranged dimers of AP1 binding consensus. The presence of either HeLa extract or K-562 extract protects the NF-E2/AP1 motif from methylation, but the footprints are different. This is most likely due to different protein-DNA contacts of the AP1-DNA complex formed in HeLa extract and the NF-E2-DNA complex in K-562 extract. In vivo methylation protection patterns of this motif parallel those observed in vitro, suggesting that it is also bound by NF-E2 in K-562 cells and by AP1 in HeLa cells. Finally, a GT-I motif binds apparently to one or more similar factors in both types of nuclear extracts, but the in vivo methylation protection patterns are not identical between living HeLa and K-562 cells. These data provide direct evidence that specific nuclear factor-DNA complexes form in vivo at functionally important sequence motifs of the 5' HS-2 enhancer in erythroid cells. The detection of conformationally different nuclear factor-DNA complexes at the same sequence motifs in HeLa and Raji cell lines also raises interesting questions regarding the origin and function of these complexes in nonerythroid cells.
Cholesterol-induced fluid-phase immiscibility in membranes.
Authors: Sankaram M B , Thompson T E
Published: May 2006

The fluid-phase behavior of binary mixtures of cholesterol with phosphatidylcholines is investigated using magnetic resonance methods. Phospholipid biradicals provide the electron spin resonance spectroscopic resolution of two immiscible fluid phases in the dipalmitoylphosphatidylcholine-cholesterol system. Isotropic chemical shifts of the phospholipid carbonyl carbons in binary mixtures with cholesterol measured using solid-state high-resolution nuclear magnetic resonance methods furnish evidence for a putative hydrogen bond between the 3 beta-hydroxyl of cholesterol and the sn-2 carbonyl of the phospholipid. The location in the bilayer of cholesterol in the two fluid phases is determined by measuring spin label-enhanced spin-lattice relaxation rates of the 13C nuclei of both the phospholipid and cholesterol molecules. These results suggest, in a time-averaged sense, that in the cholesterol-poor fluid phase the cholesterol molecule essentially spans the bilayer, whereas in the cholesterol-rich fluid phase the molecule is present in both monolayers of the bilayer.
Mouse Erk-1 gene product is a serine/threonine protein kinase that has the potential to phosphorylate tyrosine.
Authors: Crews C M , Alessandrini A A , Erikson R L
Published: May 2006

Bacterial expression of mouse gene Erk-1 yielded an active kinase with the same substrate specificity shown for ERK1 protein purified from rat cells. Although rat gene ERK1 is believed to encode a serine/threonine kinase based on sequence data and known ERK1 substrate phosphorylation sites, bacterially-produced mouse Erk-1 (bt-Erk-1) autophosphorylated on tyrosine in addition to serine and threonine residues. The bt-Erk-1 protein also had the capacity to reactivate the ribosomal protein S6 kinase (S6KII). Furthermore, treatment of bt-Erk-1 with either serine/threonine-specific phosphatase 2A or tyrosine-specific phosphatase 1B significantly decreased its kinase activity. These findings predict that autophosphorylation may play an important role in Erk-1/ERK1 regulation.
Infrared spectroscopic demonstration of a conformational change in bacteriorhodopsin involved in proton pumping.
Authors: Ormos P
Published: May 2006

Infrared spectral changes in bacteriorhodopsin (bR) were followed during the slow decay of the M intermediate in the temperature region 240-260 K. The decay of the M form is characterized by the disappearance of the ethylenic bands and the bands indicating the reprotonation of the Schiff base. The route of Schiff-base reprotonation completely changes between 240 K and 260 K. At 240 K reprotonation occurs from Asp-85, the group to which the proton was released during M formation, and there is no pumping. At 260 K Schiff-base reprotonation takes place through Asp-96 from the cytoplasmic side, in the normal sequence assumed for proton pumping. The dramatic change in the route of Schiff-base reprotonation is coupled to a protein conformational change characterized by the change of the ratio of the two amide I bands at 1658 cm-1 and 1669 cm-1. This conformational change is interpreted as the conformational switch crucial for proton pumping: a protein relaxation following M formation results in a local rearrangement of the group, in the vicinity of the Schiff base. The rearrangement changes the accessibility of the Schiff base and provides that its deprotonation and reprotonation occur on different sides. The conformational change has characteristics typical for relaxations in proteins. In addition, it is shown that at 260 K an equilibrium exists between the M and N forms.
Size of the amino acid side chain at position 158 of cytochrome b is critical for an active cytochrome bc1 complex and for photosynthetic growth of Rhodobacter capsulatus.
Authors: Atta-Asafo-Adjei E , Daldal F
Published: May 2006

