bluuebunny/crossref_metadata_2025 huggingface dataset:
Bovine brain cytosol contains three immunologically distinct forms of inositolphospholipid-specific phospholipase C.
Authors: Ryu S H , Suh P G , Cho K S , Lee K Y , Rhee S G
Published: May 2006

We previously reported that cytosolic fractions of bovine brain contain two immunologically distinct forms of phospholipase C (PLC), PLC-I and PLC-II. We now report the purification of another form of inositolphospholipid-specific phospholipase C from bovine brain cytosol, designated PLC-III, and the comparison of the catalytic properties of the three isozymes. Approximately 450 micrograms of pure PLC-III was obtained from 36 bovine brains, and it had a final specific activity of 30-40 mumol of phosphatidylinositol hydrolyzed per min per mg of enzyme in the presence of 0.1% deoxycholate. PLC-III exhibited an apparent Mr of 85,000 in NaDodSO4/PAGE, which is considerably smaller than the Mr of 150,000 for PLC-I and 145,000 for PLC-II. Neither of the two mixtures of monoclonal antibodies nor the rabbit polyclonal antibodies directed against either PLC-I or PLC-II cross-reacted with PLC-III. The catalytic properties of the three isozymes were studied by using small unilamellar vesicles prepared from either phosphatidylinositol (PtdIns) or phosphatidylinositol 4,5-bisphosphate (PtdInsP2) as substrates. Hydrolysis of both PtdIns and PtdInsP2 by the three enzymes was dependent on Ca2+. However, at low Ca2+ concentration, PtdInsP2 was the preferred substrate for all three enzymes. When PtdIns was the substrate, the three enzymes exhibited similar specific activities at their optimum pH, which was 4.8 for PLC-I, 5.0 for PLC-II, and 5.5 for PLC-III. But at neutral pH, the order of specific activity was PLC-III greater than PLC-II greater than PLC-I. In contrast, the order of specific activity was PLC-I greater than PLC-III greater than PLC-II for PtdInsP2 hydrolysis, which means that PLC-I is the most specific for PtdInsP2. The three enzymes were affected differently by bovine serum albumin: inhibition of PLC-I and activation of PLC-III were observed, whereas PLC-II was unaffected. This observation suggests that any putative protein effectors for PLC should be critically scrutinized.
CArG boxes in the human cardiac alpha-actin gene are core binding sites for positive trans-acting regulatory factors.
Authors: Miwa T , Boxer L M , Kedes L
Published: May 2006

Positively acting, rate-limiting regulatory factors that influence tissue-specific expression of the human cardiac alpha-actin gene in a mouse muscle cell line are shown by in vivo competition and gel mobility-shift assays to bind to upstream regions of its promoter but to neither vector DNA nor a beta-globin promoter. Although the two binding regions are distinctly separated, each corresponds to a cis region required for muscle-specific transcriptional stimulation, and each contains a core CC(A + T-rich)6GG sequence (designated CArG box), which is found in the promoter regions of several muscle-associated genes. Each site has an apparently different binding affinity for trans-acting factors, which may explain the different transcriptional stimulation activities of the two cis regions. Therefore, we conclude that the two CArG box regions are responsible for muscle-specific transcriptional activity of the cardiac alpha-actin gene through a mechanism that involves their binding of a positive trans-acting factor in muscle cells.
Construction of a yeast strain devoid of mitochondrial introns and its use to screen nuclear genes involved in mitochondrial splicing.
Authors: Séraphin B , Boulet A , Simon M , Faye G
Published: May 2006

We have constructed a respiring yeast strain devoid of mitochondrial introns to screen nuclear pet- mutants for those that play a direct role in mitochondrial intron excision. Intron-less mitochondria are introduced by cytoduction into pet- strains that have been made rho0; cytoductants therefrom recover respiratory competency if the original pet- mutation is required only for mitochondrial splicing. By this means, we have identified 11 complementation groups of such genes. Their total number may be estimated as about 18.
Membrane association of proline dehydrogenase in Escherichia coli is redox dependent.
Authors: Wood J M
Published: May 2006

The PutA protein, product of the Escherichia coli gene putA, has two functions essential for proline utilization and for the regulation of putP and putA expression: as the peripheral membrane flavoprotein, proline dehydrogenase (EC 1.5.99.8), it transfers electrons from proline to the respiratory chain, and, as a repressor, it controls expression of genes putP and putA in response to proline supply. Association of proline dehydrogenase with the membrane was shown to require the simultaneous presence of the soluble enzyme, membranes, and proline. The kinetics of that association, monitored by following proline oxidation in a coupled enzyme assay system, were not altered when the transmembrane proton gradient generated during proline oxidation was dissipated by a proton ionophore. However, D-lactate or NADH could replace proline as a promoter of proline dehydrogenase-membrane association under anaerobic reaction conditions. These data imply that reduction of proline dehydrogenase and/or a membrane constituent promotes enzyme-membrane association. A biochemical mechanism is suggested whereby the concentration of proline dehydrogenase associated with the respiratory chain would be determined by proline supply.
Antibody responses to Epstein-Barr virus-determined nuclear antigen (EBNA)-1 and EBNA-2 in acute and chronic Epstein-Barr virus infection.
Authors: Henle W , Henle G , Andersson J , Ernberg I , Klein G , Horwitz C A , Marklund G , Rymo L , Wellinder C , Straus S E
Published: May 2006

