bluuebunny/crossref_metadata_2025 huggingface dataset:
A 19-kDa C-terminal tryptic fragment of the alpha chain of Na/K-ATPase is essential for occlusion and transport of cations.
Authors: Karlish S J , Goldshleger R , Stein W D
Published: May 2006

Tryptic digestion of pig renal Na/K-ATPase in the presence of Rb and absence of Ca ions removes about half of the protein but leaves a stable 19-kDa membrane-embedded fragment derived from the alpha chain, a largely intact beta chain, and essentially normal Rb- and Na-occlusion capacity. Subsequent digestion with trypsin in the presence of Ca or absence of Rb ions leads to rapid loss of the 19-kDa fragment and a parallel loss of Rb occlusion, demonstrating that the fragment is essential for occlusion. The N-terminal sequence of the 19-kDa fragment is Asn-Pro-Lys-Thr-Asp-Lys-Leu-Val-Asn-Glu-Arg-Leu-Ile-Ser-Met-Ala, beginning at residue 830 and extending toward the C terminus. Membranes containing the 19-kDa fragment have the following functional properties. (i) ATP-dependent functions are absent. (ii) The apparent affinity for occluding Rb is unchanged, the affinity for Na is lower than in the control enzyme, and activation is now strongly sigmoidal rather than hyperbolic. (iii) Membranes containing the 19-kDa fragment can be reconstituted into phospholipid vesicles and sustain slow Rb-Rb exchange. Thus the transport pathway is retained. We conclude that cation occlusion sites and the transport pathway within transmembrane segments are quite separate from the ATP binding site, located on the cytoplasmic domain of the alpha chain. Interactions between cation and ATP sites, the heart of active transport, must be indirect--mediated, presumably, by conformational changes of the protein.
The noncompetitive blocker [3H]chlorpromazine labels three amino acids of the acetylcholine receptor gamma subunit: implications for the alpha-helical organization of regions MII and for the structure of the ion channel.
Authors: Revah F , Galzi J L , Giraudat J , Haumont P Y , Lederer F , Changeux J P
Published: May 2006

Labeling studies of Torpedo marmorata nicotinic acetylcholine receptor with the noncompetitive channel blocker [3H]chlorpromazine have led to the initial identification of amino acids plausibly participating to the walls of the ion channel on the alpha, beta, and delta subunits. We report here results obtained with the gamma subunit, which bring additional information on the structure of the channel. After photolabeling of the membrane-bound receptor under equilibrium conditions in the presence of agonist and with or without phencyclidine (a specific ligand for the high-affinity site for noncompetitive blockers), the purified labeled gamma subunit was digested with trypsin, and the resulting fragments were fractionated by HPLC. Sequence analysis of peptide mixtures containing various amounts of highly hydrophobic fragments showed that three amino acids are labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner: Thr-253, Ser-257, and Leu-260. These residues all belong to the hydrophobic and putative transmembrane region MII of the gamma subunit. Their distribution along the sequence is consistent with an alpha-helical organization of this segment. The [3H]chlorpromazine-labeled amino acids are conserved at homologous positions in the known sequences of other ligand-gated ion channels and may, thus, play a critical role in ion-transport mechanisms.
Introduction of specific point mutations into RNA polymerase II by gene targeting in mouse embryonic stem cells: evidence for a DNA mismatch repair mechanism.
Authors: Steeg C M , Ellis J , Bernstein A
Published: May 2006

We have introduced two specific point mutations, located 20 base pairs apart, into the endogenous murine gene that encodes the largest subunit of RNA polymerase II (RPII215). The first mutation conferred resistance to the mushroom toxin alpha-amanitin (amar), and the second mutation generated a restriction fragment length polymorphism without altering the protein sequence. Targeted amar clones were generated at a frequency of 1 in 30 totipotent embryonic stem cells that expressed stably integrated DNA vectors after electroporation. Thirty to 40% of these clones had acquired both mutations, whereas, surprisingly, the remaining clones had acquired the specific amar point mutation but lacked the restriction fragment length polymorphism. We suggest that the latter clones were generated by independent DNA mismatch repair rather than by double crossover or gene conversion. These results demonstrate that it is possible to introduce specific point mutations into an endogenous gene in embryonic stem cells. Thus it should be possible to introduce single base substitutions into other cellular genes, including nonselectable genes, by optimizing the efficiency of gene transfer and/or the sensitivity of screening for targeted clones.
Point mutations define positions in HLA-DR3 molecules that affect antigen presentation.
Authors: Mellins E , Arp B , Singh D , Carreno B , Smith L , Johnson A H , Pious D
Published: May 2006

Allelic differences in major histocompatibility complex (MHC)-encoded class II molecules affect both the binding of immunogenic peptides to class II molecules and the recognition of MHC molecule-peptide complexes by T cells. As yet, there has been no extensive mapping of these functions to the fine structure of human class II molecules. To determine sites on the HLA-DR3 molecule involved in antigen presentation to T cells, we used monoclonal antibodies specific for HLA-DR3 to immunoselect mutants of a B-lymphoblastoid line. We located the sites of single amino acid substitutions in the HLA-DR3 molecule and correlated these structural changes with patterns of recognition by HLA-DR3-restricted, antigen-specific T cells, allospecific T cells, and allospecific anti-DR3 monoclonal antibodies. We analyzed seven mutations. One mutation, at position 74 in domain 1 of the DR beta chain, affected recognition by all T cells tested, whereas others, at positions 9, 45, 73, 151, and 204 of the DR beta chain and position 115 of the DR alpha chain, altered recognition by some T cells, but not others. Each of the substitutions resulted in a unique pattern of T-cell stimulation. In addition, each T-cell clone recognized a different subset of the mutants. These results indicate that different residues of the DR3 molecule are involved in presentation of antigen to different DR3-restricted T cells. These studies further show that substitutions which most likely affect peptide binding alter recognition of DR3 molecules by an alloreactive T-cell clone and some allospecific antibodies.
lac permease of Escherichia coli: topology and sequence elements promoting membrane insertion.
Authors: Calamia J , Manoil C
Published: May 2006

