bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of type 1 human immunodeficiency virus replication by a tat antagonist to which the virus remains sensitive after prolonged exposure in vitro.
Authors: Hsu M C , Dhingra U , Earley J V , Holly M , Keith D , Nalin C M , Richou A R , Schutt A D , Tam S Y , Potash M J
Published: May 2006

The transactivator of transcription, Tat, of human immunodeficiency virus type 1 (HIV-1) is required for viral replication. Inhibition of Tat function could have the potential to keep integrated provirus in dormancy. In the presence of Tat, Ro 24-7429, an analog of Ro 5-3335, inhibited expression of indicator genes controlled by the HIV-1 long terminal repeat promoter in transient transfection assays and in a constitutive cell line at noncytotoxic concentrations. Reduction of steady-state mRNA of the indicator gene by the compound correlated with reduction of the gene product in the constitutive cell line. Ro 24-7429 has broad activity against several strains of HIV-1 in different cell lines, peripheral blood lymphocytes, and macrophages (IC90 = 1-3 microM). Importantly, Ro 24-7429 inhibited viral replication in both acute and chronic infection in vitro, a characteristic expected of a Tat antagonist and not shared by viral reverse transcriptase inhibitors. Consistent with this, the compound reduced cell-associated viral RNA and proteins and partially restored cell-surface CD4 in chronically infected cells. After 2 years of continued weekly passage of the virus in fresh CEM cells grown in the presence of the compound at 1 or 10 microM, the virus did not develop resistance to the drug. These results indicate that the compound's action might involve a cellular factor.
Treatment of chronic-relapsing experimental autoimmune encephalomyelitis with the synthetic immunomodulator linomide (quinoline-3-carboxamide).
Authors: Karussis D M , Lehmann D , Slavin S , Vourka-Karussis U , Mizrachi-Koll R , Ovadia H , Kalland T , Abramsky O
Published: May 2006

Linomide is a synthetic immunomodulator that enhances natural killer cell activity and significantly activates several lymphocytic cell subpopulations in both experimental animals and humans. In this study we examined the effect of linomide (80 mg per kg per day in drinking water) on mice with chronic-relapsing experimental autoimmune encephalomyelitis (CR-EAE), a T-cell-mediated organ-specific autoimmune disease that resembles human multiple sclerosis. None of the mice (n = 17) that were treated with linomide from day 7 after disease induction developed any clinical or histopathological signs of CR-EAE, as compared to 19 of 20 untreated controls that were severely paralyzed and had extensive demyelinating lesions in the central nervous system. Linomide-treated animals were also resistant to an induced attack by a booster injection with a murine spinal cord homogenate. When administered to mice exhibiting severe clinical signs of paralysis, linomide inhibited both spontaneous and induced relapses. Linomide treatment protected mice from passively induced CR-EAE as well, when given from the day of injection with myelin-basic-protein-specific lymphocytes. Lymphocytes obtained from linomide-treated mice had a reduced in vitro proliferative response to the myelin basic protein and to the tuberculin purified protein derivative, whereas the mitogenic response to concanavalin A was not affected. Natural killer cell and lymphokine-activated killer cell activities were enhanced. These results suggest that linomide regulates autoimmunity in the absence of systemic immunosuppression. Since linomide is very well tolerated in experimental animals and humans, it might be used in the treatment of multiple sclerosis.
Kinetic control of Ca(II) signaling: tuning the ion dissociation rates of EF-hand Ca(II) binding sites.
Authors: Renner M , Danielson M A , Falke J J
Published: May 2006

EF-hand Ca(II) binding sites share a conserved architecture and are prevalent in Ca(II) signaling pathways. The ion binding kinetics of these sites are carefully tuned to provide the physiologically appropriate activation and inactivation time scales. Here we examine kinetic tuning by the side chain at the ninth position of the EF-loop. A model is proposed in which both the size and charge of the side chain contribute to kinetic tuning. To test this model, the ninth loop position of the EF-hand-like site in the Escherichia coli D-galactose binding protein has been engineered and the Tb(III) dissociation kinetics of the resulting sites have been analyzed. Substitutions at this position are observed to generate up to 10(4)-fold changes in Tb(III) dissociation rates, with little effect on Tb(III) affinity. Furthermore, the observed pattern of rate changes confirm the model's predictions; long side chains at the ninth loop position yield slow dissociation kinetics as predicted for a steric block, whereas acidic side chains yield slow dissociation kinetics as expected for an electrostatic barrier.
Stimulus-induced dissociation of alpha subunits of heterotrimeric GTP-binding proteins from the cytoskeleton of human neutrophils.
Authors: Särndahl E , Bokoch G M , Stendahl O , Andersson T
Published: May 2006