Five distinct Epstein-Barr virus (EBV)-determined nuclear antigens (EBNA-1 to EBNA-5) were recently identified. Antibody responses to these antigens could conceivably differ, and thus prove of serodiagnostic value, in EBV-associated disease processes. As a first step, murine or human cell lines transfected with appropriate EBV DNA fragments and stably expressing either EBNA-1 or EBNA-2 were used to determine the frequency and time of emergence of antibodies to these two antigens in the course of acute and chronic infectious mononucleosis (IM) and to assess their titers in so-called chronic active EBV infections. Following IM, antibodies to EBNA-2 arose first and, after reaching peak titers, declined again in time to lower persistent or even nondetectable levels. Antibodies to EBNA-1 emerged several weeks or months after anti-EBNA-2 and gradually attained the titers at which they persisted indefinitely. The ratios between the anti-EBNA-1 and anti-EBNA-2 titers therefore were generally well below 1.0 during the first 6-12 months after IM and turned to well above 1.0 during the second year. In clear cases of chronic IM, the inversion of this ratio was delayed or prevented. In the less well-defined chronic EBV infections, low ratios were observed in only some of the patients. Because many of these illnesses were not ushered in by a proven IM and often showed EBV-specific antibody profiles within the normally expected range, a causal role of the virus in these cases remains doubtful.
Reconstitution of the lysosomal proton pump.
Authors: D'Souza M P , Ambudkar S V , August J T , Maloney P C
Published: May 2006

Lysosomal membrane proteins solubilized with octyl beta-D-glucopyranoside were reconstituted into proteoliposomes using acetone/ether-washed phospholipids from Escherichia coli. Assays of the quenching of acridine orange fluorescence showed that addition of both ATP and valinomycin to K+-loaded proteoliposomes led to the formation of a pH gradient that was acidic inside. ATP-driven acidification took place in the absence of permeant anions and was inhibited by the "protonophore", carbonylcyanide p-trifluoromethoxyphenylhydrazone, indicating that only H+ was transported actively. Proton translocation was readily blocked by N-ethylmaleimide (10 microM gave 50% inhibition of fluorescence quenching) but was unaffected by oligomycin (50 nM), orthovanadate (50 microM), or ouabain (0.5 mM); similarly, only N-ethylmaleimide affected ATP hydrolysis by proteoliposomes (88% inhibition). Other work showed that reconstitution of ATP-driven proton translocation required the presence of glycerol during protein solubilization and that optimal recovery depended on the use of both glycerol and phospholipid at this stage. We conclude that acidification of the lysosome is mediated by an ATPase capable of electrogenic H+ translocation without molecular coupling to other ionic species.
Cloning and characterization of a cDNA coding for mouse placental alkaline phosphatase.
Authors: Terao M , Mintz B
Published: May 2006

Mouse alkaline phosphatase [ALP; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] was partially purified from placenta. Data obtained by immunoblotting analysis suggested that the primary structure of this enzyme has a much greater homology to that of human and bovine liver ALPs than to the human placental isozyme. Therefore, a full-length cDNA encoding human liver-type ALP was used as a probe to isolate the mouse placental ALP cDNA. The cloned mouse cDNA is 2459 base pairs long and is composed of an open reading frame encoding a 524-amino acid polypeptide that contains a putative signal peptide of 17 amino acids. Homology at the amino acid level of the mouse placental ALP is 90% to the human liver isozyme but only 55% to the human placental counterpart. RNA blot hybridization results indicate that the mouse placental ALP is encoded by a gene identical to the gene expressed in mouse liver, kidney, and teratocarcinoma stem cells. This gene is therefore evolutionarily highly conserved in mouse and human.
Sarcoplasmic reticulum calcium pump: a model for Ca2+ binding and Ca2+-coupled phosphorylation.
Authors: Tanford C , Reynolds J A , Johnson E A
Published: May 2006

The conventional alternating access model for Ca2+ transport by the sarcoplasmic reticulum Ca2+ pump is modified, partly on the basis of the proposed MacLennan-Green domain structure for the Ca2+-pump protein. The present model divides the uptake state (E1) of the protein into three substates, differing in the condition of the Ca2+-binding domain. The domain is an open cavity in the first substate and can bind only a single Ca2+ ion. A fast "jaw-closing" (or "hinge-bending") step then partially closes the cavity to generate the second substate that has a second Ca2+-binding site. Occupation of this site is followed by another jaw-closing step that closes the binding cavity and occludes the bound ions. The subsequent translocation step (to form E2) remains unchanged from previous models. The modified model predicts a constant transport stoichiometry of two Ca2+ per pump reaction cycle. It suggests a plausible mechanism for coupling between Ca2+ binding and ATP utilization: the model predicts (in agreement with experiment) that Ca2+ binding should be a mandatory requirement for phosphorylation of the pump protein, though ATP binding per se does not require Ca2+. The model is consistent with high cooperativity in equilibrium binding of Ca2+, both in the absence and presence of ATP.
Mapping of four distinct BCR-related loci to chromosome region 22q11: order of BCR loci relative to chronic myelogenous leukemia and acute lymphoblastic leukemia breakpoints.
Authors: Croce C M , Huebner K , Isobe M , Fainstain E , Lifshitz B , Shtivelman E , Canaani E
Published: May 2006