The nonphotosynthetic mutant R126 of Rhodobacter capsulatus has a cytochrome (cyt) bc1 complex (EC 1.10.2.2) with a defective quinol oxidation Qz(o,p) site but a functional quinone reduction Qc(i,n) site. Genetic analyses of this mutant have indicated that a single-base-pair change has replaced Gly-158 of cyt b with Asp. In this work, Gly-158 was changed by oligonucleotide-mediated mutagenesis to several other amino acids to define its role on quinol oxidation catalyzed by the cyt bc1 complex. The effects of the mutations were analyzed by measuring the photosynthetic growth rate of mutants and the activity of their cyt bc1 complexes. The mutants overproduced the cyt bc1 complex, assembled its subunits, and incorporated its prosthetic groups as shown by immunoblotting and optical difference spectroscopy. Of 14 amino acid residues tested at position 158 of cyt b all but alanine and serine resulted in a marked decrease of cyt bc1 activity and failed to support photosynthetic growth of R. capsulatus. The photosynthesis-competent mutants, Gly-158----Ala and Gly-158----Ser, had lower cyt bc1 complex activities that were resistant to myxothiazol, but not to stigmatellin, quinol oxidation inhibitors. These findings indicated that the specific role of Gly-158 of cyt b on quinol oxidation and myxothiazol binding may be related to the small size of its side chain and are discussed in terms of the structure and function of the quinol oxidation site of the cyt bc1 complex.
Direct binding of secreted T-cell receptor beta chain to superantigen associated with class II major histocompatibility complex protein.
Authors: Gascoigne N R , Ames K T
Published: May 2006

The interaction of the T-cell receptor (TCR) with peptide antigen plus major histocompatibility complex (MHC) protein requires both alpha and beta chains of the TCR. The "superantigens" are a group of molecules that are recognized in association with MHC class II but that do not appear to conform to this pattern. Superantigens are defined as such because they cause the activation or thymic deletion of many or all T cells bearing specific TCR beta-chain variable region (V beta) elements. The strong association of particular V beta S with T-cell responses to superantigens suggests that their interaction with the TCR is fundamentally different from that of most antigens. We have directly investigated the involvement of the beta chain in recognition of a superantigen by using a secreted, truncated TCR beta chain and the bacterial superantigen staphylococcal enterotoxin A complexed to cell-surface MHC class II. We demonstrate that this interaction is specific for the enterotoxin and is dependent on MHC class II expression by the cell. The reaction can be inhibited by antibodies against the three components of the reaction: V beta, enterotoxin, and class II. This shows that the TCR beta chain is sufficient to mediate the interaction with a superantigen-class II complex. The TCR alpha chain and co-receptors such as CD4 are not required.
The Bradyrhizobium japonicum nolA gene and its involvement in the genotype-specific nodulation of soybeans.
Authors: Sadowsky M J , Cregan P B , Gottfert M , Sharma A , Gerhold D , Rodriguez-Quinones F , Keyser H H , Hennecke H , Stacey G
Published: May 2006

Several soybean genotypes have been identified which specifically exclude nodulation by members of Bradyrhizobium japonicum serocluster 123. We have identified and sequenced a DNA region from B. japonicum strain USDA 110 which is involved in genotype-specific nodulation of soybeans. This 2.3-kilobase region, cloned in pMJS12, allows B. japonicum serocluster 123 isolates to form nodules on plants of serogroup 123-restricting genotypes. The nodules, however, were ineffective for symbiotic nitrogen fixation. The nodulation-complementing region is located approximately 590 base pairs transcriptionally downstream from nodD2. The 5' end of pMJS12 contains a putative open reading frame (ORF) of 710 base pairs, termed nolA. Transposon Tn3-HoHo mutations only within the ORF abolished nodulation complementation. The N terminus of the predicted nolA gene product has strong similarity with the N terminus of MerR, the regulator of mercury resistance genes. Translational lacZ fusion experiments indicated that nolA was moderately induced by soybean seed extract and the isoflavone genistein. Restriction fragments that hybridize to pMJS12 were detected in genomic DNAs from both nodulation-restricted and -unrestricted strains.
T-cell and basophil activation through the cytoplasmic tail of T-cell-receptor zeta family proteins.
Authors: Letourneur F , Klausner R D
Published: May 2006