The membrane topology of Escherichia coli lac permease was analyzed using a set of 36 lac permease-alkaline phosphatase (lacY-phoA) gene fusions. The level of enzymatic activity of alkaline phosphatase fused to a cytoplasmic membrane protein appears to reflect whether the fusion junction site normally faces the cytoplasm or periplasm. The alkaline phosphatase activities of cells expressing the lacY-phoA fusions distinguish between models previously proposed for the topology of lac permease and favor one with 12 transmembrane segments. This model is fully compatible with the results of earlier biochemical and immunological studies. The properties of fusions with junctions spanning two of the transmembrane segments at 2- or 3-amino acid intervals indicate that approximately half of the residues of either segment (9-11 amino acids) suffices to promote alkaline phosphatase translocation across the membrane. The additional transmembrane segment amino acids that are not required for this membrane insertion process may normally be needed in unfused lac permease after insertion for stable association with the membrane.
In vitro expression of rat lens aldose reductase in Escherichia coli.
Authors: Old S E , Sato S , Kador P F , Carper D A
Published: May 2006

Aldose reductase (alditol:NADP+ oxidoreductase, EC 1.1.1.21), an enzyme that converts glucose to sorbitol, the first step of the polyol pathway, has been implicated in secondary complications of diabetes, such as cataracts, retinopathy, neuropathy, and nephropathy. Aldose reductase inhibitors have been observed to prevent or delay the onset of these complications; however, more potent and specific inhibitors are needed. Development of new inhibitors necessitates a better understanding of the molecular structure of this protein. To elucidate the structure-function relationships of aldose reductase and to develop methods of regulating this enzyme, large and homogeneous quantities of rat lens aldose reductase have been expressed in bacterial cells. A construction of the complete coding sequence and 3' untranslated region for rat lens aldose reductase was assembled in the expression vector pKK233-2 (Pharmacia). This construction expresses an active enzyme that has been purified and demonstrates kinetic, immunological, and inhibitory properties similar to rat lens aldose reductase.
Identification of two nuclear genes (ATP11, ATP12) required for assembly of the yeast F1-ATPase.
Authors: Ackerman S H , Tzagoloff A
Published: May 2006

Nuclear respiratory-deficient mutants of Saccharomyces cerevisiae (pet mutants) have been screened for defects in the mitochondrial ATPase. Mutants in two complementation groups were found to have 10% or less of wild-type ATPase activity. The two wild-type nuclear genes defined by the mutants have been designated ATP11 and ATP12. The proteins encoded by the two genes are not subunits of the ATPase but rather appear to exercise an important function at a late stage in the synthesis of F1 after transport of the subunits into the internal compartment of mitochondria. Mitochondria of atp11 and atp12 mutants have only marginally reduced levels of the alpha and beta subunits of F1. Both proteins are processed to their mature size but are not part of a native F1 structure or associated with the mitochondrial membrane. The most reasonable explanation for the mutant phenotype is a block in the assembly of the F1 oligomer.
Intercalators promote the binding of RecA protein to double-stranded DNA.
Authors: Thresher R J , Griffith J D
Published: May 2006

Ethidium bromide, acridine orange, 4'-(9-acridinylamino)methanesulfon-o-anisidide (o-AMSA), and m-AMSA induce the rapid binding of RecA protein to double-stranded (ds) DNA. The filaments formed appear to retain the drug and are 12.8 nm in diameter with an 8.0-nm pitch. Two classes of drugs have been distinguished: (i) those that bind to RecA protein and induce assembly at low relative concentrations (e.g., ethidium bromide) and (ii) those that do not appear to interact directly with RecA protein and must be present at relatively high drug concentrations to stimulate assembly (e.g., m-AMSA). Ethidium bromide, acridine orange, and quinacrine inhibit RecA protein binding to single-stranded DNA. Addition of ATP to the drug-induced filaments causes the protein to rapidly dissociate from dsDNA, and protein binding to dsDNA diminishes upon extended exposure to room light. We suggest that the structure of the drug-induced filaments may be more typical of the complex that initiates RecA protein assembly along DNA rather than the product of extensive polymerization as induced by adenosine 5'-[gamma-thio]triphosphate.
Bipartite mRNA for chicken alpha-fibrinogen potentially encodes an amino acid sequence homologous to beta- and gamma-fibrinogens.
Authors: Weissbach L , Grieninger G
Published: May 2006