A probe derived from the 3' region of the BCR gene (breakpoint cluster region gene) detects four distinct loci in the human genome. One of the loci corresponds to the complete BCR gene, whereas the others contain a 3' segment of the gene. After HindIII cleavage of human DNA, these four loci are detected as 23-, 19-, 13-, and 9-kilobase-pair fragments, designated BCR4, BCR3, BCR2, and BCR1, respectively, with BCR1 deriving from the original complete BCR gene. All four BCR loci segregate 100% concordantly with human chromosome 22 in a rodent-human somatic cell hybrid panel and are located at chromosome region 22q11.2 by chromosomal in situ hybridization. The BCR2 and BCR4 loci are amplified in leukemia cell line K562 cells, indicating that they fall within the amplification unit that includes immunoglobulin lambda light chain locus (IGL) and ABL locus on the K562 Philadelphia chromosome (Ph1); additionally, in chronic myelogenous leukemia-derived mouse-human hybrids retaining a Ph1 chromosome in the absence of the 9q+ and normal chromosome 22, BCR2 and BCR4 loci are retained, whereas the 3' region of BCR1 and the BCR3 locus are lost, indicating that BCR3 is distal to BCR1 on chromosome 22. Similarly, in mouse-human hybrids retaining a Ph1 chromosome derived from an acute lymphoblastic leukemia-in the absence of the 9q+ and 22, only BCR2 and BCR4 loci are retained, indicating that the breakpoint in this acute lymphoblastic leukemia, as in chronic myelogenous leukemia, is proximal to the BCR1 3' region, but distal to the IGLC locus and the BCR2 and BCR4 3' loci. Thus, the order of loci on chromosome 22 is centromere----BCR2, BCR4, and IGL----BCR1----BCR3----SIS, possibly eliminating BCR2 and BCR4 loci as candidate targets for juxtaposition to the ABL gene in the acute lymphoblastic leukemia Ph1 chromosome.
Dopaminergic neurons from embryonic mouse mesencephalon are enriched in culture through immunoreaction with monoclonal antibody to neural specific protein 4 and flow cytometry.
Authors: di Porzio U , Rougon G , Novotny E A , Barker J L
Published: May 2006

Dopaminergic neurons represent a rare neurotransmitter phenotype within the mammalian central nervous system. The mesencephalic dopaminergic neurons form the ascending dopaminergic pathways in mammals and are involved in motor and limbic functions. Here we report that about 30% of all developing mouse mesencephalic cells, including virtually all of the dopaminergic phenotype, express surface membrane determinant(s) recognized by a monoclonal antibody to neural specific protein 4 (NSP4). We have been able to isolate and culture neurons from the mesencephalon according to their expression of NSP4, using the anti-NSP4 immunoreaction in conjunction with fluorescence-activated cell sorting. Cultures of NSP4+-sorted cells showed a significant enrichment in three morphologically distinct putative dopaminergic phenotypes when compared to unsorted mesencephalic cultures, whereas the cultures of NSP4--sorted cells were virtually devoid of dopaminergic neurons. This flow cytometric enrichment in dopaminergic neurons should provide the necessary cells for multidisciplinary study of dopaminergic phenotype differentiation.
tRNA-like structures tag the 3' ends of genomic RNA molecules for replication: implications for the origin of protein synthesis.
Authors: Weiner A M , Maizels N
Published: May 2006

Single-stranded RNA viruses often have 3'-terminal tRNA-like structures that serve as substrates for the enzymes of tRNA metabolism, including the tRNA synthases and the CCA-adding enzyme. We propose that such 3'-terminal tRNA-like structures are in fact molecular fossils of the original RNA world, where they tagged genomic RNA molecules for replication and also functioned as primitive telomeres to ensure that 3'-terminal nucleotides were not lost during replication. This picture suggests that the CCA-adding activity was originally an RNA enzyme, that modern DNA telomeres with the repetitive structure CmAn are the direct descendants of the CCA terminus of tRNA, and that the precursor of the modern enzyme RNase P evolved to convert genomic into functional RNA molecules by removing this 3'-terminal tRNA-like tag. Because early RNA replicases would have been catalytic RNA molecules that used the 3'-terminal tRNA-like tag as a template for the initiation of RNA synthesis, these tRNA-like structures could have been specifically aminoacylated with an amino acid by an aberrant activity of the replicase. We show that it is mechanistically reasonable to suppose that this aminoacylation occurred by the same sequence of reactions found in protein synthesis today. The advent of such tRNA synthases would thus have provided a pathway for the evolution of modern protein synthesis.
Identification of the M-ring protein of the flagellar motor of Salmonella typhimurium.
Authors: Homma M , Aizawa S , Dean G E , Macnab R M
Published: May 2006

The M ring is a substructure of the flagellar basal body of bacteria, which lies in the cytoplasmic membrane and is therefore close to the site where the energy of the transmembrane proton potential is converted into mechanical work of rotation of the motor. The protein from which this ring is constructed has not been identified. Flagellar hook-basal body complexes from Salmonella typhimurium were used as the immunogen for the preparation of monoclonal antibodies. An antibody obtained was directed against a major basal-body component, a 65-kDa protein that from mutant studies has been assigned as the product of the flaAII.1 gene. By immunoelectron microscopy, the antibody was observed to bind the innermost feature of the basal body: the cytoplasmic-facing surface of the M ring. We conclude that the 65-kDa protein is a component--probably the main component--of this important substructure of the flagellar motor.
Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes.
Authors: Bassuk J A , Tsichlis P N , Sorof S
Published: May 2006

Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene). The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene). We report here that p14 is the liver fatty acid binding protein. The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage lambda gt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein. Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO4 gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel. Their pI values overlapped in 2-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and showed the same response to delipidation. Either polypeptide reacted with and blocked the antiserum raised against the other polypeptide. The two polypeptides bound oleic acid similarly. Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum. The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens.
Deficiency in DNA repair in mouse lymphoma strain L5178Y-S.
Authors: Evans H H , Ricanati M , Horng M F
Published: May 2006

The production and repair of radiation-induced DNA damage were measured by filter elution in strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. The induction of radiation-induced damage, as measured by filter elution at pH 12.1, 9.6, and 7.2, was similar in the resistant strain LY-R and the sensitive strain LY-S. The repair of single-strand breaks and alkali-labile sites, as measured by filter elution at pH 12.1 at various times after irradiation, was somewhat slower in strain LY-S than in strain LY-R, although after a 20-min repair period the extent of repair was equal in the two strains. However, when filter elution was performed at either pH 9.6 or pH 7.2, the repair of x-radiation-induced damage was much less extensive in strain LY-S than in strain LY-R. We have assumed that the extent of filter elution at pH 9.6 is a measure of the occurrence of frank double-strand breaks as well as closely opposing single-strand breaks and pH 9.6-labile sites (and combinations thereof), and that the extent of elution at pH 7.2 is a measure of the occurrence of frank double-strand breaks alone. If these assumptions are correct, the results suggest that the sensitivity of strain LY-S to the cytotoxic effects of ionizing radiation is caused by a deficiency in the ability of this strain to repair frank double-strand breaks and pH 9.6-labile lesions. The repair of pH 9.6-labile lesions was temperature sensitive in strain LY-S, as previously found for cellular recovery processes in this strain. Two independent radiation-resistant variants of strain LY-S, isolated after protracted exposure of LY-S cells to low-dose-rate radiation, showed a deficiency in the repair of pH 9.6-labile lesions similar to that observed in strain LY-S. However, the repair of frank double-strand breaks was more extensive in the radiation-resistant variants than in strain LY-S and was similar in extent to that occurring in strain LY-R after a 60-min postirradiation incubation. The results suggest that there is a difference in the nature of DNA damage measured by filter elution at pH 9.6 vs. pH 7.2 and that a deficiency in the repair of pH 9.6-labile lesions does not contribute to cell lethality in the case of the radiation-resistant variants. The radiation resistance of these variants in comparison to strain LY-S may be due at least in part to recovery of the ability to rejoin frank DNA double-strand breaks.
Structure of the mouse gene encoding CD4 and an unusual transcript in brain.
Authors: Gorman S D , Tourvieille B , Parnes J R
Published: May 2006

The T-cell differentiation antigen CD4 plays an important role in the function of T cells that recognize class II major histocompatibility complex proteins. Mouse CD4 (L3T4) has previously been shown to be evolutionarily related to immunoglobulin variable regions based on the predicted protein sequence from cDNA clones. The gene encoding L3T4 was found to be transcribed not only in a subset of T-lineage cells but also unexpectedly in brain, where a shorter transcript was found. In the present study the gene encoding L3T4 is shown to span 26 kilobases and to contain 10 exons. The structural organization is similar to that of other members of the immunoglobulin gene superfamily except for the striking presence of an intron in the middle of the sequence encoding the amino-terminal immunoglobulin-like homology unit. The structure of the shorter L3T4 transcript in mouse brain has been determined. This mRNA appears to be generated from a transcriptional start site within the coding sequence in exon VI. If translated, this transcript would encode a protein of 217 amino acids that lacks the usual L3T4 signal peptide and the amino-terminal 214 amino acids of the mature protein.
Antibody-enhanced thrombolysis: targeting of tissue plasminogen activator in vivo.
Authors: Runge M S , Bode C , Matsueda G R , Haber E
Published: May 2006

Tissue plasminogen activator (tPA) was modified by the unidirectional crosslinking reagent N-succinimidyl 3-(2-pyridyldithio)propionate and coupled to iminothiolane-modified anti-fibrin antibody 59D8 by the formation of disulfide bonds at neutral pH. Purification by two affinity-chromatography steps yielded tPA-anti-fibrin antibody conjugate (tPA-59D8) possessing both tPA and anti-fibrin antibody activities. In a quantitative rabbit thrombolysis model, the activity of the purified conjugate was compared with that of tPA alone and that of a conjugate between tPA and a digoxin-specific monoclonal antibody. After correction for spontaneous lysis (10.9 +/- 2.5%), tPA-59D8 was shown to be 2.8-9.6 times more potent than tPA alone. Unconjugated tPA and tPA-digoxin were equipotent. At equivalent thrombolytic concentrations, tPA-59D8 degraded less fibrinogen and consumed less alpha 2-antiplasmin than did tPA alone. This suggests that tPA can be efficiently directed to the site of a thrombus by conjugation to an anti-fibrin monoclonal antibody, resulting in both more potent and more selective thrombolysis.
Type II insulin-like growth factor receptor is present in rat serum.
Authors: Kiess W , Greenstein L A , White R M , Lee L , Rechler M M , Nissley S P
Published: May 2006