The zeta chain of the T-cell antigen receptor is the prototype of a family of proteins that exist as disulfide-linked dimers and are subunits of the T-cell antigen receptor and both IgE and IgG binding Fc receptors. Two related genes encode the zeta and gamma proteins. In this study we examine the ability of chimeric proteins consisting of the extracellular domain of the alpha chain of the interleukin 2 receptor (Tac) and the cytoplasmic domain of either zeta or gamma to activate cells when expressed in either T cells or rat basophilic leukemia cells. The zeta and gamma chimera were effective at eliciting interleukin 2 production in T cells and serotonin release in rat basophilic leukemia cells when externally cross-linked. Cytoplasmic-tail deletion mutants of zeta and gamma were constructed and used to verify the specificity of cell activation by these chimeric proteins. Signaling potencies of complementary mutants having the zeta tail truncated in position 108 or deleted from positions 66 through 114 suggested the presence of several functional domains in zeta.
Functional map of the alpha subunit of Escherichia coli RNA polymerase: two modes of transcription activation by positive factors.
Authors: Igarashi K , Hanamura A , Makino K , Aiba H , Aiba H , Mizuno T , Nakata A , Ishihama A
Published: May 2006

The role of the alpha subunit of Escherichia coli RNA polymerase in transcription activation by positive factors was investigated using two reconstituted mutant RNA polymerases (containing C-terminally truncated alpha subunits) and three positive factors [the cAMP receptor protein (CRP), OmpR, and PhoB]. The mutant RNA polymerases did not respond to transcription activation by activator proteins that bind upstream of the respective promoters. Transcription by these mutant enzymes was, however, activated in the cases where activators bind to target sites that overlap the promoter -35 region. Two different mechanisms are proposed for the positive control of transcription by activator proteins, one requiring the C-terminal domain of the alpha subunit, and the other not requiring it.
Selective induction of gene expression and second-messenger accumulation in Dictyostelium discoideum by the partial chemotactic antagonist 8-p-chlorophenylthioadenosine 3',5'-cyclic monophosphate.
Authors: Peters D J , Bominaar A A , Snaar-Jagalska B E , Brandt R , Van Haastert P J , Ceccarelli A , Williams J G , Schaap P
Published: May 2006

During development of the cellular slime mold Dictyostelium discoideum, cAMP induces chemotaxis and expression of different classes of genes by means of interaction with surface cAMP receptors. We describe a cAMP derivative, 8-p-chlorophenylthioadenosine 3',5'-cyclic monophosphate (8-CPT-cAMP), which inhibits cAMP-induced chemotaxis at low concentrations but induces chemotaxis at supersaturating concentrations. This compound, moreover, selectively activates expression of aggregative genes but not of postaggregative genes. 8-CPT-cAMP induces normal cGMP and cAMP accumulation but in contrast to cAMP, which increases inositol 1,4,5-trisphosphate levels, 8-CPT-cAMP decreases inositol 1,4,5-trisphosphate levels. The derivative induces reduced activation of guanine nucleotide regulatory proteins, which may cause its defective activation of inositol 1,4,5-trisphosphate production. Our data suggest that disruption of inositolphospholipid signaling impairs chemotaxis and expression of a subclass of cAMP-regulated genes.
Inhibition by interferon of herpes simplex virus type 1-activated transcription of tat-defective provirus.
Authors: Popik W , Pitha P M
Published: May 2006