Previous studies on the mechanism responsible for terminating the generation of second messengers induced by chemotactic factor-receptor complexes have, on one hand, suggested a direct role of a GTP-binding protein(s) (G protein), and, on the other hand, proposed that there is a lateral segregation of the ligand-receptor complexes into G protein-depleted domains of the plasma membrane. In the present investigation, which addresses these apparently contradictory findings, we found that a substantial part of the alpha subunits of the Gn protein (Gn alpha) in unstimulated neutrophils were associated with a cytoskeletal fraction and that release of these subunits occurred upon stimulation with the chemotactic factor fMet-Leu-Phe. An identical Gn alpha release could also be induced by direct activation of G proteins with guanosine 5'-[gamma-thio]triphosphate or AIF4-. In contrast, the alpha subunits of the stimulatory G protein (Gs alpha) also found associated with the cytoskeletal fraction of unstimulated cells were not released by fMet-Leu-Phe stimulation. However, they were effectively released by direct G-protein activation with guanosine 5'-[gamma-thio]triphosphate. In addition, inhibition of the fMet-Leu-Phe-stimulated modulation of the actin network by pertussis toxin did not affect the fMet-Leu-Phe-induced release of Gn alpha from the cytoskeletal fraction. These observations indicate that fMet-Leu-Phe-induced activation of neutrophils involves a specific dissociation of Gn alpha from the cytoskeleton and that this release is not a consequence of the well-known effect of fMet-Leu-Phe on the cytoskeleton of neutrophils. The present data contribute ideas concerning the transducing properties of G proteins in cellular signaling and seem to reconcile the apparently contradictory concepts of how the cytoskeleton participates in the termination of the chemotactic-factor-induced generation of second messengers in human neutrophils.
Molecular characterization of a short interspersed repetitive element from tobacco that exhibits sequence homology to specific tRNAs.
Authors: Yoshioka Y , Matsumoto S , Kojima S , Ohshima K , Okada N , Machida Y
Published: May 2006

We have characterized a family of tRNA-derived short interspersed repetitive elements (SINEs) in the tobacco genome. Members of this family of SINEs, designated TS, have a composite structure and include a region structurally similar to a rabbit tRNA(Lys), a tRNA-unrelated region, and a TTG repeat of variable length at the 3' end. Southern blot hybridization, together with a search of the GenBank data base, showed that various plants belonging to the families Solanaceae and Convolvulaceae contain sequences homologous to the TS family in the introns and flanking regions of many genes, whereas Arabidopsis in the family Cruciferae and several species of monocoytledonous plants do not. The TS family is widely involved in structural and genetic variations in the genomes of many plants that belong to the order Tubiflorae. All of nine sequences identified in a data base search are truncated at their 5' regions and lack the tRNA-related region of the TS family. We characterized the entire sequence of the members of the TS family and found that this family can be categorized as a member of a group of SINEs with a tRNA(Lys)-like structure, as can several animal SINEs. The TS family can be divided into two major subfamilies by analysis of diagnostic positions, and one of the subfamilies is clearly younger than the other. Amplification of many copies of the full sequence of the younger subfamily occurred during the recent evolution of the tobacco lineage. We also discuss mechanisms that could be involved in the generation of SINEs in animals and also in plants.
Local sources of retinoic acid coincide with retinoid-mediated transgene activity during embryonic development.
Authors: Colbert M C , Linney E , LaMantia A S
Published: May 2006

We have assessed whether retinoic acid (RA) comes from local sources or is available widely to activate gene expression in embryos. We used an RA-responsive indicator cell line, L-C2A5, to localize RA sources. In these cells, an RA-sensitive promoter/lacZ reporter construct used previously by us to produce indicator transgenic mice is induced globally by RA in medium or locally by RA released at physiological concentrations (1 nM) from AG-1X2 resin beads. Furthermore, the cells are differentially responsive to the 9-cis and all-trans isomers of RA at low concentrations. Indicator transgenic mice with the same promoter/reporter construct were used to identify regions of RA-mediated gene activation. There are distinct domains of lacZ expression in the cervical and lumbar spinal cords of embryonic indicator mice. This pattern might reflect localized RA sources or restricted spatial and temporal expression of RA receptors, binding proteins, or other factors. To resolve this issue we compared the pattern of transgene activation in indicator cell monolayers cocultured with normal embryonic spinal cords with that in transgenic spinal cords. The explants induced reporter gene expression in L-C2A5 monolayers in a pattern identical to that in transgenic mice: alar regions of the cervical and lumbar cord were positive whereas those in the thoracic and sacral regions were not. We conclude that restricted sources of RA in the developing spinal cord mediate the local activation of RA-inducible genes. Thus, region-specific gene activation in embryos can be mediated by precisely localized sources of inductive molecules like RA.
Heterologous expression of mating-type genes in filamentous fungi.
Authors: Arnaise S , Zickler D , Glass N L
Published: May 2006

Podospora anserina and Neurospora crassa, two filamentous heterothallic ascomycetes, have a single mating-type locus with two alternate forms called mat+ and mat- and A and a, respectively. Mating type controls entry into the sexual cycle, events subsequent to fertilization, and, in N. crassa, prevents the formation of mixed mating-type heterokaryons. The mating types of these two organisms display similarity in their DNA structure and in the encoded polypeptides involved in fertilization. Here we show that this molecular similarity reflects a functional homology with respect to mating identity. Transformation experiments show that the N. crassa mating-type genes can provide the fertilization functions in P. anserina strains devoid of mating specificity as well as in mat+ and mat- strains. Reciprocally, the introduction of P. anserina mating-type genes confers mating activity in N. crassa. Functional identity between the mating types is not observed for vegetative incompatibility or for post-fertilization events such as meiosis and ascosporogenesis.
A soluble divalent class I major histocompatibility complex molecule inhibits alloreactive T cells at nanomolar concentrations.
Authors: Dal Porto J , Johansen T E , Catipović B , Parfiit D J , Tuveson D , Gether U , Kozlowski S , Fearon D T , Schneck J P
Published: May 2006