Overlapping cDNAs derived from the chicken alpha-fibrinogen mRNA have been sequenced, beginning from within the coding region for the signal peptide of this subunit and terminating within the poly(A) extension. The predicted size of chicken alpha-fibrinogen is 54,187 daltons, which is the smallest of any alpha chain reported; the oligopeptide repeats that characterize the central regions of the other alpha subunits were conspicuously absent. A further unexpected finding was the presence on the mRNA of a separate, long open reading frame (752 nucleotides), beginning 312 nucleotides downstream from the alpha-fibrinogen coding sequence and containing intron-like features near its 5' end. The protein sequence predicted from this second open reading frame lacks an initiating methionine but is homologous to the C-terminal regions of all known beta- and gamma-fibrinogens as well as the C termini of two nonfibrinogen proteins: cytotactin (tenascin), an extracellular matrix protein, and pT49, a putative protein specific to cytotoxic T cells. The intron-like features of the second open reading frame immediately precede the region of common homology, and the beginnings of the corresponding homologous segments in the beta- and gamma-fibrinogen sequences are marked by aligned intron positions. Based on these findings, it is proposed that fibrinogen gene evolution included a fusion of two distinct ancestral genes.
Tyrosine phosphorylation coupled to IgE receptor-mediated signal transduction and histamine release.
Authors: Benhamou M , Gutkind J S , Robbins K C , Siraganian R P
Published: May 2006

Antigen-induced cross-linking of IgE bound to its receptors at the surface of basophils or mast cells initiates a number of biochemical events culminating in the release of histamine-containing granules. In the present study, we investigated the possible involvement of tyrosine phosphorylation in signaling by the high-affinity IgE receptor (Fc epsilon RI). Cross-linking of Fc epsilon RI in rat basophilic leukemia cells (RBL-2H3) led to the phosphorylation of several proteins on tyrosine, the most prominent having a mass of 72 kDa. Tyrosine phosphorylation was rapid, detectable 1 min after stimulation, and correlated with both the time course and antigen dose for histamine release. Reversal of Fc epsilon RI cross-linking prevented continuation of the degranulation process and resulted in rapid loss of tyrosine phosphorylation. The receptor-mediated tyrosine phosphorylation was still induced in the absence of calcium in the medium. Depletion of protein kinase C with phorbol 12-myristate 13-acetate did not dramatically affect the tyrosine phosphorylation signal or the release of histamine. In contrast, the calcium ionophore A23187 induced histamine release in the absence of a perceptible increase in protein tyrosine phosphorylation. Thus, tyrosine phosphorylation is an early signal following Fc epsilon RI aggregation, independent of the exocytotic process itself. Taken together, our findings functionally link protein phosphorylation on tyrosine residues to Fc epsilon RI-mediated signal transduction leading to histamine release.
Comparison of the sevenless genes of Drosophila virilis and Drosophila melanogaster.
Authors: Michael W M , Bowtell D D , Rubin G M
Published: May 2006

The sevenless gene of Drosophila melanogaster encodes a transmembrane tyrosine kinase receptor required for normal eye development. We report here the isolation and DNA sequence analysis of the sevenless gene from Drosophila virilis. The predicted amino acid sequences of the sevenless proteins from these two species, which diverged approximately 60 million years ago, are compared.
Reductive beta-scission of the hydroperoxides of fatty acids and xenobiotics: role of alcohol-inducible cytochrome P-450.
Authors: Vaz A D , Roberts E S , Coon M J
Published: May 2006

As shown previously in this laboratory, purified rabbit liver microsomal cytochrome P-450 form 2 (P-450 IIB4) catalyzes the reductive cleavage of hydroperoxides to yield hydrocarbons and either aldehydes or ketones. We have proposed that lipid hydroperoxides are the physiological substrates for the cleavage reaction and have shown that with 13-hydroperoxy-9,11-octadecadienoic acid the formation of pentane is roughly equimolar with respect to the NADPH consumed. In the present study, the other product was isolated and identified as 13-oxo-9,11-tridecadienoic acid. Of particular interest, the alcohol-inducible form of liver microsomal cytochrome P-450 form 3a (P-450 IIE1) is the most active of the isozymes examined in the reductive beta-scission of the 13-hydroperoxide derived from linoleic acid and the 15-hydroperoxide derived from arachidonic acid as well as the model compounds cumyl hydroperoxide (alpha, alpha-dimethylbenzyl hydroperoxide) and t-butyl hydroperoxide. In general, the forms of P-450 with lower activity, as judged by the rate of NADPH oxidation in the reconstituted system, give less of the cleavage products (hydrocarbon and oxo compound) and catalyze direct reduction of the hydroperoxides to the corresponding hydroxy compounds. The occurrence of the reductive cleavage reaction in liver microsomal membranes was demonstrated, and microsomes from animals treated with ethanol or acetone (P-450 IIE1 inducers) or phenobarbital (a P-450 IIB4 inducer) were more active than those from untreated animals. We suggest that the alcohol-inducible P-450, in addition to its known deleterious effects in chemical toxicity and chemical carcinogenesis, may enhance the reductive cleavage of lipid hydroperoxides with a resultant loss in membrane integrity.
Functional and ultrastructural evidence for intracellular formation of major histocompatibility complex class II-peptide complexes during antigen processing.
Authors: Harding C V , Unanue E R , Slot J W , Schwartz A L , Geuze H J
Published: May 2006