We previously identified in fetal rat serum a component capable of specifically binding radiolabeled insulin-like growth factor type II (IGF-II) that is considerably larger than both the fetal (40 kDa) and the adult (150 kDa) carrier proteins. We now present immunologic and affinity crosslinking data to show that this binding species is the type II IGF receptor. Rat serum was gel-filtered on a Sephadex G-200 column (0.05 M NH4HCO3, pH 8), and 125I-labeled IGF-II (125I-IGF-II) binding was measured in individual column fractions. 125I-IGF-II binding activity was found in the void volume of the column in addition to the carrier protein regions. Competitive binding studies using 125I-IGF-II and binding activity from the Sephadex G-200 void volume showed the characteristics of the type II receptor: IGF-II was more potent than IGF-I, and insulin did not compete. Moreover, a specific anti-type II IGF receptor antibody (no. 3637) completely blocked 125I-IGF-II binding. 125I-IGF-I did not bind to the void volume pool, demonstrating the absence of the type I IGF receptor in rat serum. Affinity crosslinking of 125I-IGF-II to the Sephadex G-200 void volume material demonstrated a specific band at 210 kDa without reduction and at 240 kDa after reduction of disulfide bonds. The serum type II IGF receptor size was confirmed by immunoblotting the void volume material with antiserum 3637, which revealed a band slightly smaller (approximately 10 kDa) than the type II IGF receptor from rat placental membranes. Immunoquantitation by immunoblotting using pure type II IGF receptor from rat placental membranes as standard showed a developmental pattern. In fetal rat serum (19-days gestation) and in sera from 3-, 10-, and 20-day-old rats, the concentrations of receptor protein were similar (1-5 micrograms/ml). The level of the type II IGF receptor in serum declined dramatically between age 20 and 40 days, but the receptor was still measurable at age 12 mo. We conclude that the type II IGF receptor is present in rat serum and is developmentally regulated.
Distributions of molecular forms of acetylcholinesterase and butyrylcholinesterase in nervous tissue of the cat.
Authors: Koelle G B , Massoulié J , Eugène D , Melone M A , Boulla G
Published: May 2006

We analyzed the activities of acetylcholinesterase and butyrylcholinesterase, and of the metabolic enzymes enolase and lactate dehydrogenase, in the superior cervical ganglion, ciliary ganglion, dorsal root ganglion, stellate ganglion, and caudate nucleus of the cat; we found that these tissues possess very different levels of enzymic activities. The proportions of the alpha alpha, alpha gamma, and gamma gamma enolase isozymes are also quite variable. We particularly studied the molecular forms of acetylcholinesterase and butyrylcholinesterase, in normal tissues and in preganglionically denervated SCG, in comparison with earlier histochemical findings. The results are consistent with the premise that the G1 (globular monomer) forms of both enzymes are located in the cytoplasm, the G4 (globular tetramer) forms are at the plasma membranes, and the A12 (collagen-tailed, asymmetric dodecamer) form of acetylcholinesterase is at synaptic sites.
Identification of a transposon Tn7-dependent DNA-binding activity that recognizes the ends of Tn7.
Authors: McKown R L , Waddell C S , Arciszewska L K , Craig N L
Published: May 2006

The bacterial transposon Tn7 is distinguished by its capacity for high-frequency transposition to a specific site in the Escherichia coli chromosome. tnsB is one of the five Tn7-encoded transposition genes. We have identified in vitro a tnsB-dependent DNA binding activity that interacts specifically with cis-acting transposition sequences at the Tn7 termini. Although the left and right termini of Tn7 are structurally distinct, each end contains several copies of a closely homologous 22-base-pair sequence. We present results indicating that this 22-base-pair repeat sequence is recognized by the tnsB-dependent binding activity.
Efficient introduction of plasmid DNA into Trypanosoma brucei and transcription of a transfected chimeric gene.
Authors: Eid J , Sollner-Webb B
Published: May 2006

Electroporation induces efficient transient transfection of Trypanosoma brucei, and the introduced DNA can be transcribed into RNA. When we delivered a high-voltage electric pulse to cells mixed with radiolabeled pBR322, approximately equal to 15% of the plasmid DNA was taken up by the parasites. When transfecting DNA contained a segment of T. brucei ribosomal DNA that included the 5' end of the rRNA gene, the introduced plasmid directed expression of RNA; this RNA expression was shown both by dot blots and by S1 nuclease protection assays carried out under conditions specific for probe hybridization to RNA. In the absence of the ribosomal region, analogous transcription did not occur. We optimized this trypanosomal expression system with regard to electric shock strength, concentration of input DNA, and incubation time after electric shock. This technique enabling specific trypanosome DNA expression in vivo should facilitate the molecular analysis of T. brucei gene expression.
Differences in the glycolipid membrane anchors of bovine and human erythrocyte acetylcholinesterases.
Authors: Roberts W L , Kim B H , Rosenberry T L
Published: May 2006