The herpes simplex virus type 1 (HSV-1)-mediated transactivation of human immunodeficiency virus type 1 (HIV-1) provirus was studied in cell lines containing either integrated tat-defective HIV-1 provirus (HNHIVdt4 cells) or the tat-defective HIV-1 provirus, and a plasmid in which the expression of human alpha 2 interferon (HuIFN-alpha 2) was under the control of the HIV-1 long terminal repeat (LTR) (HNHIV alpha 1 cells). In both cell lines, transcription of the HIV-1 provirus was below the limits of detection, but it could be induced effectively by transfection with a HIV-1 tat-expression plasmid. In HNHIV alpha 1 cells, HuIFN-alpha 2 was induced concomitantly with HIV-1 provirus, although these cells synthesized only low levels of IFN constitutively. In contrast, infections with HSV-1 activated transcription of HIV-1 provirus only in HNHIVdt4 cells but not in HNHIV alpha 1 cells. Similarly in a transient expression assay, HSV-1 up-regulated expression of a HIV LTR-CAT (chloramphenicol acetyltransferase gene) plasmid in HNHIVdt4 but not in HNHIV alpha 1 cells. No major differences could be detected in the expression of HSV-1 immediate-early (IE) genes IE175 and IE110 (which are essential for the activation of HIV-1 LTR) in HNHIVdt4 and HNHIV alpha 1 cells to account for the inability of HSV-1 to induce HIV-1 in HNHIV alpha 1 cells. However, major differences were observed in the binding pattern of NF-kappa B-specific nuclear proteins to the enhancer region of the HIV-1 LTR: whereas binding of the 45-kDa NF-kappa B-specific nuclear protein was detected in nuclear extracts from HNHIVdt4 cells, no protein binding was seen in extracts from HNHIV alpha 1 cells. These results suggest an alternate mechanism by which IFN may alter the expression of cellular and viral genes.
The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest.
Authors: Chien C T , Bartel P L , Sternglanz R , Fields S
Published: May 2006

We describe a method that detects proteins capable of interacting with a known protein and that results in the immediate availability of the cloned genes for these interacting proteins. Plasmids are constructed to encode two hybrid proteins. One hybrid consists of the DNA-binding domain of the yeast transcriptional activator protein GAL4 fused to the known protein; the other hybrid consists of the GAL4 activation domain fused to protein sequences encoded by a library of yeast genomic DNA fragments. Interaction between the known protein and a protein encoded by one of the library plasmids leads to transcriptional activation of a reporter gene containing a binding site for GAL4. We used this method with the yeast SIR4 protein, which is involved in the transcriptional repression of yeast mating type information. (i) We used the two-hybrid system to demonstrate that SIR4 can form homodimers. (ii) A small domain consisting of the C terminus of SIR4 was shown to be sufficient to mediate this interaction. (iii) We screened a library to detect hybrid proteins that could interact with the SIR4 C-terminal domain and identified SIR4 from this library. This approach could be readily extended to mammalian proteins by the construction of appropriate cDNA libraries in the activation domain plasmid.
DNA.RNA helicase activity of RAD3 protein of Saccharomyces cerevisiae.
Authors: Bailly V , Sung P , Prakash L , Prakash S
Published: May 2006

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of UV-damaged DNA and is essential for cell viability. The RAD3 protein exhibits a remarkable degree of sequence homology to the human excision repair protein ERCC2. The RAD3 protein is a single-stranded DNA-dependent ATPase and a DNA helicase capable of denaturing long regions of duplex DNA. Here, we demonstrate that RAD3 also possesses a potent DNA.RNA helicase activity similar in efficiency to its DNA helicase activity. The rad3 Arg-48 mutant protein, which binds but does not hydrolyze ATP, lacks the DNA.RNA unwinding activity, indicating a dependence on ATP hydrolysis. RAD3 does not show any RNA-dependent NTPase activity and, as expected, does not unwind duplex RNA. This observation suggests that RAD3 translocates on DNA in unwinding DNA.RNA duplexes. That the rad3 Arg-48 mutation inactivates the DNA and DNA.RNA helicase activities and confers a substantial reduction in the incision of UV-damaged DNA suggests a role for these activities in incision. We discuss how RAD3 helicase activities could function in tracking of DNA in search of damage sites and effect enhanced excision repair of actively transcribed genes.
Extensive genetic polymorphism in the human tumor necrosis factor region and relation to extended HLA haplotypes.
Authors: Jongeneel C V , Briant L , Udalova I A , Sevin A , Nedospasov S A , Cambon-Thomsen A
Published: May 2006

We have identified three polymorphic microsatellites (which we call TNFa, TNFb, and TNFc) within a 12-kilobase region of the human major histocompatibility complex (MHC) that includes the tumor necrosis factor (TNF) locus. TNFc is located within the first intron of the TNF-beta gene and has only 2 alleles. TNFa and TNFb are 3.5 kilobases upstream (telomeric) of the TNF-beta gene and have at least 13 and 7 alleles, respectively. TNFa, -b, and -c alleles are in linkage disequilibrium with alleles at other loci within the MHC, including class I, class II, and class III. TNFa, -b, and -c alleles are also associated with extended HLA haplotypes. These TNF polymorphisms will allow a thorough genetic analysis of the involvement of TNF in MHC-linked pathologies.
Acetyl-CoA carboxylase from Escherichia coli: gene organization and nucleotide sequence of the biotin carboxylase subunit.
Authors: Kondo H , Shiratsuchi K , Yoshimoto T , Masuda T , Kitazono A , Tsuru D , Anai M , Sekiguchi M , Tanabe T
Published: May 2006