Antigen presentation requires intracellular processing of native antigens to produce immunogenic peptides that bind to major histocompatibility complex class II (MHC-II) molecules. In functional studies of antigen processing by elicited peritoneal macrophages, MHC-II-peptide complexes were formed intracellularly. Immunogenic peptides were not released to bind surface MHC-II molecules. Ultrastructural studies employing immunogold staining in ultrathin cryosections of these macrophages showed large amounts of MHC-II molecules in intracellular sac-like vacuoles in the peripheral cytoplasm; most of these were negative for the lamp 1 lysosomal/endosomal membrane protein and cathepsin D. MHC-II molecules were also present in endosomes containing cathepsin D and lamp 1 as well as previously internalized gold-transferrin. The intracellular pool of MHC-II molecules was only slightly decreased by treatment with cycloheximide for 3 hr, indicating that it consisted mainly of endocytosed, recycling molecules, as opposed to nascent ones. These ultrastructural studies support the notion that there is endocytosis of MHC-II molecules into endocytic compartments, consistent with our earlier biochemical data. Furthermore, we have defined the distinct endocytic compartments that must mediate important functions in antigen processing, including the formation of MHC-II-peptide complexes.
Direct modulation of secretory chloride channels by arachidonic and other cis unsaturated fatty acids.
Authors: Hwang T C , Guggino S E , Guggino W B
Published: May 2006

The effect of fatty acids on Cl- channels and transepithelial Cl- secretion is investigated. Patch-clamp experiments show that arachidonic acid blocks Cl- channels in a dose-dependent manner. Kinetic analysis shows that the mean open time is decreased 10-fold with 25 microM arachidonic acid. There is a linear relationship between the reciprocal of mean open time and blocker concentration within the range of 1 to 25 microM. The reciprocal of mean blocked time does not change with arachidonic acid concentration. Other cis unsaturated fatty acids, including oleic, linoleic, and ricinoleic acids, demonstrate similar blocks. Trans unsaturated acids such as elaidic acid and saturated fatty acids, including stearic, palmitic, and myristic acids, do not inhibit the channel at 20 microM. Ricinoleic acid decreases short circuit current in T84 cells, a colonic carcinoma cell line that secretes Cl-. Our results suggest that the direct effect of arachidonic and other fatty acids on Cl- secretion is to block Cl- channel current.
Isolation of coordinately regulated genes that are expressed in discrete stages of B-cell development.
Authors: Yancopoulos G D , Oltz E M , Rathbun G , Berman J E , Smith R K , Lansford R D , Rothman P , Okada A , Lee G , Morrow M
Published: May 2006

We have utilized subtractive hybridization to isolate 16 distinct cDNA sequences representing genes expressed in pre-B-cell lines but not myeloma cell or fibroblast lines. These sequences represent RNA transcripts that vary in abundance in pre-B-cell lines from 0.001% to 0.05%. Five of these sequences were not related to any known genes. One was related to but distinct from known myosin regulatory light chain genes and another encoded a protein with lectin domains. Three represented previously identified genes encoding carbonic anhydrase type II, thymosin, and CD2; these genes were not previously known to be specifically expressed in early stages of B-cell development. Other isolated genes corresponded to pre-B-cell-specific or pre-B-cell/B cell-specific genes recently described by others. The isolated cDNA sequences may be divided into two general categories--those representing genes expressed only in the pre-B-cell stage of B-cell development and those expressed in both the pre-B-cell and B-cell stages. The in vivo expression patterns of the identified genes suggest that some function specifically in lymphocytes while others may have roles in additional lineages.
Coexpression of the platelet-derived growth factor (PDGF) B chain and the PDGF beta receptor in isolated pancreatic islet cells stimulates DNA synthesis.
Authors: Welsh M , Claesson-Welsh L , Hallberg A , Welsh N , Betsholtz C , Arkhammar P , Nilsson T , Heldin C H , Berggren P O
Published: May 2006

Suspensions rich in pancreatic beta cells were transfected by means of electroporation or by using the liposome technique with DNA constructs coding for the B chain of platelet-derived growth factor (PDGF) and the PDGF alpha and beta receptors to induce a mitotic response in this slowly replicating cell type. Transfection with the B-chain construct induced synthesis of the PDGF B-chain homodimer (PDGF-BB) as assessed by the presence of 125I-labeled PDGF-BB competing activity in the conditioned medium of the transfected islet cells. Moreover, islet cells transfected with the PDGF beta-receptor construct exhibited increased immunofluorescence staining with a PDGF beta-receptor antibody. These cells also displayed increased 125I-labeled PDGF-BB binding compared with control transfected cells. Cotransfection with the B-chain construct or the addition of 10% fetal bovine serum or purified PDGF all induced DNA synthesis in islet cells transfected with the PDGF beta-receptor construct. Islet cells transfected with the PDGF alpha-receptor construct did not respond with stimulation of [3H]thymidine incorporation to any of the PDGF isoforms (PDGF-AA, -AB, or -BB). Cotransfection of the PDGF alpha- and beta-receptor constructs resulted in a loss of the DNA synthesis response to PDGF. The beta cells exhibited elevated levels of [3H]inositol trisphosphate after transfection with the B-chain and beta-receptor constructs, indicating activation of phospholipase C. Islet cells transfected with the different receptor constructs exhibited different patterns of tyrosine phosphorylation upon ligand activation. The results demonstrate that pancreatic islet cells can be stimulated to increase DNA synthesis by transfection with the PDGF beta-receptor gene, whereas cotransfection with the alpha-receptor gene may attenuate the growth response.
Fusion of neurotransmitter vesicles with target membrane is calcium independent in a cell-free system.
Authors: Karli U O , Schäfer T , Burger M M
Published: May 2006