Acetylcholinesterases (AcChoEases; EC 3.1.1.7) from bovine (Ebo) and human (Ehu) erythrocytes were purified to apparent homogeneity by affinity chromatography. The hydrophobic portion of the glycolipid membrane anchor of each enzyme was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Several cleavage procedures demonstrated that this radiolabel was highly selective for the fatty acid portion of the anchor in both enzymes. The labeled enzymes were digested with phosphatidylinositol (PtdIns)-specific phospholipase C (EC 3.1.4.10), and label release was assessed by polyacrylamide gel electrophoresis. About 85% of the radiolabel was cleaved from Ebo AcChoEase, whereas only 5% was released from Ehu AcChoEase. This finding agrees with a report that Ebo AcChoEase was quantitatively released from intact erythrocytes by PtdIns-specific phospholipase C but Ehu AcChoEase was not [Low, M. G. & Finean, J. B. (1977) FEBS Lett. 82, 143-146]. The two AcChoEases contained comparable amounts of the anchor components ethanolamine, glucosamine, and myo-inositol, but qualitative and quantitative differences were found in the fatty acids. Thin-layer chromatography of radiolabeled fragments generated from Ebo and Ehu AcChoEases by nitrous acid deamination revealed a major difference in the membrane anchors of the two enzymes. The fragment released from Ebo AcChoEase by this procedure comigrated with PtdIns, whereas the corresponding fragment from Ehu AcChoEase had a mobility much greater than that of PtdIns even though it contained myo-inositol and fatty acids. These studies show that 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine is useful for analysis of lipid-containing compounds and indicate that, whereas Ebo AcChoEase contains PtdIns in its glycolipid anchor, Ehu AcChoEase has a different anchor structure, which is resistant to PtdIns-specific phospholipase C. This observation suggests the existence of a class of glycolipid-anchored membrane proteins resistant to this phospholipase.
Bacteriophage T4 regA protein binds to mRNAs and prevents translation initiation.
Authors: Winter R B , Morrissey L , Gauss P , Gold L , Hsu T , Karam J
Published: May 2006

The bacteriophage T4 regA protein is a translational repressor of a subset of phage mRNAs. We show here that purified regA protein binds specifically to target mRNAs near the initiating AUG and occludes binding of ribosomes. Translational repression by regA protein diminishes expression of many genes whose mRNA sequences around the initiating AUG codons are different. A comparison of nucleotide sequences from several regA-repressed mRNAs suggests that the initiating AUG is an important, but not sufficient, sequence for regA binding.
S2 episome of maize mitochondria encodes a 130-kilodalton protein found in male sterile and fertile plants
Authors: Zabala G. , O'Brien-Vedder C. , Walbot V.
Published: May 2006

The mitochondrial genome of the S-type male-sterile cytoplasm of maize contains two linear episomes, S1 (6397 base pairs) and S2 (5453 base pairs). The S2 episome contains two large unidentified open reading frames, URF1 (3512 base pairs) and URF2 (1017 base pairs). We have demonstrated that a polypeptide with an apparent molecular mass of 130 kDa is the gene product of URF1. This polypeptide was first detected in Coomassie blue-stained protein gels of
cms
-S (where
cms
= cytoplasmic male sterile) but not in those of
cms
-T,
cms
-C, or normal mitochondrial proteins. The protein product of a translational fusion containing the 5′ end of
Escherichia coli lacZ
and an internal segment from URF1 of S2 was recognized by antisera raised against the 130-kDa variant polypeptide. The mitochondria of fertile F
1
hybrids of
cms
-S × Ky21 (the male parent carrying nuclear fertility restoration genes) contain as much of the 130-kDa protein as is found in
cms
-S mitochondria of sterile plants. Spontaneous fertile cytoplasmic revertants from
cms
-S in a WF9 nuclear background also synthesized the 130-kDa polypeptide. Therefore, the mere presence or absence of the URF1 gene product of S2 does not determine the fertility status of maize plants, because male sterile and male fertile (nuclear restored and revertant) plants can contain equivalent amounts of the 130-kDa polypeptide.
Analysis of cis-active sequences involved in the leaf-specific expression of a potato gene in transgenic plants
Authors: Stockhaus Jörg , Eckes Peter , Rocha-Sosa Mario , Schell Jeff , Willmitzer Lothar
Published: May 2006

ST-LS1
is a light-inducible, single-copy gene from potato that is expressed only in photosynthetic tissues. Various sequences derived from the 5′-upstream region of this gene were fused to the coding region of the chloramphenicol acetyltransferase (CAT) gene and to the gene 7 termination region of the transfer DNA (T-DNA) from the
Agrobacterium
Ti plasmid pTiACH5 and transferred to tobacco using Ti-plasmid vectors. After regeneration of whole plants, tissues were assayed for the expression of the CAT gene. Sequences derived from the 5′-upstream region of the
ST-LS1
gene comprising positions -334 to +11 were sufficient to confer a leaf/stem-specific as well as a light-inducible expression of the CAT gene. Destruction of chloroplasts by treatment with the herbicide norfluorazon and subsequent exposure to light drastically reduced the expression of the CAT gene indicating that this upstream sequence most likely interacts with a chloroplast-dependent signal. When sequences from position -98 to position +675 were fused to a truncated inactive fragment of the cauliflower mosaic virus 35S promoter in a head-to-head manner, the corresponding chimeric genes were again expressed in photosynthetic tissues only, indicating that these sequences have enhancer-like properties.
Expression of the human B-cell surface protein CD20: alteration by phorbol 12-myristate 13-acetate.
Authors: Valentine M A , Cotner T , Gaur L , Torres R , Clark E A
Published: May 2006

