bluuebunny/crossref_metadata_2025 huggingface dataset:
Alpha 1-adrenergic agonists selectively suppress voltage-dependent K+ current in rat ventricular myocytes.
Authors: Apkon M , Nerbonne J M
Published: May 2006

The effects of alpha 1-adrenergic agonists on the waveforms of action potentials and voltage-gated ionic currents were examined in isolated adult rat ventricular myocytes by the whole-cell patch-clamp recording technique. After "puffer" applications of either of two alpha 1 agonists, phenylephrine and methoxamine, action-potential durations were increased. In voltage-clamped cells, phenylephrine (5-20 microM) or methoxamine (5-10 microM) reduced the amplitudes of Ca2+-independent voltage-activated outward K+ currents (Iout); neither the kinetics nor the voltage-dependent properties of Iout were significantly affected. The effects of phenylephrine or methoxamine on Iout were larger and longer-lasting at higher concentrations and after prolonged or repeated exposures; in all experiments, however, Iout recovered completely when puffer applications were discontinued. The suppression of Iout is attributed to the activation of alpha 1-adrenergic receptors, as neither beta- nor alpha 2-adrenergic agonists had measurable effects on Iout; in addition, the effect of phenylephrine was attenuated in the presence of the alpha antagonist phentolamine (10 microM), but not in the presence of the beta antagonist propranolol (10 microM). Voltage-gated Ca2+ currents, in contrast, were not altered measurably by phenylephrine or methoxamine and no currents were activated directly by these agents. Suppression of Iout was also observed during puffer applications of either of two protein kinase C activators, phorbol 12-myristate 13-acetate (10 nM-1 microM) and 1-oleoyl-2-acetylglycerol (60 microM). We conclude that the activation of alpha 1-adrenergic receptors in adult rat ventricular myocytes leads to action-potential prolongation as a result of the specific suppression of Iout and that this effect may be mediated by activation of protein kinase C.
Identification of a unique isoform of 1-aminocyclopropane-1-carboxylic acid synthase by monoclonal antibody
Authors: Mehta Arkesh M. , Jordan Ramon L. , Anderson James D. , Mattoo Autar K.
Published: May 2006

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14) is a key enzyme regulating ethylene biosynthesis in higher plants. A monoclonal antibody (mAb T20C) that immunoprecipitates the ACC synthase activity from tomato pericarp tissue extracts revealed that mAb T20C immunodecorates an ≈67-kDa polypeptide. On isoelectric focusing gels, ACC synthase activity in cell-free preparations was resolved into three distinct activity peaks with pI values 5.3, 7, and 9. mAb T20C specifically recognized the pI 7 form of the enzyme on electrophoretic transfer (Western) blots. When analyzed by sodium dodecyl sulfate gel electrophoresis under reducing conditions, the eluted pI 7 form was confirmed to migrate as a polypeptide of 67 kDa. The 67-kDa pI 7 isoform is a previously undescribed form of ACC synthase.
Cloning and nucleotide sequence of human gamma-glutamyl transpeptidase.
Authors: Rajpert-De Meyts E , Heisterkamp N , Groffen J
Published: May 2006

We have identified the gene for human gamma-glutamyl transpeptidase [GGT; glutamine:D-glutamyl-peptide 5-glutamyltransferase (also called gamma-glutamyltransferase), EC 2.3.2.2] in a BCR gene-related region located in band q11----qter of chromosome 22. Two cDNAs complementary to the GGT mRNA have been isolated from a human placental library constructed in phage lambda gt11. The largest cDNA has a size of 2535 base pairs (bp) and an open reading frame of 1707 nucleotides encoding 569 amino acids. By using a probe corresponding to this cDNA, a mRNA of approximately 2.4 kilobases was detected by RNA blot-hybridization analysis in mouse kidney RNA. The GGT precursor encoded by the coding sequence would have an estimated Mr of 61,400. We compared our nucleotide and deduced amino acid sequences with the published results of rat kidney cDNAs. The human and rat amino acid sequences are similar; however, a considerable discrepancy in nucleotide sequence was found within a 180-bp fragment of the heavy chain, resulting in a completely different amino acid sequence for this region. In addition, the 5' untranslated sequence of the human cDNA (669 bp) is substantially larger than that determined in the rat cDNA (227 bp). Our results may be valuable for further studies on the protein structure of human GGT as well as studies on the regulation of the enzyme.
SecA suppresses the temperature-sensitive SecY24 defect in protein translocation in Escherichia coli membrane vesicles.
Authors: Fandl J P , Cabelli R , Oliver D , Tai P C
Published: May 2006