In adrenal chromaffin cells, stimulation of Ca2+ influx leads to the secretion of neurotransmitters. The intracellular Ca2+ target involved in the fusion of secretory vesicles with the plasma membrane (PM) is still not known. We have reconstituted this fusion in vitro by using chromaffin granules (CGs) and target membrane vesicles and a Ca2(+)-dependent phospholipase A2 (PLA2). Vesicle fusion is measured by a fluorescence dequenching assay with octadecyl rhodamine B used as the marker. CGs fuse with PM vesicles only in the presence of active PLA2. The kinetics of this fusion process depend on the amount of target PM added. Once fusion competence of PM vesicles is achieved by exposure to PLA2 (primed PM vesicles), it is conserved after removal of the PLA2 even in Ca2(+)-free buffer. The kinetics of fusion between these primed PM vesicles and CGs depend on the amount of PM and on the temperature. Further incubation of the PLA2-treated PM vesicles at 30 degrees C in the absence of calcium results in an enhanced fusion competence. During this incubation, the amount of free arachidonic acid liberated by PLA2 decreases, suggesting that during a second process arachidonic acid may be processed to the terminal fusogen. The final steps of secretion can thus be subdivided into a Ca2(+)-dependent and -independent process: first, a Ca2(+)-dependent activation of PLA2 liberating fatty acids from phospholipids and second, a Ca2(+)-independent processing to the terminal fusogen and subsequent Ca2(+)-independent fusion of the CGs with the PM.
Preferential infection of CD4+ memory T cells by human immunodeficiency virus type 1: evidence for a role in the selective T-cell functional defects observed in infected individuals.
Authors: Schnittman S M , Lane H C , Greenhouse J , Justement J S , Baseler M , Fauci A S
Published: May 2006

CD4+ T cells of patients with AIDS exhibit a qualitative defect in their ability to respond to soluble antigen while their responses to mitogens remain normal. CD4+ T cells can be broadly divided phenotypically into "naive" [CD45RA+ (2H4+)] and "memory" [CD29+ (4B4+) or CD45RO+ (UCHL1+)] cell subpopulations, which represent distinct maturation stages. To determine the human immunodeficiency virus type 1 (HIV-1) infectability of memory and naive CD4+ T-cell subsets in vitro and to determine the in vivo preference of HIV-1 in these subpopulations, we obtained highly purified CD4+ T-cell subsets from normal and HIV-1-infected individuals and studied them by viral cultivation, quantitative polymerase chain reaction, and functional assays. Polymerase chain reaction studies demonstrated that the memory cell subset of CD4+ T cells is preferentially infected (4- to 10-fold more than naive T cells) by HIV-1 in vitro, and these memory cells are the principal reservoir for HIV-1 within CD4+ T cells obtained from infected individuals. Functional abnormalities attributable to CD4+ T cells in HIV-infected individuals (failure to respond in vitro to soluble antigen or to anti-CD3 monoclonal antibodies) were shown to reside primarily within these memory cells. Thus, the present study suggests that the selective functional defects present in the memory CD4+ T-cell subset of HIV-infected individuals may be a direct result of the preferential infection and consequently greater viral burden within these cells.
Yeast K1 killer toxin forms ion channels in sensitive yeast spheroplasts and in artificial liposomes.
Authors: Martinac B , Zhu H , Kubalski A , Zhou X L , Culbertson M , Bussey H , Kung C
Published: May 2006

The patch-clamp technique was used to examine the plasma membranes of sensitive yeast spheroplasts exposed to partially purified killer toxin preparations. Asolectin liposomes in which the toxin was incorporated were also examined. Excised inside-out patches from these preparations often revealed at 118 pS conductance appearing in pairs. The current through this conductance flickered rapidly among three states: dwelling mostly at the unit-open state, less frequently at the two-unit-open state, and more rarely at the closed state. Membrane voltages from -80 to 80 mV had little influence on the opening probability. The current reversed near the equilibrium potential of K+ in asymmetric KCl solutions and also reversed near O mV at symmetric NaCl vs. KCl solutions. The two levels of the conductance were likely due to the toxin protein, as treatment of spheroplasts or liposomes with extracellular protein preparations from isogenic yeasts deleted for the toxin gene gave no such conductance levels. These results show that in vivo the killer-toxin fraction can form a cation channel that seldom closes regardless of membrane voltage. We suggest that this channel causes the death of sensitive yeast cells.
Gene activation is required for developmentally programmed cell death.
Authors: Schwartz L M , Kosz L , Kay B K
Published: May 2006

The intersegmental muscles of the tobacco hawkmoth Manduca sexta die during the 36-hr period after metamorphosis. The trigger for cell death is a fall in the ecdysteroid titer. Commitment of the intersegmental muscles to degenerate involves selective repression and activation of ecdysteroid-responsive genes. When the pattern of gene expression is altered after injection of either 20-hydroxyecdysone or actinomycin D, the muscles persist. cDNA clones have been isolated for four genes that become abundantly expressed coincident with the commitment to degenerate. The data presented here indicate that programmed cell death is not due to the cessation of macromolecular synthesis in condemned cells but rather is due to the activation of a differentiative pathway.
Inhibition of Na-K-C1 cotransport in Ehrlich ascites cells by antiserum against purified proteins of the cotransporter.
Authors: Dunham P B , Jessen F , Hoffmann E K
Published: May 2006