Genetically engineered or chemically purified soluble monovalent major histocompatibility complex (MHC) molecules, which have previously been used to study T cells, have not blocked cytotoxic T-cell responses. Here we describe a genetically engineered divalent class I MHC molecule which inhibits lysis of target cells by alloreactive cytotoxic T cells. This protein, H-2Kb/IgG, was generated as a fusion protein between the extracellular domains of a murine class I polypeptide, H-2Kb, and an immunoglobulin heavy chain polypeptide. The chimeric protein has serological and biochemical characteristics of both the MHC and IgG polypeptides. Nanomolar concentrations of H-2Kb/IgG inhibited lysis of H-2Kb-expressing target cells not only by alloreactive H-2Kb-specific T-cell clones but also by alloreactive H-2Kb-specific primary T-cell cultures. A direct binding assay showed high-affinity binding between the H-2Kb/IgG molecule and an H-2Kb-specific alloreactive T-cell clone. Unlabeled H-2Kb/IgG displaced 125I-labeled H-2Kb/IgG from T cells with an IC50 of 1.2 nM.
The Saccharomyces cerevisiae PRP21 gene product is an integral component of the prespliceosome.
Authors: Arenas J E , Abelson J N
Published: May 2006

In Saccharomyces cerevisiae, the prp21 mutation causes accumulation of unspliced pre-mRNA at the nonpermissive temperature. We have cloned the PRP21 gene by complementation of its temperature-sensitive phenotype and found it to be the same as SPP91, an extragenic suppressor of the prp9 mutation previously studied in vivo by Chapon and Legrain [Chapon, C. & Legrain, P. (1992) EMBO J. 11, 3279-3288]. We have analyzed the effects of the prp21 mutation on splicing in vitro and have found that PRP21 is a splicing factor required for prespliceosome assembly. We also have analyzed the interaction of PRP21 with splicing complexes using anti-PRP21 antibodies and found that the RNA components of the prespliceosome--U1 and U2 small nuclear RNA (snRNA) particles and pre-mRNA--are specifically coimmunoprecipitated under splicing conditions in the presence of 0.2 M KCl. At higher KCl concentrations, U1 snRNP dissociates from splicing complexes; nevertheless, U2 snRNA and pre-mRNA are still efficiently immunoprecipitated. Immunoprecipitation of both U1 and U2 snRNA as well as pre-mRNA is ATP-dependent and requires a pre-mRNA capable of supporting prespliceosome assembly. Analysis of the unbound complexes in native gels confirmed that prespliceosomes are specifically immunoprecipitated by anti-PRP21 antibodies. These results demonstrate that PRP21 is an integral component of the prespliceosome and establishes a stable interaction with U2 snRNP and/or pre-mRNA in that complex.
Crystal structures of native and inhibited forms of human cathepsin D: implications for lysosomal targeting and drug design.
Authors: Baldwin E T , Bhat T N , Gulnik S , Hosur M V , Sowder R C , Cachau R E , Collins J , Silva A M , Erickson J W
Published: May 2006

Cathepsin D (EC 3.4.23.5) is a lysosomal protease suspected to play important roles in protein catabolism, antigen processing, degenerative diseases, and breast cancer progression. Determination of the crystal structures of cathepsin D and a complex with pepstatin at 2.5 A resolution provides insights into inhibitor binding and lysosomal targeting for this two-chain, N-glycosylated aspartic protease. Comparison with the structures of a complex of pepstatin bound to rhizopuspepsin and with a human renin-inhibitor complex revealed differences in subsite structures and inhibitor-enzyme interactions that are consistent with affinity differences and structure-activity relationships and suggest strategies for fine-tuning the specificity of cathepsin D inhibitors. Mutagenesis studies have identified a phosphotransferase recognition region that is required for oligosaccharide phosphorylation but is 32 A distant from the N-domain glycosylation site at Asn-70. Electron density for the crystal structure of cathepsin D indicated the presence of an N-linked oligosaccharide that extends from Asn-70 toward Lys-203, which is a key component of the phosphotransferase recognition region, and thus provides a structural explanation for how the phosphotransferase can recognize apparently distant sites on the protein surface.
cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization.
Authors: Shetty K T , Link W T , Pant H C
Published: May 2006

A protein kinase that phosphorylates a specific KSP sequence [K(S/T)PXK], which is abundant in high molecular weight neurofilament (NF) proteins, was identified and isolated from rat spinal cord. Characterization of this enzyme activity revealed a close relationship with p34cdc2 kinase with respect to its molecular mass (32.5 kDa by SDS/PAGE) and substrate specificities. It could phosphorylate a synthetic peptide analog of the simian virus 40 large tumor antigen, reportedly a specific substrate for p34cdc2 kinase. Histone (H1) and peptide analogs of the KSP sequence present in the C-terminal end of rat and mouse neurofilament proteins were phosphorylated. This kinase did not phosphorylate alpha-casein and peptide substrates of other known second messenger-dependent or -independent kinases. Dephosphorylated rat NF protein NF-H was strongly phosphorylated by the purified enzyme; NF proteins NF-M and native NF-H, but not NF-L, were slightly phosphorylated. Studies on synthetic peptide analogs of KSP repeats with substitution of specific residues, known to be present in the C-terminal regions of NF-H, revealed a consensus sequence of X(S/T)PXK, characteristic of the p34cdc2 kinase substrate. On Western blots, the enzyme was immunoreactive with antibody against the C-terminal end of cdc2 kinase (mouse) and neuronal cdc2-like kinase from rat but not with an antibody against the conserved PSTAIRE region of the p34cdc2 kinase. The antibody against the C-terminal end of cdc2 kinase could immunoprecipitate (immunodeplete) the purified kinase activity. Since the adult nervous system is composed primarily of postmitotic cells, the present observations indicate a nonmitotic role for this cdc2-like kinase activity. The effective phosphorylation of NF-H by this kinase suggests a function in axonal structure.
The functional form of the erythropoietin receptor is a 78-kDa protein: correlation with cell surface expression, endocytosis, and phosphorylation.
Authors: Sawyer S T , Hankins W D
Published: May 2006