Genetic analysis of protein secretion in Escherichia coli has identified secY/prlA and secA as components of the secretory apparatus. We have examined the roles of the secY(prlA) gene product (an integral membrane protein) and the soluble secA gene product in translocation of OmpA and alkaline phosphatase precursors in an in vitro system. The protein translocation defect of the secY24 mutation was recently demonstrated in vitro as was its suppression by an S300 extract. We show here that the extract was essentially inactive in SecY24 suppression when SecA protein was removed from it by immunoaffinity chromatography. Furthermore, purified SecA protein suppressed the SecY24 defect. Preincubation of the inactivated SecY24 membrane vesicles either with S300 containing SecA or with purified SecA protein reconstituted the membranes and restored the translocation activity when assayed in the absence of additional soluble proteins. These results suggest that the SecY24 translocation defect is suppressed by SecA interacting, directly or indirectly, with SecY24 on the cytoplasmic membrane.
Deletion of transfected oncogenes from NIH 3T3 transformants by inhibitors of poly(ADP-ribose) polymerase.
Authors: Nakayasu M , Shima H , Aonuma S , Nakagama H , Nagao M , Sugimura T
Published: May 2006

a1-1 cells, a transformant line obtained by transfection of NIH 3T3 cells with human c-Ha-rasT24 (hc-Ha-rasT24), were converted to morphologically normal flat cells following a 2-week culture in the presence of benzamide (BA), an inhibitor of poly(ADP-ribose) polymerase [ADP-ribosyltransferase (polymerizing); EC 2.4.2.30]. Concomitant with these morphological changes was the loss of the exogenous hc-Ha-rasT24 sequence. When cells were cultured without transfer, multiple clusters of flat revertant cells surrounded by transformed cells within single colonies of a1-1 cells were observed. This, together with the slow growth rate of flat cells in the presence of BA, indicated that flat revertants were induced rather than selected by BA. Flat cells isolated from mixed colonies completely lost the exogenous and amplified hc-Ha-rasT24 gene. In contrast, the endogenous mouse c-Ha-ras in flat revertant cells was not lost during culture with BA. Similarly, the endogenous hc-Ha-rasT24 in human bladder carcinoma T24 cells was not affected by BA. By using various chemicals, it was suggested that inhibition of poly(ADP-ribose) polymerase induces an efficient and specific loss of the exogenous transforming genes including Ki-ras, N-ras, c-raf, and ret-II.
cAMP-mediated phosphorylation of the low-Km cAMP phosphodiesterase markedly stimulates its catalytic activity.
Authors: Grant P G , Mannarino A F , Colman R W
Published: May 2006

Treatment of intact human platelets with the adenylate cyclase agonist forskolin (100 microM) resulted in an increase in cAMP phosphodiesterase activity in freeze-thaw lysates. When the low-Km (high affinity), cGMP-inhibited cAMP phosphodiesterase was isolated from such lysates by blue dextran-Sepharose chromatography, the specific activity of the enzyme was increased an average of 11-fold over similarly processed control platelets. The increase in the low-Km, cGMP-inhibited cAMP phosphodiesterase activity was inhibited when platelets were incubated with the protein kinase inhibitor H-8 prior to treatment with forskolin, suggesting that the stimulation of cAMP phosphodiesterase activity involved a cAMP-dependent phosphorylation. When intact platelets that had been prelabeled with 32Pi were treated with forskolin and the low-Km, cGMP-inhibited phosphodiesterase was isolated by blue dextran-Sepharose chromatography, a protein of 110,000 kDa was phosphorylated. By using a monospecific antiserum to the purified phosphodiesterase, this protein was shown to be the low-Km, cGMP-inhibited cAMP phosphodiesterase by electrophoretic transfer blot (Western blot) analysis and by immunoprecipitation. The stable prostacyclin analog iloprost also stimulated the low-Km cAMP phosphodiesterase activity about 2-fold and caused phosphorylation of the enzyme. These results suggest that phosphorylation of the low-Km, cGMP-inhibited phosphodiesterase may be an important regulatory mechanism for this enzyme in platelets.
Evidence for a single rolling circle in the replication of potato spindle tuber viroid
Authors: Branch Andrea D. , Benenfeld Bonnie J. , Robertson Hugh D.
Published: May 2006

We analyzed
in vivo
-labeled RNA to determine which of the two proposed rolling-circle models is more likely to depict the replication cycle of potato spindle tuber viroid. A key feature distinguishing the two models is the presence of a circular monomeric minus strand in one and not the other. Chromatography on cellulose CF11 was used to purify a fraction containing the replication intermediates free from single-stranded progeny. Heat denaturation followed by gel electrophoresis was used to seek possible circular templates—species required for rolling-circle replication to take place. Upon heating, a
32
P-labeled RNA was released. Limited nuclease digestion (“nicking”) revealed that this was a unitlength circular RNA. Fingerprinting identified it as a plus strand. No circular minus strands were detected in this population or in nuclease-treated samples containing RNase T1-resistant cores of the replication complex. Thus, potato spindle tuber viroid appears to use an asymmetric pathway in which minus strands are synthesized by rolling-circle copying, but plus strands are not. More details of the replication pathways used by various viroid-like RNAs are needed and will help to establish the evolutionary relationships among these infectious agents.
Chromosomal translocation in T-cell leukemia line HUT 78 results in a MYC fusion transcript.
Authors: Finger L R , Huebner K , Cannizzaro L A , McLeod K , Nowell P C , Croce C M
Published: May 2006

Primary cultures and established cell lines derived from human T-cell leukemias were analyzed for genomic rearrangements in the region 3' of the MYC locus. A T-cell leukemia line, HUT 78, whose 3' MYC region is rearranged, carries a chromosome t(2;8) juxtaposition; i.e., a locus derived from chromosome region 2q34 is attached to the 3' end of one MYC allele. The t(2;8) rearrangement in the HUT 78 cell line results in expression of a fused transcript encompassing the MYC gene and a locus designated TCL4 (T-cell leukemia/lymphoma 4), which normally resides on chromosome 2. The steady-state level of MYC-TCL4 fusion transcripts in HUT 78 cells is significantly higher than the MYC RNA level found in several other B- and T-cell lines. The production of fused MYC-TCL4 transcripts in a leukemic cell line raises the possibility that other B- and T-cell leukemias may express MYC fusion transcripts as an integral step in their pathogenesis.
Effect of mutations and variations of HLA-A2 on recognition of a virus peptide epitope by cytotoxic T lymphocytes.
Authors: McMichael A J , Gotch F M , Santos-Aguado J , Strominger J L
Published: May 2006