Two proteins were purified earlier from solubilized membranes of Ehrlich ascites cells by using a bumetanide-Sepharose affinity column. These proteins were proposed to be constituents of the Na-K-C1 cotransporter. However, the specificity of binding of bumetanide to the cotransporter was insufficient evidence for this proposal. We now have direct evidence that the purified protein contains components of the cotransporter. Antiserum raised against the bumetanide-binding proteins strongly inhibits Na-K-C1 cotransport measured by two independent methods. Cotransport was induced by hypertonic challenge and was measured as the bumetanide-sensitive portion of unidirectional C1 influx and as regulatory cell volume increase. In both assays, cotransport was strongly inhibited by the antiserum. Fab fragments of the antibodies inhibited cotransport to a similar extent.
Identification of the albino mutation of mouse tyrosinase by analysis of an in vitro revertant.
Authors: Jackson I J , Bennett D C
Published: May 2006

From within an albino melanocyte line grown in vitro we identified and cloned cells that apparently had reverted to wild type. We sequenced a part of the tyrosinase gene, encompassing a candidate mutation, from wild-type, albino, and revertant cell DNAs. The revertant cells contain, on one chromosome, a perfect base reversion to the wild-type sequence of this candidate mutation, proving that this is the sole defect in the tyrosinase gene of albino mutant mice. The revertant cells readily regain the albino phenotype after freezing and thawing. Taking advantage of a Dde I restriction site created by the albino mutation, we demonstrated that the regained phenotype is due to allele loss involving the wild-type chromosome. The Dde I site also allowed us to show that all inbred albino mice carry the same mutation and so must be derived from the same progenitor.
CD3 zeta subunit can substitute for the gamma subunit of Fc epsilon receptor type I in assembly and functional expression of the high-affinity IgE receptor: evidence for interreceptor complementation.
Authors: Howard F D , Rodewald H R , Kinet J P , Reinherz E L
Published: May 2006

The high-affinity receptor for IgE (Fc epsilon RI) is a four-subunit structure consisting of three distinct polypeptides: the IgE-binding alpha chain, the four-fold membrane-spanning beta chain, and the disulfide-linked gamma-gamma homodimer. cDNAs encoding each subunit have previously been isolated. Here we show that microinjection of Xenopus oocytes with a mixture of in vitro transcribed RNAs encoding each subunit results in expression of IgE receptors at the oocyte surface as detected by binding of IgE or anti-Fc epsilon RI alpha subunit monoclonal antibody to intact oocytes. Surface expression of Fc epsilon RI requires injection of all three subunits (alpha, beta, and gamma) RNAs. In particular, omission of Fc epsilon RI gamma RNA from the mixtures abolishes surface binding of either IgE or anti-Fc epsilon RI alpha monoclonal antibody to microinjected oocytes. However, addition of CD3 zeta RNA to Fc epsilon RI alpha and Fc epsilon RI beta RNAs restores IgE receptor surface expression when this combination is microinjected into oocytes. Metabolic labeling and immunoprecipitation of oocyte microinjected with a mixture of CD3 zeta plus Fc epsilon RI alpha and Fc epsilon RI beta RNAs reveals a noncovalent association between the CD3 zeta-zeta disulfide-linked homodimer and Fc epsilon RI alpha-beta. These results provide direct evidence for the functional relatedness of CD3 zeta and Fc epsilon RI gamma.
PD134308, a selective antagonist of cholecystokinin type B receptor, enhances the analgesic effect of morphine and synergistically interacts with intrathecal galanin to depress spinal nociceptive reflexes.
Authors: Wiesenfeld-Hallin Z , Xu X J , Hughes J , Horwell D C , Hökfelt T
Published: May 2006

The effects of systemic PD134308 [0.1-3 mg/kg; an antagonist of the cholecystokinin (CCK) type B receptor], morphine, and intrathecal (i.t.) galanin (GAL) on the excitability of the spinal nociceptive flexor reflex and in the hot plate test were examined in rats. PD134308 caused a weak naloxone-reversible depression of the flexor reflex and a moderate antinociceptive effect in the hot plate test. However, PD134308 significantly potentiated the antinociceptive effect of morphine as well as its depressive effect on the flexor reflex. PD134308 and i.t. GAL synergistically depressed the flexor reflex, an effect that was reversed by naloxone. Finally, the magnitude and duration of the depression of the flexor reflex by morphine were synergistically increased by coadministering PD134308 and GAL i.t. The results demonstrated that a CCK antagonist directed to the central CCK type B receptor potentiates the analgesic effects of opioids and nonopioid drugs at the spinal level, thus supporting the notion that CCK in the central nervous system may be an endogenous, physiological opioid antagonist.
Interactions of neural networks: models for distraction and concentration.
Authors: Wang L P , Ross J
Published: May 2006