An abundant 70- to 78-kDa form of the erythropoietin receptor (EPOR) was observed in HC-D57 murine erythroleukemia cells deprived of erythropoietin (EPO). In contrast to the 64- and 66-kDa EPOR proteins, these high molecular mass forms of EPOR (hmm-EPOR) correlated well with the number of binding sites and endocytosis of EPO. The hypothesis that hmm-EPOR are more highly glycosylated forms of the EPOR, appear on the cell surface, and represent at least one component of the biologically active EPOR was tested. Consistent findings were as follows. (i) Only hmm-EPOR increased following withdrawal of EPO from HC-D57 cells, correlating with a 10-fold increase in binding of 125I-labeled EPO. In addition, the EPO-dependent downregulation of 125I-EPO binding and disappearance of hmm-EPOR occurred in parallel while the amount of 66-kDa EPOR did not change. (ii) The 78-kDa EPOR was detected in COS cells expressing EPOR cDNA. (iii) Probing of the intact surface of these cells with anti-NH2-terminal antibody recovered only the 78-kDa EPOR. (iv) Enzymatic deglycosylation and dephosphorylation showed that hmm-EPOR apparently resulted from additional N-linked glycosylation of a 62-kDa EPOR. (v) The hmm-EPOR turnover in HC-D57 cells was accelerated 12-fold in the presence of EPO (half-life changed from 3 hr to 15 min). (vi) Anti-phosphotyrosine antiserum detected an EPO-dependent phosphorylation of the 78-kDa EPOR. The kinetics of tyrosine phosphorylation of a 97-kDa protein correlated with the occupancy and internalization of hmm-EPOR. In summary, we suggest that the 78-kDa EPOR is directly involved in the initial biological actions of EPO.
PEBP2/PEA2 represents a family of transcription factors homologous to the products of the Drosophila runt gene and the human AML1 gene.
Authors: Ogawa E , Maruyama M , Kagoshima H , Inuzuka M , Lu J , Satake M , Shigesada K , Ito Y
Published: May 2006

cDNAs representing the alpha subunit of polyomavirus enhancer binding protein 2 (PEBP2; also called PEA2) were isolated. The products of the cDNAs are highly homologous to that of Drosophila segmentation gene runt (run) for an N-proximal 128-amino acid region showing 66% identity. The run homology region encompasses the domain capable of binding to a specific nucleotide sequence motif and of dimerizing with the companion beta subunit. The human AML1 gene related to t(8;21) acute myeloid leukemia also had a run homology region. Together with the beta subunit, which increases the affinity of the alpha subunit to DNA without binding to DNA by itself, PEBP2 represents a newly discovered family of transcription factor. The major species of PEBP2 alpha mRNA was expressed in T-cell lines but not in B-cell lines tested. Evidence indicated that PEBP2 functions as a transcriptional activator and is involved in regulation of T-cell-specific gene expression.
Allele-specific B pocket transplant in class I major histocompatibility complex protein changes requirement for anchor residue at P2 of peptide.
Authors: Colbert R A , Rowland-Jones S L , McMichael A J , Frelinger J A
Published: May 2006

To investigate the role of an anchoring pocket in allele-specific peptide presentation by a major histocompatibility complex class I molecule, we "transplanted" a B pocket from HLA-A*0201 into HLA-B*2705 by site-directed mutagenesis. The resulting protein, designated B27.A2B, binds a different set of endogenous peptides than B*2705 as evidenced by complete loss of allorecognition as well as restored expression in the antigen processing-defective mutant cell line T2. B27.A2B also fails to present an HLA-B27-restricted influenza virus peptide [nucleoprotein (383-391)] to cytotoxic T lymphocytes (CTLs). However, substitution of leucine, the predominant P2 anchor residue in A*0201-restricted peptides, for arginine, the P2 anchor in nucleoprotein-(383-391) and other B*2705-restricted peptides, restores recognition of B27.A2B by the same B*2705-restricted peptide-specific CTLs. These results demonstrate that a dominant polymorphic pocket in a class I molecule, through interaction with the anchor residue of an antigenic peptide, can distinguish among peptides differing by only a single amino acid and thus determine the allelic specificity of peptide presentation.
Reconstitution of retinoid X receptor function and combinatorial regulation of other nuclear hormone receptors in the yeast Saccharomyces cerevisiae.
Authors: Hall B L , Smit-McBride Z , Privalsky M L
Published: May 2006