The monoclonal antibody 1F5 recognizes human B-cell surface protein CD20 and can activate resting B cells; with this antibody we found CD20 to be a 35/37-kDa non-disulfide-linked protein. The protein has a pI of 7.5-8.0 and is phosphorylated in B-cell lines, tonsillar B cells, and peripheral blood B cells. Both CD20 surface expression and phosphorylation are increased on buoyant tonsillar B cells activated in vivo. Because phorbol 12-myristate 13-acetate (PMA) supports the activation signal initiated by monoclonal antibody 1F5, we studied the effect of PMA on CD20 expression. After brief incubation with mitogenic levels of PMA, the number of dense tonsillar B cells positive for CD20 protein transiently decreased. Paradoxically, the cells remaining positive had more surface CD20 than did control cells, and these remaining surface CD20 molecules were hyperphosphorylated. Furthermore, PMA not only induced phosphorylation of CD20 protein on Raji cells but also increased the internalization of CD20 molecules; both phosphorylation and internalization of CD20 molecules were decreased with the protein kinase C inhibitor palmitoyl carnitine. Conditions that increase CD20 phosphorylation are shown also to increase surface mobility of the molecule, suggesting that CD20 protein internalization may be a critical early event for B-cell entry into the G1 phase of the cell cycle.
12(R)-hydroxyicosatetraenoic acid: a cytochrome-P450-dependent arachidonate metabolite that inhibits Na+,K+-ATPase in the cornea.
Authors: Schwartzman M L , Balazy M , Masferrer J , Abraham N G , McGiff J C , Murphy R C
Published: May 2006

When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, four polar metabolites (compounds A-D) were formed. Synthesis of these metabolites could be inhibited by carbon monoxide, SKF 525A, and anti-cytochrome c reductase antibodies. One of the metabolites, compound C, was found to inhibit partially purified Na+,K+-ATPase from the corneal epithelium in a dose-dependent manner with an ID50 of approximately 50 nM. After compound C was purified by TLC and HPLC, it was found to have a UV absorption spectrum with a maximum absorbance at 236 nm suggesting the presence of a conjugated diene. Mass spectrometric analysis using positive- and negative-ionization modes was carried out on derivatized compound C that had been synthesized from a mixture of specifically labeled ([5,6,8,9,11,12,14,15-2H8]arachidonic acid) and unlabeled arachidonic acid. Abundant fragment ions were consistent with compound C being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon-12 of the icosanoid backbone; all deuterium atoms from [2H8]arachidonate were retained in the structure. Oxidative ozonolysis yielded products indicating double bonds between carbons at positions 10 and 11 and positions 14 and 15 of the 20-carbon chain. Compound C was, therefore, characterized as a 12-hydroxyicosatetraenoic acid. However, only 12(R) isomer was found to be an inhibitor of the Na+,K+-ATPase from the corneal epithelium, suggesting that the biologically active compound C was 12(R)-hydroxy-5,8,10,14-icosatetraenoic acid. Such an inhibitor of Na+,K+-ATPase synthesized in the cornea may have an important role in regulating ocular transparency and aqueous human secretion.
Trypanosome mRNAs have unusual "cap 4" structures acquired by addition of a spliced leader.
Authors: Perry K L , Watkins K P , Agabian N
Published: May 2006

A single capped oligonucleotide is released from Trypanosoma brucei poly(A)+ RNA upon digestion with RNase T2. This observation supports the hypothesis that all T. brucei mRNAs share a common leader sequence. Digestion of the T2-resistant species with nucleotide pyrophosphatase shows that the capping nucleotide is 7-methylguanosine 5'-monophosphate (pm7G). Additional characterization of the T2-resistant fragment indicates that modifications are present on the first four transcribed nucleotides; the 5' termini of T. brucei mRNAs can, therefore, be described as "cap 4" structures. Identical 5'-cap structures are found on the T. brucei spliced leader (SL) RNA; an observation compatible with the hypothesis that the small SL RNA acts as a donor of the SL for the mRNA. However, we find that within a population of purified SL RNAs are species that are capped but incompletely modified. The presence of these unmodified and partially modified species allowed us to analyze the 5' sequence of the SL RNA transcript. The results indicate that transcription begins four nucleotides upstream of the reported 5' end. Therefore, the T. brucei SL transcript is actually 39 rather than 35 nucleotides long. We have also analyzed the capped oligonucleotide of a distantly related Trypanosomatid, Leptomonas collosoma and find it to be identical to that of T. brucei. The potential significance of these results is discussed in light of observations of trypanosome gene expression.
Nucleotide sequence of the gene from the cyanobacterium Anacystis nidulans R2 encoding the Mn-stabilizing protein involved in photosystem II water oxidation.
Authors: Kuwabara T , Reddy K J , Sherman L A
Published: May 2006

The gene for the Mn-stabilizing protein (MSP; the so-called extrinsic 33-kDa protein) that is involved in photosystem II water oxidation was cloned and sequenced from the genome of the cyanobacterium Anacystis nidulans R2. The gene (here designated woxA) was shown to be present in a single copy. The deduced amino acid sequence indicated that the translation product consisted of 277 amino acid residues with a Mr of 29,306. The comparison of the sequence with that of mature MSP from spinach chloroplasts suggested that the translation product is a precursor whose amino-terminal 28 amino acid residues represent the signal peptide for the protein to cross the thylakoid membrane into the lumen. The length of the putative signal peptide was less than half that of the transit peptide for thylakoid-lumenal proteins of higher plants, whereas the structural profile of the putative signal peptide was similar to that of the carboxyl-terminal portion of the higher plant transit peptides. The amino acid sequence of the mature A. nidulans R2 MSP showed rather low homology (48-49%) to higher plant MSPs, but the conserved amino acid residues appeared to be clustered. Five clusters were tentatively assigned, in which the homology values were in a range of 66-70%. Domains essential for the functioning of MSP are expected to be situated in these clusters. It is of note that the two cysteine residues in MSP were conserved, and the disulfide linkage between them may play an important role in maintaining the tertiary structure of MSP.
Rat cardiac ventricle has two Na+,K+-ATPases with different affinities for ouabain: developmental changes in immunologically different catalytic subunits.
Authors: Sweadner K J , Farshi S K
Published: May 2006