We present a model of neural group interactions, which are projections from one neural network (network B) of McCulloch-Pitts neurons connected via a Hebbian rule, to another network (network A) of the same structure. We first consider the case in which the projecting network B is in a pattern different from the initial attracting state of network A. A critical projecting strength lambda c is found such that for lambda below this value there exists a noise threshold sigma lambda corresponding to each lambda. For the case where lambda less than lambda c and the noise level sigma less than sigma lambda, there are two possible retrievals, with different probabilities: the initial attracting state of network A and the projecting pattern. If lambda less than lambda c and sigma greater than sigma lambda, stable states of network A disappear. In the case lambda greater than lambda c, network A is pulled out of its initial basin of attraction and into that of the projecting pattern. This analysis provides a model for distraction. Second-order interactions reduce the distraction. When the projecting network B is in the same pattern as the initial attracting state of network A, the projection acts as an external reinforcement, which enables network A to retrieve in highly noisy conditions. Sharp noise thresholds for nonzero retrievals are shown to be eliminated by the projection. Higher-order connectivity improves the retrieval ability of the network. The second case serves as a model of concentration. We discuss the model of distraction and concentration (i) in connection with common experience of expectation of recognition and (ii) in connection with recent T-maze experiments on infant rats; finally, we suggest a refined version of the Bruner-Potter experiment to test our prediction of the disappearance of hysteresis.
Failure to reverse long-term potentiation by coupling sustained presynaptic activity and N-methyl-D-aspartate receptor blockade.
Authors: Goldman R S , Chavez-Noriega L E , Stevens C F
Published: May 2006

The proposal that long-term potentiation (LTP) is a mechanism underlying memory in the mammalian brain rests on a number of properties of LTP that parallel characteristics of memory defined by behavior. A prominent feature of behaviorally defined memory is its reversibility. LTP is induced at synapses that correlate in their activity, and the signal for induction is calcium influx through N-methyl-D-aspartate (NMDA) receptor channels. By analogy to the reversibility of behaviorially defined memory, uncorrelated synaptic activity might be expected to reverse LTP, an anti-Hebbian effect called long-term depression, which has only recently been described in the hippocampus [Stanton, P. K. & Sejnowski, T. J. (1989) Nature (London) 339, 215-218]. Because the extent to which synaptic activity is correlated is represented by postsynaptic calcium concentrations, it seemed likely to us that long-term depression is related to the failure of calcium to pass through the NMDA channel. One way to block the calcium influx that signals correlated synaptic activity is with the NMDA receptor antagonist D-(-)-2-amino-5-phosphonovalerate. We performed a series of experiments in rat hippocampal slices designed to estimate the amount of synaptic depression per afferent test pulse under these conditions. Schaffer collateral-commissural afferents to field CA1 were repetitively stimulated in the presence of 2-amino-5-phosphonovalerate. No enduring synaptic depression nor reversal of LTP could be detected. We conclude that some other mechanism underlies long-term depression in the hippocampus.
The effect of excess beta-chain synthesis on cell-surface expression of allele-mismatched class II heterodimers in vivo.
Authors: Gilfillan S , Aiso S , Michie S A , McDevitt H O
Published: May 2006

We have recently described 12 lines of H-2s/s mice carrying from 1 to 65 copies of an A beta k transgene. The transgene was coexpressed with the endogenous allele, and A beta K mRNA expression correlated well with transgene copy number. Overexpression of the transgene was associated with a variety of defects, including a significant reduction in I-A cell-surface expression. In this paper, we assess the effect of increased levels of A beta k mRNA synthesis on I-A cell-surface expression in these mice. Crossing representatives from several lines of A beta k mice to A alpha k transgenic mice demonstrated that the A beta k mRNA was translated and expressed at high levels on the cell surface in association with A alpha k. In H-2s/s (A alpha s /A beta s) mice carrying greater than 10 copies of the A beta k transgene, excess A beta k mRNA and protein synthesis did drive cell-surface expression of the less favored A alpha s/A beta k heterodimers. However, the highest levels of A beta k detected on the cell surface were only 50-70% of those observed in [B10.A(4R) x nontransgenic]F1 controls. Maximum levels of A alpha s/A beta k cell-surface expression were accompanied by a significant reduction in A alpha s/A beta s expression. Unpaired and improperly paired complexes were not detected intracellularly and appeared to be degraded quite rapidly. Thus, only a fraction of the chains competing for pairing reached the cell surface under conditions of asymmetric chain synthesis in these mice. This markedly reduced total Ia cell-surface levels in mice carrying greater than 10 copies of the A beta k transgene.
Protein dissociation from GRP78 and secretion are blocked by depletion of cellular ATP levels.
Authors: Dorner A J , Wasley L C , Kaufman R J
Published: May 2006

Secretory proteins expressed in Chinese hamster ovary (CHO) cells interact to various degrees with glucose-regulated protein 78 (GRP78), a resident protein of the endoplasmic reticulum. von Willebrand factor (vWF) and wild-type tissue plasminogen activator (tPA) are efficiently secreted and exhibit a slight transient association with GRP78. Factor VIII and unglycosylated tPA are inefficiently secreted and display a more stable association with GRP78. We have studied the effect of ATP depletion mediated by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment on GRP78 association and secretion of factor VIII and vWF that are coexpressed in CHO cells. Low concentrations of CCCP in the medium prevented disassociation of factor VIII from GRP78 and blocked its secretion. In the same cells, higher concentrations of CCCP were required to block secretion of vWF. Thus, the block to factor VIII secretion at low CCCP concentrations did not result from a general defect in secretion. Secretion of unglycosylated tPA but not wild-type tPA from CHO cells was also blocked by low concentrations of CCCP. The increased sensitivity to CCCP concentration observed for secretion of factor VIII and unglycosylated tPA compared to wild-type tPA and vWF correlates with their degree of interaction with GRP78. In vivo, dissociation from GRP78 may be a primary ATP-dependent step in transport from the endoplasmic reticulum. ATP requirements for secretion of various proteins may vary.
Overexpression of c-src enhances beta-adrenergic-induced cAMP accumulation.
Authors: Bushman W A , Wilson L K , Luttrell D K , Moyers J S , Parsons S J
Published: May 2006