The nuclear hormone receptor family of transcription factors regulates gene expression via a complex combinatorial network of interactions. Of particular interest is the ability of retinoid X receptors (RXRs) to form heterodimers with retinoic acid receptors (RARs) and thyroid hormone receptors (TRs), thereby modifying their activities. We report here that RXR, RAR, and TR function can be reconstituted in the yeast Saccharomyces cerevisiae and demonstrate that the combinatorial regulation seen in vertebrate cells can be reproduced in the yeast background. Using this system, we have shown that RARs respond to a wide variety of retinoid ligands but that RXRs are specific for the 9-cis isomer of retinoic acid. RXR enhanced the activity of RARs and TRs on a variety of hormone response elements without demonstrably altering their DNA specificity. Interestingly, the ability of RXR to potentiate gene activation by RARs and by TRs varied for different receptor isoforms.
Dominant transposition-deficient mutants of maize Activator (Ac) transposase.
Authors: Kunze R , Behrens U , Courage-Franzkowiak U , Feldmar S , Kühn S , Lütticke R
Published: May 2006

The maize transposable element Activator (Ac) encodes a transposase (TPase) protein, whose DNA-binding domain is located in a basic region around aa 200. The N-terminal 102 aa of the TPase are not required for the transposition reaction. In transfected petunia protoplasts, we analyzed the protein levels of the N-terminally truncated TPase and mutants thereof and the corresponding transposition frequencies. The TPase protein forms large insoluble aggregates at high expression levels. There is no proportionality observed between TPase levels and transposition frequency. Twenty-one mutations (of 26), which are distributed over the whole length of the protein, inactivate the TPase completely. By coexpressing inactive mutant and active truncated TPase, it was found that several mutations have a trans-dominant inhibitory effect. Among those are two DNA-binding-deficient mutants, indicating that inhibition of the active TPase is not caused by competition for the binding sites on the transposon. Accordingly, Ac TPase acts as an oligo- or multimer formed by protein-protein interactions. Peculiarly, two mutants lacking 53 and 98 aa from the C terminus that are themselves transpositionally inactive lead to an increased excision frequency when they are coexpressed with the active truncated TPase.
Identification, cloning, and nucleotide sequencing of the ornithine decarboxylase antizyme gene of Escherichia coli.
Authors: Canellakis E S , Paterakis A A , Huang S C , Panagiotidis C A , Kyriakidis D A
Published: May 2006

The ornithine decarboxylase antizyme gene of Escherichia coli was identified by immunological screening of an E. coli genomic library. A 6.4-kilobase fragment containing the antizyme gene was subcloned and sequenced. The open reading frame encoding the antizyme was identified on the basis of its ability to direct the synthesis of immunoreactive antizyme. Antizyme shares significant homology with bacterial transcriptional activators of the two-component regulatory system family; these systems consist of a "sensor" kinase and a transcriptional regulator. The open reading frame next to antizyme is homologous to sensor kinases. Antizyme overproduction inhibits the activities of both ornithine and arginine decarboxylases without affecting their protein levels. Extracts from E. coli bearing an antizyme gene-containing plasmid exhibit increased antizyme activity. These data strongly suggest that (i) the cloned gene encodes the ornithine decarboxylase antizyme and (ii) antizyme is a bifunctional protein serving as both an inhibitor of polyamine biosynthesis as well as a transcriptional regulator of an as yet unknown set of genes.
Hepsin, a putative cell-surface serine protease, is required for mammalian cell growth.
Authors: Torres-Rosado A , O'Shea K S , Tsuji A , Chou S H , Kurachi K
Published: May 2006

Hepsin was previously identified as a putative cell-surface serine protease. When hepatoma cells were treated with anti-hepsin antibodies, their growth was substantially arrested, suggesting the requirement of hepsin molecules present at the cell surface for normal cell growth. This was further supported by a gross inhibition of cell growth with hepsin-specific antisense oligonucleotides. Upon treatment of cells with antisense oligonucleotides, rapid reduction in cellular hepsin was observed. This reduction in cellular hepsin levels was accompanied by drastic morphological changes. Various tissues in the developing mouse embryo showed greatly elevated hepsin levels in regions of active proliferation. These results indicate that hepsin plays an essential role in cell growth and maintenance of cell morphology.
Identification of a precursor genomic segment that provided a sequence unique to glycophorin B and E genes.
Authors: Onda M , Kudo S , Rearden A , Mattei M G , Fukuda M
Published: May 2006

Human glycophorin A, B, and E (GPA, GPB, and GPE) genes belong to a gene family located at the long arm of chromosome 4. These three genes are homologous from the 5'-flanking sequence to the Alu sequence, which is 1 kb downstream from the exon encoding the transmembrane domain. Analysis of the Alu sequence and flanking direct repeat sequences suggested that the GPA gene most closely resembles the ancestral gene, whereas the GPB and GPE genes arose by homologous recombination within the Alu sequence, acquiring 3' sequences from an unrelated precursor genomic segment. Here we describe the identification of this putative precursor genomic segment. A human genomic library was screened by using the sequence of the 3' region of the GPB gene as a probe. The genomic clones isolated were found to contain an Alu sequence that appeared to be involved in the recombination. Downstream from the Alu sequence, the nucleotide sequence of the precursor genomic segment is almost identical to that of the GPB or GPE gene. In contrast, the upstream sequence of the genomic segment differs entirely from that of the GPA, GPB, and GPE genes. Conservation of the direct repeats flanking the Alu sequence of the genomic segment strongly suggests that the sequence of this genomic segment has been maintained during evolution. This identified genomic segment was found to reside downstream from the GPA gene by both gene mapping and in situ chromosomal localization. The precursor genomic segment was also identified in the orangutan genome, which is known to lack GPB and GPE genes. These results indicate that one of the duplicated ancestral glycophorin genes acquired a unique 3' sequence by unequal crossing-over through its Alu sequence and the further downstream Alu sequence present in the duplicated gene. Further duplication and divergence of this gene yielded the GPB and GPE genes.
Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank.
Authors: Zwaal R R , Broeks A , van Meurs J , Groenen J T , Plasterk R H
Published: May 2006