Cytotoxic T lymphocytes (CTL) specific for influenza A virus were prepared from 15 donors. Those with HLA-A2 recognized autologous or HLA-A2-matched B-lymphoblastoid cells in the presence of synthetic peptide representing residues 55-73 or 56-68 of the virus matrix protein sequence. Influenza A virus-specific CTL from donors without HLA-A2 or with an HLA-A2 variant type failed to respond to this peptide. CTL lines specific for HLA-A2 plus peptide did not lyse peptide-treated target cells from HLA-A2 variant donors. They also failed to lyse peptide-treated cells with point mutations that had been inserted into HLA-A2 at positions 62-63, 66, 152, and 156 and, in some instances, mutations at positions 9 and 70. CTL lysed peptide-treated target cells with mutations in HLA-A2 at positions 43, 74, and 107. The results imply that this defined peptide epitope therefore interacts with HLA-A2 in the binding groove so that the long alpha-helices of HLA-A2 make important contact with the peptide at positions 66, 152, and 156. Different amino acids at position 9, which is in the floor of the peptide binding groove of HLA-A2 and the closely related position 70, modulate the peptide interaction so that some T-cell clones react and some do not.
Juxtaposition of the T-cell receptor alpha-chain locus (14q11) and a region (14q32) of potential importance in leukemogenesis by a 14;14 translocation in a patient with T-cell chronic lymphocytic leukemia and ataxia-telangiectasia.
Authors: Davey M P , Bertness V , Nakahara K , Johnson J P , McBride O W , Waldmann T A , Kirsch I R
Published: May 2006

We describe a t(14;14)(q11;q32) translocation in a patient with T-cell chronic lymphocytic leukemia and ataxia-telangiectasia (AT). By using a battery of joining (J)-segment probes from the T-cell receptor (TCR) alpha-chain locus TCRA, three distinct J alpha rearrangements were observed. One rearrangement reflected a normal TCRA variable (V) region V alpha-to-J alpha recombination. The second rearrangement was caused by the translocation even itself, which joined a DNA segment from 14q32 centromeric to the immunoglobulin heavy chain locus (IGH) and a J alpha gene located approximately 75 kilobases (kb) 5' of the TCRA constant region gene (C alpha). A third rearrangement involved a 17-kb internal deletion 3' to the translocation, a rearrangement within the J alpha locus that has been observed once before in a patient with AT. Analysis of these three rearrangements underscores the increase in aberrant locus-specific recombination in lymphocytes from patients with AT. Furthermore, these studies support the view that a growth-effecting gene is present in the 14q32 region that participates in the leukemogenic process.
NifA-dependent in vivo protection demonstrates that the upstream activator sequence of nif promoters is a protein binding site.
Authors: Morett E , Buck M
Published: May 2006

Primer-extension analysis of the Klebsiella pneumoniae nifH promoter was used to determine changes in the accessibility of the promoter DNA to methylation after exposure of growing cells to dimethyl sulfate. Four guanine residues present in the nifH upstream activator sequence (UAS), the proposed NifA binding site, were protected from methylation and two guanine residues were hypermethylated when the transcriptional activator protein NifA was present in the cells. The interaction detected at the nifH UAS was independent of the alternative sigma factor NtrA required for transcription of the nifH and other nif promoters. Mutations within the nifH UAS that diminish NifA-dependent transcriptional activation reduced the interaction at the UAS. It seems likely that the pattern of methylation protection observed in the nifH UAS is the result of NifA binding.
Hydrogen burst associated with nitrogenase-catalyzed reactions.
Authors: Liang J , Burris R H
Published: May 2006

We have used a membrane-leak mass spectrometer to follow the time courses of H2 evolution and substrate reduction by nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. In the absence of added substrates, dinitrogenase passes all of its electrons to protons to form H2, but when a reducible substrate is added the electrons from dinitrogenase are shared between protons and the added substrate so that the steady-state rate of H2 production is decreased. If a reducible substrate is added before the nitrogenase reaction is initiated, a pre-steady-state burst of H2 is evident upon initiation of the reaction. This burst is associated with all the substrates of nitrogenase examined--i.e., N2, N2O, C2H2, NaN3, and NaCN. The H2 burst is stoichiometric with dinitrogenase, but not with dinitrogenase reductase. In the H2 burst phase, 1 H2 is evolved per dinitrogenase molybdenum. Although a change in the ratio of nitrogenase components changed the initial rate of the H2 burst, the stoichiometry was not affected. Production of H2 by the burst in the presence of a high concentration of substrate is terminated after production of 1 H2 per dinitrogenase molybdenum, and a steady-state rate of H2 production is established. This response suggests that the H2 burst is not a catalytic event but a result of a once-only activation process.
Non-heme protein in the giant extracellular hemoglobin of the earthworm Lumbricus terrestris.
Authors: Fushitani K , Riggs A F
Published: May 2006