During our investigations into the physiological role of c-src tyrosine kinase in normal cells, we found that clonal transfectants of C3H10T1/2 murine fibroblasts overexpressing chicken c-src exhibited strikingly elevated levels of cAMP accumulation in response to adrenergic stimulation as compared to control cells. Enhanced cAMP accumulations were detected when cells were treated with the beta-agonists, epinephrine, isoproterenol, or terbutaline and were blocked by treatment with the beta-specific antagonist propranolol, indicating action through beta-adrenergic receptors. The hyperresponsiveness was not observed in cells overexpressing kinase-defective c-src. No differences in basal levels of cAMP, agonist concentration dependence, or kinetics of cAMP accumulation were detected between cells containing elevated levels of wild-type or kinase-defective c-src protein and control cells. To determine if the degree of c-src overexpression could influence the response, multiple clones, transfected with DNA encoding genes for wild-type or kinase-defective c-src plus neomycin resistance or neomycin resistance alone, were derived in parallel and assayed for the amounts of c-src protein produced and the levels of cAMP accumulated in response to epinephrine. Only clones with abundant wild-type c-src protein (greater than 10-fold above endogenous) exhibited enhanced cAMP accumulation, averaging 3.3-fold above control cells. We conclude, therefore, that the enhanced degree of cAMP accumulation in cells overexpressing c-src is dependent upon activation of beta-adrenergic receptors and upon a threshold level of pp60c-src that retains full tyrosine kinase activity.
Cloning of the cDNA for human 12-lipoxygenase.
Authors: Izumi T , Hoshiko S , Rådmark O , Samuelsson B
Published: May 2006

A full-length cDNA clone encoding 12-lipoxygenase (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme.
Antibody caging of a nuclear-targeting signal.
Authors: Halleck M S , Rechsteiner M
Published: May 2006

We have developed a technique for reversibly masking a peptide-targeting signal. A fluoresceinated derivative of the simian virus 40 large tumor antigen nuclear-targeting signal was synthesized and cross-linked to bovine serum albumin. The conjugated protein was efficiently transported into rat liver nuclei unless the peptide-targeting signal was sterically hindered by binding of an anti-fluorescein antibody. Addition of free 5-aminofluorescein competed for antibody binding and rapidly restored nuclear accumulation of the derivatized bovine serum albumin. General use of hapten derivatization and anti-hapten antibodies for caging portions of macromolecular surfaces can be extended to a variety of proteins, including antibodies themselves.
Regulation of human renin expression in chorion cell primary cultures.
Authors: Duncan K G , Haidar M A , Baxter J D , Reudelhuber T L
Published: May 2006

The human renin gene is expressed in the kidney, placenta, and several other sites. The release of renin or its precursor, prorenin, can be affected by several regulatory agents. In this study, primary cultures of human placental cells were used to examine the regulation of prorenin release and renin mRNA levels and of the transfected human renin promotor linked to chloramphenicol acetyltransferase reporter sequences. Treatment of the cultures with a calcium ionophore alone, calcium ionophore plus forskolin (that activates adenylate cyclase), or forskolin plus a phorbol ester increased prorenin release and renin mRNA levels 1.3- to 6-fold, but several classes of steroids did not affect prorenin secretion or renin RNA levels. The transfected renin promoter (584 or 100 base pairs of 5'-flanking DNA) initiated at the correct start site in these cells and forskolin increased its expression 2.5- to 4-fold. Constructs containing renin 5'-flanking DNA linked to a heterologous promoter cotransfected into HeLa cells with either glucocorticoid or estrogen receptor expression vectors were not regulated by dexamethasone or 17 beta-estradiol. These results suggest that (i) the first 584 base pairs of the renin gene 5'-flanking DNA do not contain functional glucocorticoid or estrogen response elements, (ii) placental prorenin release and renin mRNA are regulated by calcium ion and by the combinations of cAMP with either C kinase or calcium ion, and (iii) the first 100 base pairs of the human renin 5'-flanking DNA direct accurate initiation of transcription and can be regulated by cAMP. Thus, some control of renin release in the placenta (and by inference in other tissues) occurs via transcriptional influences on its promoter.
Levels of mRNA for a putative kainate receptor are affected by seizures.
Authors: Gall C , Sumikawa K , Lynch G
Published: May 2006

In situ hybridization and RNA blot-hybridization techniques were used (i) to examine the regional distribution of mRNA for a putative kainate receptor in adult rat brain and ii) to test the possibility that seizures affect expression of the receptor gene. The highest densities of hybridization were distributed within hippocampal pyramidal and granule cells, medial habenula, Purkinje cells and the molecular layer of cerebellum, and olfactory bulb. Recurrent limbic seizures caused a massive, delayed, and reversible reduction in levels of the kainate receptor mRNA in dentate gyrus; lesser decreases were found in pyramidal cell fields of hippocampus and superficial cortex. These findings provide evidence that unusual patterns of physiological activity can alter genomic expression for a subclass of glutamate receptors in brain.