To understand how genotype determines the phenotype of the animal Caenorhabditis elegans, one ideally needs to know the complete sequence of the genome and the contribution of genes to phenotype, which requires an efficient strategy for reverse genetics. We here report that the Tc1 transposon induces frequent deletions of flanking DNA, apparently resulting from Tc1 excision followed by imprecise DNA repair. We use this to inactivate genes in two steps. (i) We established a frozen library of 5000 nematode lines mutagenized by Tc1 insertion, from which insertion mutants of genes of interest can be recovered. Their address within the library is determined by PCR. (ii) Animals are then screened, again by PCR, to detect derivatives in which Tc1 and 1000-2000 base pairs of flanking DNA are deleted, and thus a gene of interest is inactivated. We have thus far isolated Tc1 insertions in 16 different genes and obtained deletion derivatives of 6 of those.
Striatal Fos expression is indicative of dopamine D1/D2 synergism and receptor supersensitivity.
Authors: LaHoste G J , Yu J , Marshall J F
Published: May 2006

Immediate-early genes, such as c-fos, are responsive to dopaminergic stimulation in the brain and can have prolonged effects on the transcription of other genes. Thus, they may mediate some of the long-term consequences of altered dopaminergic transmission on striatal neurons, such as the supersensitivity to dopamine and its agonists that occurs in response to dopamine denervation. The two dopamine receptor families, D1 and D2, interact synergistically under normal conditions but independently after treatments that induce pronounced supersensitivity to dopamine agonists. Using immunocytochemical methods in rats treated with directly acting selective dopamine agonists, we have determined that dopamine-mediated expression of Fos and Fos-like antigens in the striatum normally requires concomitant stimulation of D1 and D2 receptors. Separate administration of a high dose of a selective D1 (SKF 38393; 20 mg/kg) or D2 (quinpirole; 3 mg/kg) agonist induced Fos-like immunoreactivity in few neurons, whereas combined administration of the D1 and D2 agonists produced patches of intensely stained immunoreactive nuclei in the caudate-putamen. Repeated administration of reserpine (1 mg/kg per day for 5 days), which causes supersensitivity to dopamine agonists and a breakdown in D1/D2 synergism behaviorally, also causes a change in control of c-fos, such that independent stimulation of D1 receptors by SKF 38393 (20 mg/kg) elicited pronounced Fos-like immunoreactivity in the striatum; combined treatment with SKF 38393 (20 mg/kg) and quinpirole (3 mg/kg) in reserpine-treated rats elicited Fos-like expression in no more neurons than did D1 agonism alone. These data demonstrate that dopamine-mediated Fos expression in the striatum is indicative of the state of D1/D2 synergism and receptor supersensitivity.
Human mitochondrial carbonic anhydrase: cDNA cloning, expression, subcellular localization, and mapping to chromosome 16.
Authors: Nagao Y , Platero J S , Waheed A , Sly W S
Published: May 2006

A full-length cDNA clone encoding human mitochondrial carbonic anhydrase (CA), CA V, was isolated from a human liver cDNA library. The 1123-bp cDNA includes a 55-bp 5' untranslated region, a 915-bp open reading frame, and a 153-bp 3' untranslated region. Expression of the cDNA in COS cells produced active enzyme. The 34-kDa precursor and 30-kDa mature form of CA V were identified on Western blots of COS-cell homogenates by a CA V-specific antibody raised to a synthetic peptide corresponding to the C-terminal 17 aa of CA V. Both 34-kDa and 30-kDa bands were also present in mitochondria isolated from transfected COS cells, whereas only the 30-kDa band was present in mitochondria isolated from normal human liver. The N-terminal sequence determined directly on the 30-kDa soluble CA purified from transfected COS cells indicated that processing of the precursor to mature human CA V involves removal of a 38-aa mitochondrial leader sequence. The 267-aa sequence deduced for mature human CA V shows 30-49% similarity to amino acid sequences of previously characterized human CAs (CA I-CA VII) and 76% similarity to the corresponding amino acid sequence deduced from the mouse cDNA. PCR analysis of DNAs from human-rodent somatic cell hybrids localized the gene for CA V to human chromosome 16, the same chromosome to which CA VII has previously been mapped.
Photostimulation using caged glutamate reveals functional circuitry in living brain slices.
Authors: Callaway E M , Katz L C
Published: May 2006