The protein/heme mass ratio for the extracellular hemoglobin of the earthworm Lumbricus terrestris has been redetermined. We find a value of 19,000 g of protein per mol of heme. Four major, heme-containing chains (a, b, c, and d), present in equal proportions, have a total molecular mass, with four hemes, of 69,664 Da based on their sequences. The intact hemoglobin comprises 12 subunits that form a two-layered hexagonal structure of about 3.8 MDa. This value, together with our determination of the protein/heme ratio, requires that 4 abcd units are present in each 1/12th subunit and that 192 heme-containing chains are present in the hemoglobin molecule. Our data indicate that approximately 2200 g of non-heme protein is present for each mole of heme-containing chain, or about 35,200 g per 1/12th subunit. This conclusion is consistent with the observation that chains of 31-37 kDa are present. On this basis the intact molecule would have 12 non-heme chains and 204 chains in all to give a total molecular mass of 3.77 MDa, close to that observed.
Molecular polymorphism of a cell surface proteoglycan: distinct structures on simple and stratified epithelia.
Authors: Sanderson R D , Bernfield M
Published: May 2006

Epithelial cells are organized into either a single layer (simple epithelia) or multiple layers (stratified epithelia). Maintenance of these cellular organizations requires distinct adhesive mechanisms involving many cell surface molecules. One such molecule is a cell surface proteoglycan, named syndecan, that contains both heparan sulfate and chondroitin sulfate chains. This proteoglycan binds cells to fibrillar collagens and fibronectin and thus acts as a receptor for interstitial matrix. The proteoglycan is restricted to the basolateral surface of simple epithelial cells, but is located over the entire surface of stratified epithelial cells, even those surfaces not contacting matrix. We now show that the distinct localization in simple and stratified epithelia correlates with a distinct proteoglycan structure. The proteoglycan from simple epithelia (modal molecular size, 160 kDa) is larger than that from stratified epithelia (modal molecular size, 92 kDa), but their core proteins are identical in size and immunoreactivity. The proteoglycan from simple epithelia has more and larger heparan sulfate and chondroitin sulfate chains than the proteoglycan from stratified epithelia. Thus, the cell surface proteoglycan shows a tissue-specific structural polymorphism due to distinct posttranslational modifications. This polymorphism likely reflects distinct proteoglycan functions in simple and stratified epithelia, potentially meeting the different adhesive requirements of the cells in these different organizations.
Human T-cell-receptor delta chain: genomic organization, diversity, and expression in populations of cells.
Authors: Loh E Y , Cwirla S , Serafini A T , Phillips J H , Lanier L L
Published: May 2006

The locus of the delta chain of the human T-cell receptor has been isolated and examined. Three D (diversity) regions and two J (joining) regions are present on the 5' side of the C (constant) region. The closest V (variable) region to the constant region is V delta 2, which in the germ line is found on the 3' side of the constant region in an inverted direction. The genomic structure of the human locus closely parallels its mouse counterpart. Several cDNA sequences and a series of rearranged genomic sequences are compared which demonstrate an enormous potential diversity in the junctional region, between the variable region and the joining region. We find the predominant utilization of the PEER variable region in thymic polyclonal gamma delta cell lines and in some peripheral blood gamma delta cell lines. Thus, the delta chain may have relatively limited variable-region diversity but a large junctional-region diversity. The implications of this observation are discussed.
Cytotoxic activity of an interleukin 6-Pseudomonas exotoxin fusion protein on human myeloma cells.
Authors: Siegall C B , Chaudhary V K , FitzGerald D J , Pastan I
Published: May 2006

A chimeric toxin composed of human interleukin 6 (IL-6) attached to a portion of Pseudomonas exotoxin (PE) devoid of its own cell recognition domain has been produced in Escherichia coli. The fusion protein (IL-6-PE40) is cytotoxic to a human myeloma cell line expressing IL-6 receptors but has no effect on IL-6 receptor-negative cells. The specificity of IL-6-PE40 cytotoxicity was demonstrated through competition with excess IL-6 and neutralization with an antibody to IL-6. IL-6-PE40 may be useful in the selective elimination of myeloma cells and other cells with high numbers of IL-6 receptors.
Human immunodeficiency virus type 2 long terminal repeat: analysis of regulatory elements.
Authors: Arya S K , Gallo R C
Published: May 2006

The long terminal repeats (LTRs) of the human immunodeficiency virus type 2 (HIV-2) and a related simian immunodeficiency virus (SIVmac) contain cis-acting positive regulatory elements upstream and the major transactivator gene (tat) response element and a possible negative regulatory element downstream of the transcriptional initiation site. The tat response element of HIV-2 and of SIVmac was more complex than that of HIV-1. Two structurally similar subelements within the HIV-2 tat response element could be identified. Both of these subelements were required for optimal transactivation by the HIV-2 tat gene product. Either of these subelements, however, was sufficient for transactivation by the HIV-1 tat gene product. These observations provide an explanation for the poor transactivation of HIV-1 LTR-directed gene expression by the HIV-2 tat gene product since the HIV-1 LTR contains an analog of only one of the HIV-2 subelements. The HIV-2 tat gene product also affected the function of the upstream elements, including enhancer activity. The response of these cis elements of HIV-2 to transactivation by HIV-2/SIVmac and HIV-1 tat gene differed somewhat in virus-infected and tat gene transfected cells, probably related to the differences in the effective concentration of the tat gene products and/or other viral or cellular factors. The steady-state levels of HIV-2 LTR-linked gene transcripts were much higher in the presence of HIV-2, SIVmac, and HIV-1 tat genes than in their absence, suggesting transcriptional modulation as a mechanism for tat gene function.
Rare male mating advantage in a natural population of Drosophila pseudoobscura.
Authors: Salceda V M , Anderson W W
Published: May 2006