The mechanism of the inotropic effect of cardiac glycosides on the heart has long been controversial. Inotropic effects at low concentrations of cardiac glycosides indicate more than one class of receptor or more than one cellular mechanism. In the brain of the rat, high- and low-affinity cardiac glycoside receptors have been shown to be associated with two structurally different isoforms of the catalytic subunit of the Na+,K+-ATPase, termed alpha and alpha(+). Evidence is presented here that the high- and low-affinity sites in rat cardiac ventricle are associated with Na+,K+-ATPase catalytic subunit forms similar to the alpha(+) and alpha forms in the brain. Membranes from the rat ventricle contained polypeptides with the electrophoretic mobilities of alpha and alpha(+), which could be stained by isoform-specific anti-Na+,K+-ATPase antibodies on electrophoretic blots. Both polypeptides also displayed Na+-stimulated phosphorylation with [gamma-32P]ATP. Inhibition of Na+,K+-ATPase activity by ouabain demonstrated the presence of both high- and low-affinity ATPases proportional to the presence of the alpha(+) and alpha polypeptides. The ratios of the two isoforms changed with postnatal maturation, paralleling known changes in cardiac physiology and cardiac glycoside sensitivity. Cardiac glycoside sensitivity can evidently be regulated at the level of gene expression by developmental signals.
Antineoplastic bryostatins are multipotential stimulators of human hematopoietic progenitor cells.
Authors: May W S , Sharkis S J , Esa A H , Gebbia V , Kraft A S , Pettit G R , Sensenbrenner L L
Published: May 2006

The bryostatins are macrocyclic lactones, extracted from the marine bryozoan Bugula neritina, and have been reported to be potent antineoplastic agents. Results described here demonstrate that the bryostatins may also be useful as stimulators of normal human hematopoietic cells since they can (i) directly stimulate bone marrow progenitor cells to form colonies in vitro and (ii) functionally activate neutrophils. Structure-activity studies with bryostatin congeners indicate that these stimulatory properties may be dependent on the chain length and the unsaturated nature of the acylated group at carbons 20 and 7 of the bryostatin molecule. These stimulatory properties demonstrate that the naturally occurring bryostatins can mimic many of the biological effects of multipotential granulocyte-macrophage colony-stimulating factor. Thus, the coupling of antineoplastic activity with stimulatory growth properties for normal hematopoietic cells makes this agent an excellent probe to dissect the mechanism(s) of normal hematopoiesis. In addition, bryostatin may represent a clinically attractive agent useful for treating bone marrow failure states.
The upstream activation site of a Ty2 element of yeast is necessary but not sufficient to promote maximal transcription of the element.
Authors: Liao X B , Clare J J , Farabaugh P J
Published: May 2006

The Ty elements of yeast are a family of retrovirus-like transposons that are highly transcribed, accounting for about 10% of total mRNA. We have mapped two sites to the nontranscribed region of the element upstream of the transcription start site that are required for maximal gene expression and are similar to sites previously defined in other genes. One, the TATA site, is located 74 base pairs upstream of the transcription start site and has the canonical sequence TATAAAA. This site is required for normal rates of initiation; deletion of the site greatly reduces the amount of Ty917 mRNA without changing its 5' end. A second site is located in a region from 140 to 110 base pairs upstream of the start site. Unlike other upstream activation site (UAS) elements previously defined, the Ty917 UAS is not sufficient to promote any transcription in the absence of downstream transcription regulatory sites. Thus the UAS is necessary but not sufficient for maximal transcription. Comparison of constructs lacking either the UAS or the downstream enhancer or both shows evidence of synergistic interaction between the sites since the effect of the sites on the rate of transcription initiation is more than additive.
Partition site of the P1 plasmid.
Authors: Martin K A , Friedman S A , Austin S J
Published: May 2006

We have defined a minimal partition site, parS, from the plasmid P1. It contains sufficient cis-acting information to direct accurate segregation of low-copy-number plasmids that contain it as long as the two essential P1 Par proteins are supplied in trans. The site is, at most, 34 base pairs and contains a perfect 13-base-pair inverted repeat. Site-directed mutations were made within the repeat sequence that abolished activity whether or not the symmetry of the palindrome was maintained. Partition appears to be a competitive process, as differentially marked plasmids carrying the same type of partition site are not independently segregated but are randomly distributed with respect to each other. We have studied competition between plasmids carrying various fragments encompassing the parS site. As expected, two plasmids carrying the minimal parS site compete with each other. However, a sequence that lies to the left of the minimal parS site acts as a major modulator of this competition, changing the specificity of the competitive effect completely. Thus, this adjacent sequence appears to be an important determinant of the specificity of the wild-type P1 partition system without being necessary for its efficient function.