An approach for high-spatial-resolution mapping of functional circuitry in living mammalian brain slices has been developed. The locations of neurons making functional synaptic connections to a single neuron are revealed by photostimulation of highly restricted areas of the slice (50-100 microns in diameter) while maintaining a whole-cell recording of the neuron of interest. Photostimulation is achieved by bathing brain slices in a molecularly caged form of the neurotransmitter glutamate [L-glutamic acid alpha-(4,5-dimethoxy-2-nitrobenzyl) ester], which is then converted to the active form by brief pulses (< 1 ms in duration) of ultraviolet irradiation. Direct activation of receptors on recorded neurons in rat hippocampus and ferret visual cortex demonstrates that photostimulation is reliable and reproducible and can be repeated at the same site at least 30 times without obvious decrement in neuronal responsiveness. Photostimulation of presynaptic neurons at sites distant to the recorded neuron evoked synaptic responses in hippocampal and cortical cells at distances of up to several millimeters from the recorded neuron. Stimulation of 25-100 distinct presynaptic sites while recording from a single postsynaptic neuron was easily achieved. Caged glutamate-based photostimulation eliminates artifacts and limitations inherent in conventional stimulation methods, including stimulation of axons of passage, desensitization, and poor temporal resolution of "puffer" pipettes, and current artifacts of iontophoretic application. This approach allows detailed physiological investigation and manipulation of the complex intrinsic circuitry of the mammalian brain.
A physical model for the translocation and helicase activities of Escherichia coli transcription termination protein Rho.
Authors: Geiselmann J , Wang Y , Seifried S E , von Hippel P H
Published: May 2006

Transcription termination protein Rho of Escherichia coli interacts with newly synthesized RNA chains and brings about their release from elongation complexes paused at specific Rho-dependent termination sites. Rho is thought to accomplish this by binding to a specific Rho "loading site" on the nascent RNA and then translocating preferentially along the transcript in a 5'-->3' direction. On reaching the elongation complex, Rho releases the nascent RNA by a 5'-->3' RNA.DNA helicase activity. These translocation and helicase activities are driven by the RNA-dependent ATPase activity of Rho. In this paper we propose a mechanism for these processes that is based on the structure and properties of the Rho protein. Rho is a hexamer of identical subunits that are arranged as a trimer of asymmetric dimers with D3 symmetry. The binding of ATP and RNA to Rho also reflects this pattern; the Rho hexamer carries three strong and three weak binding sites for each of these entities. The asymmetric dimers of Rho correspond to functional dimers that can undergo conformational transitions driven by ATP hydrolysis. We propose that the quaternary structure of Rho coordinates the ATP-driven RNA binding and release processes to produce a biased random walk of the Rho hexamer along the RNA, followed by RNA.DNA helicase activity and transcript release. The proposed model may have implications for other hexameric DNA.DNA, RNA.DNA, and RNA.RNA helicases that function in replication and transcription.
Two different forms of metarhodopsin II: Schiff base deprotonation precedes proton uptake and signaling state.
Authors: Arnis S , Hofmann K P
Published: May 2006

Rhodopsin is a retinal protein and a G-protein-coupled receptor; it shares with both of these families the seven helix structure. To generate the G-interacting helix-loop conformation, generally identified with the 380-nm absorbing metarhodopsin II (MII) photoproduct, the retinal Schiff base bond to the apoprotein must be deprotonated. This occurs as a key event also in the related retinal proteins, sensory rhodopsins, and the proton pump bacteriorhodopsin. In MII, proton uptake from the aqueous phase must be involved as well, since its formation increases the pH of the aqueous medium and is accelerated under acidic conditions. In the native membrane, the pH effect matches MII formation kinetically, suggesting that intramolecular and aqueous protonation changes contribute in concert to the protein transformation. We show here, however, that proton uptake, as indicated by bromocresol purple, and Schiff base deprotonation (380-nm absorption change) show different kinetics when the protein is solubilized in suitable detergents. Our data are consistent with a two-step reaction:
Inheritance of freezing resistance in tuber-bearing Solanum species: evidence for independent genetic control of nonacclimated freezing tolerance and cold acclimation capacity.
Authors: Stone J M , Palta J P , Bamberg J B , Weiss L S , Harbage J F
Published: May 2006

Frost or winter survival is regarded as a complex trait with polygenic inheritance. Two major components of this survival in crop plants are freezing tolerance in the nonacclimated state and cold acclimation capacity. To date researchers have not distinguished the two components as separate heritable traits. The mode of inheritance of these two traits was investigated in F1 and backcross populations of two wild diploid potato species (Solanum commersonii and Solanum cardiophyllum) exhibiting extremes of freezing tolerance and acclimation capacity. Precise assessment of these two traits allowed distinction of small but significant differences among genotypes. The two traits were not correlated in segregating populations, suggesting independent genetic control. Analyses of generation means indicate that all of the variance for acclimation capacity and a major proportion of the variance for the nonacclimated freezing tolerance can be best explained by an additive-dominance model with both traits being partially recessive. Recovery of parental phenotypes in limited populations suggests that both traits are controlled by relatively few genes. To our knowledge this is the first study demonstrating independent genetic control of the two main traits associated with frost or winter survival. Our results show that it should be possible to incorporate these traits from wild germ plasm into cultivated crop plants by independent selection. These results help explain the lack of progress in improving winter survival through field selection. Furthermore, our study demonstrates relative simplicity of the inheritance of cold acclimation, thus providing avenues for understanding the link between biochemical and genetic aspects of low-temperature stress in crop plants.
Secretion of a single-gene-encoded immunoglobulin from myeloma cells.
Authors: Shu L , Qi C F , Schlom J , Kashmiri S V
Published: May 2006