The natural selection acting on chromosomal inversions was studied in a natural population of Drosophila pseudoobscura. Females from this population were allowed to produce offspring from their matings in nature. They were then remated to males from a laboratory strain and again allowed to produce offspring. Offspring were also produced from matings of males from nature to laboratory females. Diagnosis of salivary chromosomes in these several sets of larval offspring allowed us to deduce the karyotypes of adult females and males from nature as well as the karyotypes of the offspring of these females by their matings in nature. We reason that the males collected with the females are a reasonable sample of those that mated the females and deposited the sperm they carried on capture. Chromosome frequencies in the offspring of wild females by their matings in nature were decomposed into male and female parental contributions. Changes in chromosome frequency due to male mating success were calculated by comparing chromosomal frequencies in adult males with those in the chromosomes they contributed to their offspring. These changes were sizable and provide direct evidence that male sexual selection is an important component of selection on the inversions in this natural population. We proceeded further to classify karyotypes on the basis of their frequencies and to calculate the fraction of offspring fathered by rare or common males. Rare male karyotypes as a group had a selective value nearly twice that of the common male karyotypes.
The rhesus rotavirus gene encoding protein VP3: location of amino acids involved in homologous and heterologous rotavirus neutralization and identification of a putative fusion region.
Authors: Mackow E R , Shaw R D , Matsui S M , Vo P T , Dang M N , Greenberg H B
Published: May 2006

The complete gene 4 nucleotide sequence was determined for rhesus rotavirus and each of 11 viral variants selected by neutralizing monoclonal antibodies. Gene 4 is 2362 bases in length and encodes a protein, VP3, of 776 amino acids with a calculated Mr of 86,500. A conserved trypsin cleavage site, located at amino acid 247, divides VP3 into VP8 and VP5. Neutralizing monoclonal antibodies directed at VP3 were used to select variants that escaped neutralization. Each variant contains a single gene 4 mutation that permits viral growth in the presence of the antibody. Variant mutations were identified in six distinct neutralization regions in VP8 and VP5. Five of the six neutralization regions were found in VP8. The VP8 regions were primarily associated with strain-specific or limited heterotypic rotavirus neutralization. One region was identified in VP5 by three monoclonal antibodies that neutralize a broad range of rotavirus serotypes. The VP5 neutralization region is largely hydrophobic and is similar to putative fusion sequences of Sindbis and Semliki Forest viruses.
Human 67-kDa calelectrin contains a duplication of four repeats found in 35-kDa lipocortins.
Authors: Südhof T C , Slaughter C A , Leznicki I , Barjon P , Reynolds G A
Published: May 2006

The 67-kDa calelectrin is the largest member of a family of Ca2+-binding proteins that associate with membranes and phospholipids in a Ca2+-dependent manner. Oligonucleotide probes based on peptide sequences obtained from purified bovine 67-kDa calelectrin were used to screen a human retina cDNA library, and the complete primary structure of human 67-kDa calelectrin was deduced by DNA sequence analysis. The protein consists of eight 68-amino acid repeats separated by linking sequences of variable lengths. It is highly similar to the human lipocortin I and II sequences, each of which contains four such repeats. The amino termini of the three proteins show no sequence similarity; however, in the repeated regions the proteins are 42-45% identical in sequence. Analysis of the 16 repeats from the three proteins provides insights into the structural basis for Ca2+-dependent phospholipid binding. These data place the calelectrins and the lipocortins into the same gene family and suggest that these proteins have similar functions and have evolved from a common ancestor.
Nuclear proteins interacting with the promoter region of the human granulocyte/macrophage colony-stimulating factor gene.
Authors: Shannon M F , Gamble J R , Vadas M A
Published: May 2006

The gene for human granulocyte/macrophage colony-stimulating factor (GM-CSF) is expressed in a tissue-specific as well as an activation-dependent manner. The interaction of nuclear proteins with the promoter region of the GM-CSF gene that is likely to be responsible for this pattern of GM-CSF expression was investigated. We show that nuclear proteins interact with DNA fragments from the GM-CSF promoter in a cell-specific manner. A region spanning two cytokine-specific sequences, cytokine 1 (CK-1, 5' GAGATTCCAC 3') and cytokine 2 (CK-2, 5' TCAGGTA 3') bound two nuclear proteins [nuclear factor (NF)-GMa and NF-GMb] from GM-CSF-expressing cells in gel retardation assays. NF-GMb was inducible with phorbol 12-myristate 13-acetate and accompanied induction of GM-CSF message. NF-GMb was absent in cell lines not producing GM-CSF, some of which had other distinct binding proteins. NF-GMa and NF-GMb eluted from a heparin-Sepharose column at 0.3 and 0.6 M KCl, respectively. We hypothesize that the sequences CK-1 and CK-2 bind specific proteins and regulate GM-CSF transcription.
Enzyme stimulation upon fertilization is revealed in electrically permeabilized sea urchin eggs.
Authors: Swezey R R , Epel D
Published: May 2006