Biotin carboxylase [biotin-carboxyl-carrier-protein:carbon-dioxide ligase (ADP-forming), EC 6.3.4.14] is the enzyme mediating the first step of the acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] reaction. We screened an Escherichia coli DNA library and a DNA fragment carrying the biotin carboxylase gene fabG, and its flanking regions were cloned. The gene for biotin carboxyl carrier protein was found 13 base pairs upstream of the fabG gene. Nucleotide sequencing of the recombinant plasmids revealed that the fabG codes for a 449-amino acid residue protein with a calculated molecular weight of 49,320, a value in good agreement with that of 51,000 determined by SDS/polyacrylamide gel electrophoresis of the purified enzyme. The deduced amino acid sequence of biotin carboxylase is also consistent with the partial amino acid sequence determined by Edman degradation. The primary structure of this enzyme exhibits a high homology with those of other biotin-dependent enzymes and carbamoyl-phosphate synthetase [carbon-dioxide:L-glutamine amino-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5]; therefore, all these enzymes probably function through the same mechanism of reaction.
Inverse changes in erythroid cell volume and number regulate the hematocrit in newborn genetically hypertensive rats.
Authors: Boylan J W , Van Liew J B , Feig P U
Published: May 2006

Erythrocytosis and microcytosis have been described in strains of genetically hypertensive rats and in essentially hypertensive humans. Published discussion of these phenomena has centered around their relationship to observed alterations in ionic transport and the pathogenesis of hypertension. In presenting data for another strain of spontaneously hypertensive rats in which these findings are exhibited, we note that erythroid cell size decreases concurrently with the increase in cell numbers so that the hematocrit and the mean corpuscular hemoglobin concentration remain constant. Data from the literature support the hypothesis that erythroid cell size is inversely proportional to cell count in a large number of species. Erythrocytosis, as it develops in the neonatal rat, is a consequence of the marked immaturity of this species at birth. Erythrocytosis in the spontaneously hypertensive rat is not due to a difference in the affinity of its hemoglobin for oxygen or to significant tissue anorexia. Microcytosis in the spontaneously hypertensive rat is the consequence of a continuation of the linear volume decrease with age of its erythroid cells seen in the normotensive animals and may be accounted for by the production of smaller cells with concomitant regulation of individual cell volume.
Direct assignment of vitamin K1 as the secondary acceptor A1 in photosystem I.
Authors: Snyder S W , Rustandi R R , Biggins J , Norris J R , Thurnauer M C
Published: May 2006

The characteristic electron spin polarized electron paramagnetic resonance (ESP EPR) signal observed in photosystem I (PSI) has been previously assigned to a radical pair composed of the oxidized primary donor and a reduced vitamin K1. Under conditions in which Bottin, H. & Setif, P. [(1991), Biochim. Biophys. Acta 105, 331-336] proposed that A1 is doubly reduced, the ESP EPR signal was not observed. Therefore, the ESP EPR signal can be directly attributed to A-1, and vitamin K1 can be assigned as this PSI acceptor. The ESP EPR signal was partially restored by removal of the chemical reductants.
Interplay between glutamate and gamma-aminobutyric acid transmitter systems in the physiological regulation of brain-derived neurotrophic factor and nerve growth factor synthesis in hippocampal neurons.
Authors: Zafra F , Castrén E , Thoenen H , Lindholm D
Published: May 2006