We describe construction of a single gene encoding a single-chain immunoglobulin-like molecule. This single-gene approach circumvents inefficiencies inherent in delivering two genes into a mammalian cell and in the assembly of a functional immunoglobulin molecule. It would also facilitate ex vivo transfection of cells for gene-therapy protocols. SP2/0 murine myeloma cells transfected with the single gene SG delta CLCH1 expressed a single-chain protein, SC delta CLCH1, comprising approximately 60 kDa of the anti-carcinoma monoclonal antibody (mAb) CC49. The single-chain protein consisted of the heavy- and light-chain variable (VH and VL) domains of the mAb covalently joined through a short linker peptide, while the carboxyl end of the VL domain was linked to the amino terminus of the human gamma 1 Fc region through the hinge region. The single-chain protein assembled into a dimeric molecule, termed SCA delta CLCH1, of approximately 120 kDa and was secreted into the tissue culture fluid. SDS/PAGE analysis of the secreted immunoglobulin purified by protein G affinity chromatography confirmed the size of the molecule. The native mAb CC49 and SCA delta CLCH1 of CC49 showed similar binding to the tumor-associated glycoprotein TAG-72, and the chimeric mAb CC49 and SCA delta CLCH1 showed similar cytotoxic activity. This single-gene construct approach provides a way of generating an immunoglobulin-like molecule which retains the specificity, binding properties, and cytolytic activity of the chimeric mAb CC49. The immunoglobulin-like molecule SCA delta CLCH1 is potentially a therapeutic and diagnostic reagent against a range of human carcinomas.
Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.
Authors: Burns J C , Friedmann T , Driever W , Burrascano M , Yee J K
Published: May 2006

The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived vectors in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus. Such vectors can be concentrated by ultracentrifugation to titers > 10(9) colony-forming units/ml and can infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo. The availability of these pseudotyped vectors will also facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system, through the efficient introduction and expression of foreign genes.
Binding of human apolipoprotein E to synthetic amyloid beta peptide: isoform-specific effects and implications for late-onset Alzheimer disease.
Authors: Strittmatter W J , Weisgraber K H , Huang D Y , Dong L M , Salvesen G S , Pericak-Vance M , Schmechel D , Saunders A M , Goldgaber D , Roses A D
Published: May 2006

Apolipoprotein E (apoE), a plasma apolipoprotein that plays a central role in lipoprotein metabolism, is localized in the senile plaques, congophilic angiopathy, and neurofibrillary tangles of Alzheimer disease. Late-onset familial and sporadic Alzheimer disease patients have an increased frequency of one of the three common apoE alleles, epsilon 4, suggesting apoE4 is associated with increased susceptibility to disease. To follow up on this suggestion, we compared the binding of synthetic amyloid beta (beta/A4) peptide to purified apoE4 and apoE3, the most common isoform. Both isoforms bound synthetic beta/A4 peptide, the primary constituent of the plaque and angiopathy, forming a complex that resisted dissociation by boiling in SDS. Oxygen-mediated complex formation was implicated because binding was increased in oxygenated buffer, reduced in nitrogen-purged buffer, and prevented by reduction with dithiothreitol or 2-mercaptoethanol. Binding of beta/A4 peptide was saturable at 10(-4) M peptide and required residues 12-28. Examination of apoE fragments revealed that residues 244-272 are critical for complex formation. Both oxidized apoE4 and apoE3 bound beta/A4 peptide; however, binding to apoE4 was observed in minutes, whereas binding to apoE3 required hours. In addition, apoE4 did not bind beta/A4 peptide at pH < 6.6, whereas apoE3 bound beta/A4 peptide from pH 7.6 to 4.6. Together these results indicate differences in the two isoforms in complexing with the beta/A4 peptide. Binding of beta/A4 peptide by oxidized apoE may determine the sequestration or targeting of either apoE or beta/A4 peptide, and isoform-specific differences in apoE binding or oxidation may be involved in the pathogenesis of the intra- and extracellular lesions of Alzheimer disease.
DNA minor groove-binding ligands: a different class of mammalian DNA topoisomerase I inhibitors.
Authors: Chen A Y , Yu C , Gatto B , Liu L F
Published: May 2006

A number of DNA minor groove-binding ligands (MGBLs) are known to exhibit antitumor and antimicrobial activities. We show that DNA topoisomerase (Topo) I may be a pharmacological target of MGBLs. In the presence of calf thymus Topo I, MGBLs induced limited but highly specific single-strand DNA breaks. The 3' ends of the broken DNA strands are covalently linked to Topo I polypeptides. Protein-linked DNA breaks are readily reversed by a brief heating to 65 degrees C or the addition of 0.5 M NaCl. These results suggest that MGBLs, like camptothecin, abort Topo I reactions by trapping reversible cleavable complexes. The sites of cleavage induced by MGBLs are distinctly different from those induced by camptothecin. Two of the major cleavage sites have been sequenced and shown to be highly A + T-rich, suggesting the possible involvement of a Topo I-drug-DNA ternary complex at the sites of cleavage. Different MGBLs also exhibit varying efficiency in inducing Topo I-cleavable complexes, and the order of efficiency is as follows: Hoechst 33342 and 33258 >> distamycin A > berenil > netropsin. The lack of correlation between DNA binding and cleavage efficiency suggest that, in addition to binding to the minor grooves of DNA, MGBLs must also interact with Topo I in trapping Topo I-cleavable complexes.