Sea urchin eggs and embryos subjected to high-voltage electric discharge in a medium mimicking the intracellular milieu retain their structural integrity and remain permeable, permitting substrates to enter the cytoplasm and thus assay of enzyme activity. At saturating concentrations of substrates, five of six enzymes assayed for more active (three to fifteen times) in permeabilized embryos than in permeabilized eggs, but no fertilization-related differences are seen in homogenates prepared from these same permeabilized cells. Furthermore, enzyme activity in homogenates always exceeds that in the permeabilized cell suspensions. This difference in enzyme reaction rates between unfertilized eggs and fertilized eggs is not due to differences in the diffusibility of substrates into the permeabilized cells. The activity of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in permeabilized cells was studied in greater detail and has the following characteristics. (i) Regulation of activity persists during early development. (ii) This regulation is not mediated by diffusible allosteric agents. (iii) Stimulation at fertilization is initiated by a rise in intracellular calcium and is further promoted by cytoplasmic alkalinization. (iv) The microenvironment experienced by this enzyme intracellularly differs from that of the enzyme in homogenates as evidenced by markedly different pH vs. activity profiles. These results indicate that the regulatory status of enzymes is preserved in electrically permeabilized cells and suggest that this regulation depends on some cell structural feature(s) that is (are) destroyed upon homogenization.
Antisense RNA inhibits endogenous gene expression in mouse preimplantation embryos: lack of double-stranded RNA "melting" activity.
Authors: Bevilacqua A , Erickson R P , Hieber V
Published: May 2006

beta-Glucuronidase activity increases 60-fold from the 4-cell to the blastocyst stage during in vitro development of mouse preimplantation embryos, secondary to a 13-fold increase in beta-glucuronidase mRNA. Injections of antisense RNA from a beta-glucuronidase cDNA clone lacking the 5'-untranslated region and the coding sequences for approximately equal to 150 N-terminal amino acids were effective in partially blocking the appearance of beta-glucuronidase activity. Injection of the same RNA, capped with guanosine(5')triphospho(5')guanosine (GpppG), into each blastomere at the 4-cell stage yielded 75% inhibition of enzyme activity at the blastocyst stage. Injections of the sense strand or of an unrelated RNA did not alter the normal increase in activity of the enzyme. These results are in accord with our inability to detect RNA-duplex "melting" activity in 1-cell mouse embryos. We suggest that it may be possible to analyze genetics of mammalian development by antisense techniques.
rbcS genes in Solanum tuberosum: conservation of transit peptide and exon shuffling during evolution.
Authors: Wolter F P , Fritz C C , Willmitzer L , Schell J , Schreier P H
Published: May 2006

Five genes of the rbcS gene family of Solanum tuberosum (potato) were studied. One of these is a cDNA clone; the other four are located on two genomic clones representing two different chromosomal loci containing one (locus 1) and three genes (locus 2), respectively. The intron/exon structure of the three genes in locus 2 is highly conserved with respect to size and position. These genes contain two introns, whereas the gene from locus 1 contains three introns. Although in most cases the amino acid sequences in the transit peptide part of different rbcS genes from the same species varied considerably more than the corresponding mature amino acid sequences, one exception found in tomato and potato indicates that the transit peptide of rbcS could have a special function. A comparison of the rbcS genes of higher plants with those of prokaryotes offers suggestive evidence that introns first served as spacer material in the process of exon shuffling and then were removed stepwise during the evolution of higher plants.
Binding of photoreactive lysozyme peptides to murine histocompatibility class II molecules.
Authors: Luescher I F , Allen P M , Unanue E R
Published: May 2006

We have studied the interaction of six photoreactive conjugates of the immunogenic hen egg-white lysozyme peptides HEL(46-61) or HEL(49-61) with the murine histocompatibility class II molecules I-Ak, I-Ad, I-Ek, and I-Ed. All compounds tested selectively labeled the alpha chain of the class II molecules. This was true when testing class II molecules on cell membranes or solubilized in detergents. The COOH-terminal conjugate of HEL(49-61) with (4-azidobenzoyl)cystine preferentially labeled I-Ak. However, addition of hydroxyl or iodine substituents to the photoreactive moiety increased the labeling efficiency and resulted in labeling of the other class II molecules. The data suggest that the photoreactive groups enhanced the binding affinities of these peptides to class II molecules, reflected by the increased labeling efficiencies. Conversely, introduction of an iodine substitution into the tyrosine residue of HEL(46-61) or HEL(49-61) strongly decreased the photoaffinity labeling, possibly due to steric interference with ligand binding to class II molecules. Judicious use of photoaffinity probes that conserve binding specificity of the peptide should be useful for mapping the antigen-binding site of a class II molecule.
Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages.
Authors: García E , García J L , García P , Arrarás A , Sánchez-Puelles J M , López R
Published: May 2006