In the central nervous system brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are predominantly located in neurons. Here we demonstrate that the balance between the activity of the glutamatergic and gamma-aminobutyric acid (GABA)ergic systems controls the physiological levels of BDNF and NGF mRNAs in hippocampal neurons in vitro and in vivo. The blockade of the glutamate receptors and/or stimulation of the GABAergic system reduces BDNF and NGF mRNAs in hippocampus and NGF protein in hippocampus and septum. The reduction of NGF in the septum reflects the diminished availability of NGF in the projection field of NGF-dependent septal cholinergic neurons. These neurons do not synthesize NGF themselves but accumulate it by retrograde axonal transport. The refined and rapid regulation of BDNF and NGF synthesis by the glutamate and GABA transmitter systems suggests that BDNF and NGF might be involved in activity-dependent synaptic plasticity.
Effect of CP-96,345, a nonpeptide substance P receptor antagonist, on salivation in rats.
Authors: Snider R M , Longo K P , Drozda S E , Lowe J A , Leeman S E
Published: May 2006

CP-96,345 [(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl) methyl]-1-azabicyclo[2.2.2]octan-3-amine) antagonism of substance P-stimulated salivation was investigated in pentobarbital-anesthetized rats. Administered either intraperitoneally or orally, CP-96,345 produced dose-dependent inhibition of the sialogogic response elicited by substance P, with a median effective dose of 12-24 mumol/kg (5-10 mg/kg) of body weight, but had no effect on acetylcholine-stimulated salivation. CP-96,345 produced concentration-dependent inhibition of [3H]substance P binding to rat submaxillary gland membranes, with a median effective concentration of 34 +/- 3.6 nM. These biological activities were confined to CP-96,345 in that the 2R,3R enantiomer (CP-96,344) was without effect.
cAMP-dependent regulation of proenkephalin by JunD and JunB: positive and negative effects of AP-1 proteins.
Authors: Kobierski L A , Chu H M , Tan Y , Comb M J
Published: May 2006

We demonstrate that JunD, a component of the AP-1 transcription factor complex, activates transcription of the human proenkephalin gene in a fashion that is completely dependent upon the cAMP-dependent protein kinase, protein kinase A. Activation of proenkephalin transcription by JunD is dependent upon a previously characterized cAMP-, phorbol ester-, and Ca(2+)-inducible enhancer, and JunD is shown to bind the enhancer as a homodimer. Another component of the AP-1 transcription complex, JunB, is shown to inhibit activation mediated by JunD. As a homodimer JunB is unable to bind the enhancer; however in the presence of c-Fos, high-affinity binding is observed. Furthermore, JunD is shown to activate transcription of genes linked to both cAMP and phorbol ester response elements in a protein kinase A-dependent fashion, further blurring the distinction between these response elements. These results demonstrate that the transcriptional activity of an AP-1-related protein is regulated by the cAMP-dependent second-messenger pathway and suggest that JunD and other AP-1-related proteins may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways.
Expression of acidic and basic fibroblast growth factors in the substantia nigra of rat, monkey, and human.
Authors: Bean A J , Elde R , Cao Y H , Oellig C , Tamminga C , Goldstein M , Pettersson R F , Hökfelt T
Published: May 2006

The distribution of acidic (aFGF) and basic (bFGF) fibroblast growth factor mRNA and protein were examined in mesencephalon by immunohistochemistry, immunoblot analysis, in situ hybridization histochemistry, and RNA analysis. Coexistence of aFGF or bFGF with tyrosine hydroxylase protein in nigral cells was observed with immunohistochemistry. Both aFGF and bFGF mRNAs were found in the substantia nigra. Unilateral 6-hydroxydopamine lesions of nigrostriatal neurons resulted in a loss of aFGF and tyrosine hydroxylase [L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] mRNA-positive neurons on the lesioned side. The distribution of aFGF mRNA in monkey brain was similar to that seen in the rat. RNA and immunoblot analysis confirmed the presence of both aFGF and bFGF mRNAs and proteins in the substantia nigra of rat, monkey, and human.
High correlation between pentosidine protein crosslinks and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis.
Authors: Nagaraj R H , Sell D R , Prabhakaram M , Ortwerth B J , Monnier V M
Published: May 2006