A 2.9-kilobase Acc I fragment of the DNA of the pneumococcal bacteriophage Cp-1, containing the cpl gene, hybridizes with the lytA gene encoding the pneumococcal amidase. The nucleotide sequence of the cpl gene of Cp-1, encoding a muramidase (CPL), has been determined. The 3' regions of the cpl and lytA coding sequences show considerable nucleotide sequence homology and the carboxyl-terminal domains of the deduced amino acid sequences of these lysins are quite similar: 73 of the carboxyl-terminal 142 amino acid residues are identical, and of the 69 substitutions, 55 are conservative. Comparisons between CPL, the pneumococcal amidase, and the muramidase of the fungus Chalaropsis sp. (an enzyme that also degrades the pneumococcal cell wall) strongly suggest that the carboxyl-terminal domains of CPL and of the amidase might be responsible for the specific recognition of choline-containing cell walls, as well as for the noncompetitive inhibition of the catalytic activity of these enzymes by the pneumococcal lipoteichoic acid or by high concentrations of choline. In addition, the active center of these enzymes should be located in their amino-terminal domains. Our results suggest an evolutionary relationship between phage and host lysins.
Anaerobic induction of Escherichia coli formate dehydrogenase (hydrogenase-linked) is enhanced by gyrase inactivation.
Authors: Axley M J , Stadtman T C
Published: May 2006

Escherichia coli synthesizes a hydrogenase-linked formate dehydrogenase (FDHH) under anaerobic conditions in the absence of nitrate. In striking contrast to many other anaerobic-specific genes, which require DNA to be negatively supercoiled for expression, we have found that inhibition of DNA gyrase activity enhances expression from the gene (fdhF) encoding the selenopolypeptide of FDHH. Fusions of the 5' flanking region of fdhF and the structural gene of lacZ were used to determine fdhF expression under varying conditions. Chemical inhibitors and a temperature-sensitive mutant allowed in vivo inhibition of gyrase activity. In each case, concomitant with gyrase inhibition there was a substantial increase in the induction of fusion protein synthesis. This enhancement of expression is observed for the intact fdhF gene residing on the chromosome as well as the fusion gene in a multicopy plasmid. Inhibition of gyrase activity will partially overcome the inhibition of fdhF expression due to nitrate but does not allow fusion protein synthesis in the presence of oxygen.
Recombinant vaccinia virus vaccine against the human melanoma antigen p97 for use in immunotherapy.
Authors: Estin C D , Stevenson U S , Plowman G D , Hu S L , Sridhar P , Hellström I , Brown J P , Hellström K E
Published: May 2006

We have constructed a recombinant vaccinia virus, v-p97NY, which expresses the human melanoma-associated glycoprotein p97. Immunization with v-p97NY could induce humoral and cell-mediated immunity to p97, including delayed-type hypersensitivity, in mice and in two of two monkeys (Macaca fascicularis). The fact that an immune response was induced also in monkeys is important because normal cells from monkeys, but not from mice, express a low level of cross-reactive p97. Mice immunized with v-p97NY rejected transplants of syngeneic mouse melanoma expressing p97. A rejection response could be detected also when immunization was started 2 days after tumor transplantation, irrespective of whether the transplanted cells grew subcutaneously or as lung metastases. Evidence was obtained that melanoma cells lacking p97 may be killed as "bystanders" at the site of an immune response to melanoma cells expressing p97.
Voltage gating of conductance in lipid bilayers induced by porin from outer membrane of Neisseria gonorrhoeae.
Authors: Mauro A , Blake M , Labarca P
Published: May 2006

Porins, polypeptides of approximately 35 kDa, are present as integral membrane proteins in the outer membranes of a variety of Gram-negative bacteria. As reported previously for a purified porin from Escherichia coli, voltage gating of conductance was found to be induced in a lipid bilayer by the solubilized purified porin, protein I, from Neisseria gonorrhoeae. The unitary response to an applied potential showed a cascade of current from an initial level through at least three levels, more or less equal, to a persisting lower level. The initial level of current corresponded to 1.0-1.3 nS for 0.2 M NaCl on either side of the bilayer. Briefly reducing the potential to zero restored the current to its initial level. Interpretation of the unitary response is suggested by electron microscopic data obtained on negatively stained outer membranes of E. coli indicating the presence of "pores" appearing in triplets. Moreover, low-resolution x-ray and neutron diffraction studies on crystals obtained with an E. coli porin show that three polypeptides associate to form a unit. Combining such structural data with the present electrical data lends support for the hypothesis that the unitary response results from three pores acting as a unit in response to an applied potential. Evidence obtained with the patch-clamp technique is mounting for a similar mechanism of many channels operating as a unit in a variety of cell membranes. The porin channel holds promise as a concrete model for the analysis of voltage gating of ionic conductance.
Induction of fibronectin gene transcription and mRNA is a primary response to growth-factor stimulation of AKR-2B cells.
Authors: Blatti S P , Foster D N , Ranganathan G , Moses H L , Getz M J
Published: May 2006

A cDNA library, prepared from poly(A)+ RNA isolated from quiescent AKR-2B cells 4 hr after stimulation with epidermal growth factor in the presence of cycloheximide, was screened to identify RNA transcripts whose abundance is specifically increased as a primary response to growth stimulation. Approximately 40% of the inducible clones detected by this procedure corresponded to either cytoskeletal beta- or gamma-actin genes. One nonactin clone, designated c99, was found to be derived from an 8.5-kilobase RNA whose abundance began to increase as early as 30 min after stimulation. DNA sequencing established the identity of this RNA as fibronectin. Several additional mitogens were then tested and found to efficiently induce fibronectin mRNA. These included fetal calf serum, platelet-derived growth factor, and transforming growth factor type beta. For at least one inducer, fetal calf serum, the increase in mRNA was preceded by an increase in fibronectin gene transcription. This increase was rapid, reaching maximal levels within 10 min, and was accompanied by near-coordinate increases in both c-fos and beta-actin transcription. These results indicate that fibronectin is a member of a class of "early-response" genes, typified by c-fos and including beta-actin, whose rapid expression may be important in mediating cellular responses to peptide growth factors.
Prenatal lethality in a transgenic mouse line is the result of a chromosomal translocation.
Authors: Mahon K A , Overbeek P A , Westphal H
Published: May 2006

We have produced a line of transgenic mice that is characterized by prenatal lethality. These mice bear a chimeric plasmid containing the long terminal repeat of the Rous sarcoma virus linked to the coding region of the chloramphenicol acetyltransferase gene (pRSV-CAT). Mice heterozygous for the pRSV-CAT integration site are semisterile, producing litters approximately equal to 40% of the average size when crossed to normal mice. Approximately 50% of the progeny from such a cross bear the pRSV-CAT sequences and also produce litters of smaller size. An analysis of embryogenesis revealed that normal numbers of embryos implanted, but 60% failed to develop past day 7. Eight other independent transgenic lines containing RSV-CAT show no evidence of embryonic lethality; thus, it is unlikely that the defect observed is due to the direct effects of RSV-CAT expression. We have found that carrier mice bear a reciprocal translocation between chromosomes 6 and 17, T(6A2-6A3;17D-17E1), that can explain the apparent dominant embryonic lethality seen in this line. The site of integration has been localized by in situ hybridization at or near the translocation breakpoint in one of the translocated chromosomes (6(17)). Because the foreign DNA is present in one of the translocated chromosomes, we propose that this rearrangement was elicited by the introduction of foreign DNA.
Repression of nicotinic acetylcholine receptor expression by antisense RNAs and an oligonucleotide.
Authors: Sumikawa K , Miledi R
Published: May 2006

Four antisense RNAs, synthesized from cDNA clones coding for the four subunits of the acetylcholine receptor of Torpedo electroplaques, were used to study their effect on the expression of functional Torpedo acetylcholine receptors in Xenopus oocytes. All antisense RNAs inhibited the appearance of functional receptors in the oocyte's surface membrane for at least 1 week. This inhibition was specific because the antisense RNAs did not block the expression of the Cl- channels, also encoded by Torpedo electroplaque mRNA. Experiments with incomplete antisense RNAs and a synthetic oligonucleotide indicate that covering the ribosome binding site or the initiation codon in the mRNA is not a necessary requirement for efficient blocking. Thus, the use of antisense RNAs combined with the Xenopus oocyte system provides a novel approach to screen cDNA libraries for the genes coding for multisubunit neurotransmitter receptors.
Gel retardation at low pH resolves trp repressor-DNA complexes for quantitative study.
Authors: Carey J
Published: May 2006

The affinity and stoichiometry of DNA binding by Escherichia coli trp repressor were studied by electrophoresis in nondenaturing gels. The ability of trp repressor to retard the electrophoretic mobility of an operator DNA fragment depends on the pH of the gel system. Above the pI of the protein, little retardation of DNA is observed, although complex formation can be detected by other assays. As the pH of the gel is lowered, retardation is enhanced. The apparent dissociation constant for the interaction between trp repressor and trpEDCBA operator fragments is 0.5 nM under the conditions used here. Nonspecific binding occurs with only about 200-fold weaker affinity. The stoichiometries of specific and nonspecific complexes were determined directly by using trp repressor labeled in vivo. High-affinity operator binding requires a single dimer of trp repressor. DNase I-protection analysis ("footprinting") was used to confirm the dissociation constants and to locate the binding site.
Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence.
Authors: DeWitt D L , Smith W L
Published: May 2006

Prostaglandin G/H synthase (8,11,14-icosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1) catalyzes the first step in the formation of prostaglandins and thromboxanes, the conversion of arachidonic acid to prostaglandin endoperoxides G and H. This enzyme is the site of action of nonsteroidal anti-inflammatory drugs. We have isolated a 2.7-kilobase complementary DNA (cDNA) encompassing the entire coding region of prostaglandin G/H synthase from sheep vesicular glands. This cDNA, cloned from a lambda gt 10 library prepared from poly(A)+ RNA of vesicular glands, hybridizes with a single 2.75-kilobase mRNA species. The cDNA clone was selected using oligonucleotide probes modeled from amino acid sequences of tryptic peptides prepared from the purified enzyme. The full-length cDNA encodes a protein of 600 amino acids, including a signal sequence of 24 amino acids. Identification of the cDNA as coding for prostaglandin G/H synthase is based on comparison of amino acid sequences of seven peptides comprising 103 amino acids with the amino acid sequence deduced from the nucleotide sequence of the cDNA. The molecular weight of the unglycosylated enzyme lacking the signal peptide is 65,621. The synthase is a glycoprotein, and there are three potential sites for N-glycosylation, two of them in the amino-terminal half of the molecule. The serine reported to be acetylated by aspirin is at position 530, near the carboxyl terminus. There is no significant similarity between the sequence of the synthase and that of any other protein in amino acid or nucleotide sequence libraries, and a heme binding site(s) is not apparent from the amino acid sequence. The availability of a full-length cDNA clone coding for prostaglandin G/H synthase should facilitate studies of the regulation of expression of this enzyme and the structural features important for catalysis and for interaction with anti-inflammatory drugs.