Pentosidine is a recently discovered protein crosslink, involving lysine and arginine residues linked together in an imidazo [4,5,6] pyridinium ring formed by a 5-carbon sugar during nonenzymatic browning (Maillard reaction). The presence of high ascorbate levels in the human lens and its ability to undergo nonenzymatic browning led us to investigate pentosidine formation in the aging human lens. Incubation of lens crystallins with ascorbate and its oxidation products dehydroascorbate and 2,3-diketogulonate leads progressively to the formation of pentosidine crosslinks in the presence of oxygen. Under nitrogen, however, pentosidine forms only from 2,3-diketogulonate or xylosone, a degradation product of 2,3-diketogulonate. A high correlation between pentosidine crosslinks and the degree of lens pigmentation is noted in cataractous lenses. Pentosidine is found to be primarily associated with alpha-crystallin fractions of 300-5000 kDa. These results suggest that redox imbalance in cellular senescent systems such as the ocular lens may lead to irreversible ascorbate oxidation and protein crosslinking by xylosone. This mechanism may play an important role in the pathogenesis of "brunescent" cataracts.
Four decades of inversion polymorphism in Drosophila pseudoobscura.
Authors: Anderson W W , Arnold J , Baldwin D G , Beckenbach A T , Brown C J , Bryant S H , Coyne J A , Harshman L G , Heed W B , Jeffery D E
Published: May 2006

We report data that continue the studies of Dobzhansky and others on the frequencies of third-chromosome inversions in natural populations of Drosophila pseudoobscura in North America. The common gene arrangements continue to be present in frequencies similar to those described four decades ago, and the broad geographic patterns also remain unchanged. There is only one pronounced trend over time: the increase in frequency of the Tree Line inversion in Pacific coast populations.
Poliovirus thiol proteinase 3C can utilize a serine nucleophile within the putative catalytic triad.
Authors: Lawson M A , Semler B L
Published: May 2006

The picornavirus 3C proteinases are substrate-specific thiol proteases that have been shown by secondary structure predictions and protein modeling studies to be similar to the trypsin-like serine proteases. We have examined several mutations of the 3C proteinase at putative active site and non-active site residues. The effect on 3C-mediated protein processing supports the model of serine protease similarity. In particular, we have shown that 3C can utilize a serine at position 147, which is predicted to supply the nucleophilic residue of the catalytic triad. We suggest that picornavirus 3C proteinases may represent a class of enzymes that have maintained the catalytic mechanism characteristic of a proposed enzyme ancestral to the highly divergent class of serine proteases.
Peritoneal B cells regulate the numbers of allotype-matched pre-B and B cells in bone marrow.
Authors: Marcos M A , Sundblad A , Malenchère E , Coutinho A
Published: May 2006

The mechanisms regulating growth and differentiation of B-cell precursors in adult bone marrow (BM) and/or selecting immunocompetent cells for peripheral export are poorly understood. We report here that small numbers of activated peritoneal B cells selectively suppress the numbers of small pre-B (B220+IgM-) and B (B220+IgM+) cells in BM, if transferred into syngeneic adult mice. No significant alterations are detected in other BM cell lineages or in peripheral lymphocytes of recipient mice. Both CD5+ and CD5- peritoneal B cells display this activity, but the same or higher numbers of similarly activated splenic B cells have no effect. Suppression of B-lineage cells is independent of T lymphocytes but requires that both donor and recipient are matched for immunoglobulin allotypes. These findings provide evidence for regulation of BM B-cell production by peripheral B cells, especially when located in the peritoneal cavity, and ascribe regulatory roles to the peritoneal B-cell compartment. They also could contribute to understanding the control of total B-lymphocyte numbers in the organism.
p55CDC25 is a nuclear protein required for the initiation of mitosis in human cells.
Authors: Millar J B , Blevitt J , Gerace L , Sadhu K , Featherstone C , Russell P
Published: May 2006

The cdc25+ gene of fission yeast encodes a phosphotyrosine phosphatase that dephosphorylates tyrosine-15 of p34cdc2 and thereby activates p34cdc2/cyclin to bring about entry into M phase. We have recently cloned a human homolog, CDC25, which rescues the M-phase initiation defect of yeast cdc25 temperature-sensitive mutants. Antibodies raised against the CDC25 gene product specifically recognize human proteins of approximately 55 and approximately 52 kDa. Microinjection of affinity-purified anti-CDC25 antibodies into HeLa cells inhibits entry into mitosis. These observations suggest that the CDC25 gene products are essential for the initiation of mitosis in human cells, similar to their homologs in fission yeast and Drosophila. CDC25 gene products, like p34CDC2, are localized primarily in the nucleus during interphase, suggesting that activation of p34CDC2/cyclin by p52/p55CDC25 occurs within the nucleus.
Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.
Authors: Scanlon K J , Jiao L , Funato T , Wang W , Tone T , Rossi J J , Kashani-Sabet M
Published: May 2006

The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression.