bluuebunny/crossref_metadata_2025 huggingface dataset:
Organ injury associated with extrathymic induction of immune tolerance in doubly transgenic mice.
Authors: Fields L E , Loh D Y
Published: May 2006

The developmental fate of autoreactive T cells encountering extrathymically expressed self-antigen was studied in a doubly transgenic mouse model system where pancreatic acinar cells expressed H-2Ld and T cells expressed an antigen receptor (2C TCR) specific for H-2Ld. Thymocytes bearing 2C TCR differentiated normally. They were positively selected without evidence of intrathymic clonal deletion. Survival of H-2Ld-bearing skin allografts was significantly prolonged in pancreatic H-2Ld singly and doubly transgenic mice, consistent with an in vivo state of T-cell tolerance. The mechanism of tolerance induction was determined and found to have two components. First, up to 80% of peripheral CD8+2C TCR+ T cells were eliminated. Second, those T cells which escaped elimination had a significantly reduced proliferative response to H-2Ld. Thus, autoreactive T cells can be made self-tolerant through interaction with self-antigen located extrathymically. This is accomplished by a reduction in the percentage of autoreactive T cells as well as by a reduction in the functional capacity of residual T cells. Surprisingly, although pancreatic lymphocytic infiltration and organ injury were absent in exocrine tissue of singly transgenic mice, it was present in doubly transgenic mice. This suggests that when the percentage of autoreactive T cells is high, tolerance induction can be associated with an inflammatory infiltrate in extrathymic tissue where self-antigen is presented.
Intracellular amplification and expression of a synthetic analog of rotavirus genomic RNA bearing a foreign marker gene: mapping cis-acting nucleotides in the 3'-noncoding region.
Authors: Gorziglia M I , Collins P L
Published: May 2006

cDNAs were constructed to encode plus- or minus-sense analogs of gene 9 RNA of porcine rotavirus strain OSU in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene was flanked by the 5'-terminal 44 nucleotides (nt) and 3'-terminal 35 nt of the authentic rotavirus gene. Transfection of plus-sense gene-9-CAT RNA into rotavirus-infected cells resulted in its amplification and in the efficient expression of CAT; this was greatly enhanced by the presence of a 5' cap structure. Amplification was ablated by omitting the rotavirus superinfection or by removing the 3'-terminal 35-nt rotavirus sequence from the RNA. This result indicated that amplification depended both on rotavirus proteins supplied in trans and on cis-acting rotavirus sequences. Minus-sense or double-stranded gene-9-CAT RNA was essentially inactive, indicating that synthetic RNAs can be introduced into the rotavirus replicative cycle in vivo only when provided in the plus sense. However, incorporation of the CAT-bearing RNA into infectious rotavirus was not detected. Two heterologous rotaviruses, the simian RRV and chicken Ch2 strains, efficiently complemented the OSU-based gene-9-CAT RNA, even though the Ch2 strain was only 50%-66% related in the noncoding regions. Mutational analysis of the 35-nt 3'-noncoding region showed that the 3'-terminal 12 or 17 nt were sufficient for reduced (12% or 23%, respectively) levels of amplification, whereas inclusion of the 3'-terminal 19 nt fully restored amplification. Thus, the 3'-terminal cis-acting signals required for amplification include the 7-nt-terminal consensus sequence together with 12 nt of adjoining, less-well-conserved sequence.
Human chromosomal localization of genes encoding the gamma 1 and gamma 2 subunits of the gamma-aminobutyric acid receptor indicates that members of this gene family are often clustered in the genome.
Authors: Wilcox A S , Warrington J A , Gardiner K , Berger R , Whiting P , Altherr M R , Wasmuth J J , Patterson D , Sikela J M
Published: May 2006

The gamma-aminobutyric acid (GABA) receptors are the major inhibitory neurotransmitter receptors in the brain and the site of action of a number of important pharmacological agents including barbiturates, benzodiazepines, and ethanol. The gamma 1 and gamma 2 subunits have been shown to be important in mediating responses to benzodiazepines, and a splicing variant of the gamma 2 subunit, gamma 2L, has been shown to be necessary for ethanol actions on the receptor, raising the possibility that the gamma 2 gene may be involved in human genetic predisposition to the development of alcoholism. We have assigned the human genes encoding the gamma 1 and gamma 2 subunits of the GABAA receptor to chromosomes 4 and 5, respectively, by PCR amplification of human-specific products from human-hamster somatic cell hybrid DNAs. Using panels of chromosome-specific natural deletion hybrids, we have further localized the gamma 1 gene (GABRG1) to 4p14-q21.1 and the gamma 2 gene (GABRG2) to 5q31.1-q33.2. These data indicate that the gamma 1 gene may be clustered together with the previously mapped alpha 2 and beta 1 genes on chromosome 4 and that the gamma 2 gene may be close to the previously localized alpha 1 gene on chromosome 5. To further examine the latter possibility the alpha 1 gene was mapped using the chromosome 5 deletion hybrids and shown to be within the same region as the gamma 2 gene, 5q31.1-q33.2. A PCR-based screening strategy was used to isolate a 450-kilobase human genomic yeast artificial chromosome clone containing both the alpha 1 and gamma 2 genes. Pulsed-field gel restriction mapping of the yeast artificial chromosome indicates that the two genes are within 200 kilobases of each other. The data presented here provide further evidence for the nonrandom organization of the human genome by demonstrating that members of the GABAA receptor gene family often occur in small gene clusters widely distributed in the genome.
Design and synthesis of a CD4 beta-turn mimetic that inhibits human immunodeficiency virus envelope glycoprotein gp120 binding and infection of human lymphocytes.
Authors: Chen S , Chrusciel R A , Nakanishi H , Raktabutr A , Johnson M E , Sato A , Weiner D , Hoxie J , Saragovi H U , Greene M I
Published: May 2006

Poor bioavailability, rapid degradation, antigenicity, and high cost often limit the use of proteinaceous pharmaceuticals. One goal of structural biochemistry is the reduction of complex molecules to small functional units that are amenable to high-resolution structural analysis and rapid modification. The dissection of complex proteins into small synthetic conformationally restricted components is an important step in the design of low molecular weight nonpeptides that mimic the activity of the native protein. We have developed a reverse-turn mimetic system to explore peptide and protein structure-function relationships. We now report the design and synthesis of a small molecule (M(r) 810, as its trifluoroacetate salt), water soluble, proteolytically stable mimetic of residues Gln40-Thr45 of the complementarity-determining 2-like region of CD4. This mimetic has a low micromolar Kd for human T-lymphotropic virus type IIIB gp120 and reduces syncytium formation.
Oxytocin receptor distribution reflects social organization in monogamous and polygamous voles.
Authors: Insel T R , Shapiro L E
Published: May 2006

The neuropeptide oxytocin has been implicated in the mediation of several forms of affiliative behavior including parental care, grooming, and sex behavior. Here we demonstrate that species from the genus Microtus (voles) selected for differences in social affiliation show contrasting patterns of oxytocin receptor expression in brain. By in vitro receptor autoradiography with an iodinated oxytocin analogue, specific binding to brain oxytocin receptors was observed in both the monogamous prairie vole (Microtus ochrogaster) and the polygamous montane vole (Microtus montanus). In the prairie vole, oxytocin receptor density was highest in the prelimbic cortex, bed nucleus of the stria terminalis, nucleus accumbens, midline nuclei of the thalamus, and the lateral aspects of the amygdala. These brain areas showed little binding in the montane vole, in which oxytocin receptors were localized to the lateral septum, ventromedial nucleus of the hypothalamus, and cortical nucleus of the amygdala. Similar differences in brain oxytocin receptor distribution were observed in two additional species, the monogamous pine vole (Microtus pinetorum) and the polygamous meadow vole (Microtus pennsylvanicus). Receptor distributions for two other neurotransmitter systems implicated in the mediation of social behavior, benzodiazepines, and mu opioids did not show comparable species differences. Furthermore, in the montane vole, which shows little affiliative behavior except during the postpartum period, brain oxytocin receptor distribution changed within 24 hr of parturition, concurrent with the onset of maternal behavior. We suggest that variable expression of the oxytocin receptor in brain may be an important mechanism in evolution of species-typical differences in social bonding and affiliative behavior.
Binding of the Bacillus subtilis spoIVCA product to the recombination sites of the element interrupting the sigma K-encoding gene.
Authors: Popham D L , Stragier P
Published: May 2006

The gene encoding sigma K, a transcription factor controlling mother-cell-specific gene expression at a late stage of sporulation, is interrupted by the skin element in Bacillus subtilis. The skin element is excised from the mother cell chromosome by a DNA rearrangement that depends on the spoIVCA gene product. This protein has no other role in sporulation than promoting skin excision and exhibits sequence similarity to a family of bacterial site-specific recombinases. An expression library of B. subtilis DNA in lambda gt11 was screened for the presence of a gene encoding a protein able to bind in vitro to an oligonucleotide matching the inverted repeat sequences present at the ends of the skin element. Several bacteriophages were found to contain the spoIVCA gene. A cell extract containing the SpoIVCA protein protected the inverted repeats and their neighboring sequences from DNase I digestion and methylation. SpoIVCA decreased the electrophoretic mobility of a DNA fragment containing its binding sequence and simultaneously bent the DNA. A single molecule of SpoIVCA bound initially to the repeat sequence followed by binding of a second molecule to create a complex straddling the recombination site.
The endoplasmic reticulum-sarcoplasmic reticulum connection: distribution of endoplasmic reticulum markers in the sarcoplasmic reticulum of skeletal muscle fibers.
Authors: Volpe P , Villa A , Podini P , Martini A , Nori A , Panzeri M C , Meldolesi J
Published: May 2006

The skeletal muscle sarcoplasmic reticulum (SR) was investigated for the presence of well-known endoplasmic reticulum (ER) markers: the lumenal protein BiP and a group of membrane proteins recognized by an antibody raised against ER membrane vesicles. Western blots of SR fractions revealed the presence of BiP in fast- and slow-twitch muscles of the rabbit as well as in rat and chicken muscles. Analyses of purified SR subfractions, together with cryosection immunofluorescence and immunogold labeling, revealed BiP evenly distributed within the longitudinal SR and the terminal cisternae. Within the terminal cisternae BiP appeared not to be mixed with calsequestrin but to be distributed around the aggregates of the latter Ca2+ binding protein. Of the various membrane markers only calnexin (91 kDa) was found to be distributed within both SR subfractions, whereas the other markers (apparent molecular masses of 64 kDa and 58 kDa and a doublet around 28 kDa) were concentrated in the terminal cisternae. These results suggest that the SR is a specialized ER subcompartment in which general markers, such as the ones we have investigated, coexist with the major SR proteins specifically responsible for Ca2+ uptake, storage, and release. The differential distribution of the ER markers reveals new aspects of the SR molecular structure that might be of importance for the functioning of the endomembrane system.
Oncogenic point mutations in exon 20 of the RB1 gene in families showing incomplete penetrance and mild expression of the retinoblastoma phenotype.
Authors: Onadim Z , Hogg A , Baird P N , Cowell J K
Published: May 2006

The retinoblastoma-predisposition gene, RB1, segregates as an autosomal dominant trait with high (90%) penetrance. Certain families, however, show an unusual low-penetrance phenotype with many individuals being unaffected, unilaterally affected, or with evidence of spontaneously regressed tumors. We have used single-strand conformation polymorphism analysis and PCR sequencing to study two such families. Mutations were found in exon 20 of RB1 in both cases. In one family a C----T transition in codon 661 converts an arginine (CGG) to a tryptophan (TGG) codon. In this family, incomplete penetrance and mild phenotypic expression were observed in virtually all patients, possibly indicating that single amino acid changes may modify protein structure/function such that tumorigenesis is not inevitable. In the second family the mutation in codon 675 is a G----T transversion that converts a glutamine (GAA) to a stop (TAA) codon. However, this mutation also occurs near a potential cryptic splice acceptor site, raising the possibility of alternative splicing resulting in a less severely disrupted protein.
Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr.
Authors: Cheung A L , Koomey J M , Butler C A , Projan S J , Fischetti V A
Published: May 2006

A single insertion of transposon Tn917LTV1 into the chromosome of a Staphylococcus aureus clinical isolate, strain DB, resulted in a pleiotropic effect on the expression of a number of extracellular and cell-wall-associated proteins. Detailed comparison of phenotypes associated with the mutant, 11D2, and the parent, DB, indicated that the chromosomal locus inactivated as a result of transposon mutagenesis differs from the S. aureus accessory gene regulator locus (agr). In particular, the expression of alpha-hemolysin, which is not detectable in Agr- mutants, was enhanced in mutant 11D2, while it remained at a low level in strain DB. Likewise, protease activity was significantly enhanced in 11D2 compared with DB. In addition, most of the cell-bound proteins were expressed at lower levels in the mutant than the parent strain. This pattern is contrary to that found in switching from Agr+ to Agr- phenotypes. Southern blot hybridization with an agr probe indicated that the inactivated chromosomal locus is distinct from agr. Transduction experiments demonstrated that the phenotypes associated with mutant 11D2 could be transferred to the parental strain DB as well as to RN450, an S. aureus strain with a genetic background similar to strain 8325-4. This locus on the S. aureus chromosome, possibly regulatory in nature, has been designated sar for staphylococcal accessory regulator.
Plausible structure of the iron-molybdenum cofactor of nitrogenase.
Authors: Madden M S , Krezel A M , Allen R M , Ludden P W , Shah V K
Published: May 2006

A plausible structure of the iron-molybdenum cofactor of nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1] is presented based on altered substrate reduction properties of dinitrogenase containing homocitrate analogs within the cofactor. Alterations on each carbon of the four-carbon homocitrate backbone were correlated with altered substrate reduction properties of dinitrogenase containing these analogs. Altered substrate reduction properties are the basis for a model in which homocitrate is oriented about two cubane metal clusters.
Cloning and chromosomal location of human alpha 1(XVI) collagen.
Authors: Pan T C , Zhang R Z , Mattei M G , Timpl R , Chu M L
Published: May 2006

We have characterized cDNA clones that encode a newly discovered collagenous polypeptide. A 4-kilobase (kb) cDNA clone was initially isolated by screening a human fibroblast cDNA library with a probe encoding the collagenous domain of the human alpha 3(VI) collagen. Subsequent screening of another fibroblast cDNA library yielded overlapping clones having a total length of 5.4 kb, which contained an open reading frame of 1603 amino acids including a 21-amino acid signal peptide. The predicted polypeptide consists of 10 collagenous domains 15-422 amino acids long, which were interspersed with 11 noncollagenous (NC) domains. Except for a large N-terminal NC11 domain of 312 residues, most of the NC domains were short (11-39 residues) and cysteine-rich. The overall structural arrangement differed significantly from other known collagen chains. Further analysis indicated that the deduced polypeptide exhibited several structural features characteristically seen in members of the fibril-associated collagen, types IX, XII, and XIV. In addition, the cysteine-rich motifs in the NC domains resembled those found in the cuticle collagen of Caenorhabditis elegans. Northern blot analyses showed hybridization of the cDNA to a 5.5-kb mRNA in human fibroblasts and keratinocytes. The gene was localized by in situ hybridization to band p34-35 of human chromosome 1. The data clearly support the conclusion that the cDNA encodes a collagen chain that has not been previously described. We suggest that the cDNA clones encode the alpha 1 chain of type XVI collagen.
Wood contains a cell-wall structural protein.
Authors: Bao W , O'Malley D M , Sederoff R R
Published: May 2006

A pine extensin-like protein (PELP) has been localized in metabolically active cells of differentiating xylem and in mature wood of loblolly pine (Pinus taeda L.). This proline-rich glycosylated protein was purified from cell walls of differentiating xylem by differential solubility and gel electrophoresis. Polyclonal rabbit antibodies were raised against the deglycosylated purified protein (dPELP) and purified antibody was used for immunolocalization. Immunogold and alkaline phosphatase secondary antibody staining both show antigen in secondary cell walls of earlywood and less staining in latewood. Immunoassays of milled dry wood were developed and used to show increased availability of antigen after hydrogen fluoride or cellulase treatment and decreased antigen after chlorite treatment. The specificity of the antigen-antibody reaction was confirmed by competition assays and by preadsorption of antibody to the purified protein. We propose that extensin-like protein is present in xylem cell walls during lignification and that the protein remains as a structural component of cell walls in wood for many years after xylogenesis. We suggest that such structural proteins play important roles in the differentiation of xylem and thereby could affect the properties of wood.
Comparison of the nitration of polyfluoronitrobenzenes by nitronium salts in superacidic and aprotic media: activation of the nitronium ion by protosolvation.
Authors: Olah G A , Laali K K , Sandford G
Published: May 2006

The reactivity of nitronium tetrafluoroborate in the nitration of deactivated di- and trifluoronitrobenzenes is enhanced in superacidic trifluoromethanesulfonic (triflic) acid compared with aprotic methylene chloride and sulfolane solutions. The enhanced reactivity is discussed in terms of better solubility and higher dissociation of the nitronium salts, as well as protosolvation of NO2+ by superacids.
Expression of platelet-derived growth factor and its receptor in AIDS-related Kaposi sarcoma in vivo suggests paracrine and autocrine mechanisms of tumor maintenance.
Authors: Stürzl M , Roth W K , Brockmeyer N H , Zietz C , Speiser B , Hofschneider P H
Published: May 2006

As previously described, proliferation of Kaposi sarcoma (KS)-derived cells in vitro is dependent on the presence of platelet-derived growth factor (PDGF). To test the hypothesis that PDGF may also be a major growth factor for KS cells in vivo, we performed in situ hybridization and immunohistochemical staining for PDGF and PDGF receptors in tissue sections of AIDS-related KS. The data suggest that KS consists of two types of tumor cells. (i) The main population are spindle-shaped cells with elongated nuclei (KS-s cells). They reveal a strong expression of PDGF beta receptors but do not express the PDGF-A and PDGF-B isoforms. (ii) A minor population of KS cells express PDGF beta receptor as well as PDGF-A and PDGF-B (KS-p cells). These cells are often grouped in whorls and surrounding vascular slits. They reveal spherical nuclei with evenly distributed chromatin and inconspicuous nucleoli. PDGF alpha receptor is not expressed in either form of KS cells. The results suggest that the isoforms of PDGF and the PDGF beta receptor are differentially expressed in two different cell types in KS and that PDGF isoforms may contribute to the pathogenesis of KS.
A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects.
Authors: Apfel C , Bauer F , Crettaz M , Forni L , Kamber M , Kaufmann F , LeMotte P , Pirson W , Klaus M
Published: May 2006

Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RAR alpha, RAR beta, and RAR gamma. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RAR alpha antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyclonal activation. Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions.
Overexpressed full-length human BCL2 extends the survival of baculovirus-infected Sf9 insect cells.
Authors: Alnemri E S , Robertson N M , Fernandes T F , Croce C M , Litwack G
Published: May 2006

Full-length and truncated human BCL2 lacking the entire C-terminal hydrophobic domain have been overexpressed in Spodoptera frugiperda insect cells with the baculovirus expression system. Immunoblot analysis with BCL2-specific antibodies revealed that both full-length and truncated BCL2 are expressed as multiple immunoreactive species, suggesting posttranslational modifications. The expression of the full-length but not the truncated BCL2 extended the survival of baculovirus-infected cells by preventing virus-induced DNA cleavage. This result is consistent with the reported protective effect of BCL2 against apoptosis in mammalian lymphocytes and suggests a conserved function in evolution. Subcellular fractionation and indirect immunofluorescence studies in intact cells demonstrated that the recombinant full-length and truncated BCL2 proteins were expressed predominantly as nuclear membrane-associated proteins. These results imply that BCL2 must utilize hydrophobic domains other than the deleted domain for its association with the subcellular membranes. Metabolic labeling of insect cells expressing the full-length and the truncated form of BCL2 with 32P(i) demonstrated that BCL2 is a phosphoprotein.
Hepatocyte nuclear factor 1 alpha is expressed in a hamster insulinoma line and transactivates the rat insulin I gene.
Authors: Emens L A , Landers D W , Moss L G
Published: May 2006

Systematic mutational analysis previously identified two primary regulatory elements within a minienhancer (-247 to -198) of the rat insulin I promoter that are critical for transcriptional activity. The Far box (-241 to -232) and the FLAT element (-222 to -208) synergistically upregulate transcription and, together, are sufficient to confer tissue-specific and glucose-responsive transcriptional activity on a heterologous promoter. Detailed analysis of the FLAT element further revealed that, in addition to the positive regulatory activity it mediates in tandem with the Far box, it is a site for negative regulatory control. A portion of the FLAT element bears considerable sequence similarity to the consensus binding site for hepatocyte nuclear factor 1 alpha (HNF1 alpha; LF-B1) a liver-enriched homeodomain-containing transcription factor. Here we show that the HNF1-like site within the FLAT element exhibited positive transcriptional activity in both HepG2 and HIT cells and bound similar, but distinguishable, nuclear protein complexes in the respective nuclear extracts. Screening of a hamster insulinoma cDNA library with a PCR-derived probe encompassing the DNA-binding domain of rat HNF1 alpha resulted in isolation of a hamster HNF1 alpha (hHNF1 alpha) cDNA homolog. Specific antiserum identified the HNF1 alpha protein as one component of a specific FLAT-binding complex in HIT nuclear extracts. Expression of the hHNF1 alpha cDNA in COS cells resulted in transactivation of reporter constructs containing multimerized segments of the rat insulin I minienhancer. Thus, HNF1 alpha, one component of a DNA-binding complex involved in transcriptional regulation of the rat insulin I gene, may play a significant role in nonhepatic as well as hepatic gene transcription.
Rapid adaptation of single mechanosensitive channels in Xenopus oocytes.
Authors: Hamill O P , McBride D W
Published: May 2006

Mechanosensitive (MS) channels are expressed in a wide range of cell types and have been implicated in diverse functions, including osmoregulation and mechanoreception. The majority of previous studies on single MS channels have been carried out on nonsensory cells and have dealt with the steady-state properties of the channel. Here we measure the dynamic or nonstationary properties of the MS channel in Xenopus laevis oocytes. MS channels open transiently in response to a step change in suction applied to the membrane patch. This adaptive behavior occurs because of a reduction in open channel probability rather than a decrease in channel conductance. Double-step suction protocols indicate that adapted MS channels can be reactivated by application of stronger stimulation, consistent with a change in gating sensitivity rather than channel inactivation. Adaptation is highly voltage dependent, being most evident at resting or hyperpolarized potentials and absent at strongly positive potentials. Neither adaptation nor its voltage sensitivity requires the presence of extracellular Ca2+. Adaptation is fragile, dependent on patch history, and can be irreversibly abolished by moderate suction applied to the patch while MS channel activity is retained. Further suction can abolish MS channel activity without compromising the seal. We propose that the selective loss of adaptation and MS channel activity is due to different stages of membrane-cytoskeleton decoupling caused by the mechanical stresses associated with patch clamp recording.
Identification of a peroxisome proliferator-responsive element upstream of the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase.
Authors: Zhang B , Marcus S L , Sajjadi F G , Alvares K , Reddy J K , Subramani S , Rachubinski R A , Capone J P
Published: May 2006

Ciprofibrate, a hypolipidemic drug that acts as a peroxisome proliferator, induces the transcription of genes encoding peroxisomal beta-oxidation enzymes. To identify cis-acting promoter elements involved in this induction, 5.8 kilobase pairs of promoter sequence from the gene encoding rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (EC 4.2.1.17/EC 1.1.1.35) was inserted upstream of a luciferase reporter gene. Transfection of this expression vector into rat hepatoma H4IIEC3 cells in the presence of ciprofibrate resulted in a 5- to 10-fold, cell type-specific increase in luciferase activity as compared to cells transfected in the absence of drug. A peroxisome proliferator-responsive element (PPRE) was localized to a 196-nucleotide region centered at position -2943 from the transcription start site. This PPRE conferred ciprofibrate responsiveness on a heterologous promoter and functioned independently of orientation or position. Gel retardation analysis with nuclear extracts demonstrated that ciprofibrate-treated or untreated H4IIEC3 cells, but not HeLa cells or monkey kidney cells, contained sequence-specific DNA binding factors that interact with the PPRE. These results have implications for understanding the mechanisms of coordinated transcriptional induction of genes encoding peroxisomal proteins by hypolipidemic agents and other peroxisome proliferators.
Stochastic rearrangement of immunoglobulin variable-region genes in chicken B-cell development.
Authors: Benatar T , Tkalec L , Ratcliffe M J
Published: May 2006

The molecular mechanism by which immunoglobulin (Ig) gene rearrangement occurs is highly conserved between mammalian and avian species. However, in avian species, an equivalent to the mammalian pre-B cell, which has undergone Ig heavy-chain gene rearrangement and expresses mu heavy chains in the absence of Ig light-chain rearrangement, has not been convincingly demonstrated. It is consequently unclear whether an ordered progression of gene rearrangement events leading to functional Ig expression occurs in avian species. To examine the sequence of Ig gene rearrangement events in chicken B-cell development, we transformed day 12 embryo bursal cells with the REV-T(CSV) retrovirus. More than 100 clones were analyzed by Southern blotting and polymerase chain reaction for the presence of Ig gene rearrangements. The majority of these clones contained only germline Ig sequences. Several clones contained complete heavy- and light-chain rearrangements and 13 clones contained only heavy-chain rearrangements analogous to stages of mammalian B-cell development. However, 5 clones contained rearrangements of light-chain genes in the absence of complete heavy-chain rearrangement. Consequently, we conclude that rearrangement of chicken Ig light-chain genes does not require heavy-chain variable-region rearrangement. This observation suggests that chicken Ig gene rearrangement events required for Ig expression occur stochastically rather than sequentially.
Long-term expression of human adenosine deaminase in rhesus monkeys transplanted with retrovirus-infected bone-marrow cells.
Authors: van Beusechem V W , Kukler A , Heidt P J , Valerio D
Published: May 2006

Gene transfer into hemopoietic stem cells could offer a lasting cure for a variety of congenital disorders. As a preclinical test for such a gene therapy, rhesus monkeys were transplanted with autologous bone-marrow cells infected with helper-free recombinant retroviruses carrying the human adenosine deaminase gene. The in vivo regenerative capacity of the infected bone marrow could be conserved, suggesting survival of repopulating hemopoietic stem cells. In the hemopoietic system of transplanted animals the foreign gene could be observed for as long as the animals were analyzed (in two monkeys greater than 1 yr after transplantation). Genetically modified cell types and tissues included peripheral blood mononuclear cells, granulocytes, bone-marrow cells of various densities, and spleen and lymph nodes. The presence of the provirus in the short-living granulocytes greater than 1 yr after bone-marrow transplantation provided evidence for the transduction of very primitive hemopoietic progenitors. Moreover, the gene transfer resulted in sustained production of functional human adenosine deaminase enzyme in peripheral blood mononuclear cells. These results demonstrate the feasibility of bone-marrow gene-therapy approaches, in particular for treating adenosine deaminase deficiency.
Circulating estrogens in a male songbird originate in the brain.
Authors: Schlinger B A , Arnold A P
Published: May 2006

Gonadal steroids act on brain to regulate the development and expression of vertebrate reproductive behavior. In addition, steroid targets in brain are an integral part of the feedback regulation of gonadal steroidogenesis. The actions of androgens in brain are often mediated by enzymatic activation or inactivation of circulating hormone, including local conversion of androgen to estrogen. We now report that in zebra finches, the brain synthesizes large quantities of estrogen from androgen and releases this estrogen into blood. Since there appears to be no other significant source of estrogen synthesis in adult male zebra finches, it is likely that the brain directly controls plasma levels of this steroid by contributing most or all of the high levels of estrogen found in the circulation.
Chloroplast DNA inversions and the origin of the grass family (Poaceae).
Authors: Doyle J J , Davis J I , Soreng R J , Garvin D , Anderson M J
Published: May 2006

The phylogenetic affinities of the grass family (Poaceae) have long been debated. The chloroplast genomes of at least some grasses have been known to possess three inversions relative to the typical gene arrangement found in most flowering plants. We have surveyed for the presence of these inversions in grasses and other monocots by polymerase chain reaction amplification with primers constructed from sequences flanking the inversion end points. Amplification phenotypes diagnostic for the largest inversion (28 kilobase pairs) were found in genera representing all grass subfamilies, and in the nongrass families Restionaceae, Ecdeiocoleaceae, and Joinvilleaceae, but not in any other monocots--notably, Flagellariaceae, Anarthriaceae, Cyperaceae, or Juncaceae. This finding is consistent with one of the two principal views of grass phylogeny in suggesting that Poaceae and Cyperaceae (sedges) are not closest relatives. A second (approximately 6 kilobases) inversion appears to occur in a subset of the families possessing the 28-kilobase inversion and links Joinvilleaceae and Poaceae, while the smallest inversion appears unique to grasses. These inversions thus provide a nested set of phylogenetic characters, indicating a hierarchy of relationships in the grasses and allies, with Joinvilleaceae identified as the likely sister group to the Poaceae.
Correlating genetic variation in carbon isotopic composition with complex climatic gradients.
Authors: Comstock J P , Ehleringer J R
Published: May 2006

Genetic variation in both carbon isotope discrimination and the proportions of leaf and photosynthetic twig tissues were observed in ecotypes of Hymenoclea salsola T.G., a common shrub in the deserts of the western United States, when grown under common garden conditions. These variations were correlated with climatic conditions in the habitats of origin through a model that described the leaf-to-air water vapor gradients experienced by plants during the growing season. Both carbon isotope discrimination and the proportion of leaves in the canopy were lower in plants derived from habitats with higher leaf-to-air vapor gradients, despite the fact that some of these sites received relatively high amounts of annual precipitation. These patterns were consistent with the notion that plants are able to maintain substantial control of water-use efficiency over large environmental gradients of temperature and moisture availability.
Role of rap1B and p21ras GTPase-activating protein in the regulation of phospholipase C-gamma 1 in human platelets.
Authors: Torti M , Lapetina E G
Published: May 2006

Thrombin activates phospholipase C in human platelets, but the specific isoenzymes activated and the signal pathway used are unknown. Using specific antibodies, we found that phospholipase C-gamma 1 and the p21ras GTPase-activating protein, rasGAP, are present in human platelets. Furthermore, phospholipase C-gamma 1 was detectable, based on enzyme activity and Western blot analysis, in immunoprecipitates of rasGAP, suggesting that these two proteins form tight complexes. The pool of phospholipase C-gamma 1 associated with rasGAP was phosphorylated but not through tyrosine phosphorylation. Although thrombin stimulation had no effect on the level of phosphorylation of phospholipase C-gamma 1 and only slightly increased the tyrosine phosphorylation of rasGAP, the agonist induced the association of rasGAP with rap1B, as indicated by the appearance of rap1B on a Western blot of rasGAP immunoprecipitates. Our results suggest the formation of a signaling complex involving rasGAP, phospholipase C-gamma 1, and rap1B that might be important in the cascade leading to platelet activation.
Induction of cytotoxic T lymphocytes with peptides in vitro: identification of candidate T-cell epitopes in human papilloma virus.
Authors: Stauss H J , Davies H , Sadovnikova E , Chain B , Horowitz N , Sinclair C
Published: May 2006

A set of overlapping peptides corresponding to the L1, E6, and E7 proteins of human papilloma virus 16 was tested for their ability to bind to major histocompatibility complex class I molecules and to stimulate cytotoxic T-lymphocyte (CTL) responses in vitro. A class I binding assay using intact RMA-S cells showed that 20 of the 99 human papilloma virus peptides bound to H-2Kb and/or Db molecules. Fifteen of the 20 class I-binding peptides stimulated primary CTL responses, whereas peptides that were negative in the binding assay failed to do so. Peptide-induced CTLs recognized the immunizing peptide very efficiently, requiring no more than 1-10 nM peptide for target cell lysis. However, two observations were made that have important implications for the design of peptide-based vaccines for inducing CTLs. (i) Not all major histocompatibility complex-binding peptides that contained known motifs characteristic of naturally processed peptides induced CTLs. (ii) The efficiency of CTL lysis was strongly decreased when the size of the target peptide differed by only one amino acid residue from that of the immunizing peptide. We conclude that peptides chosen for vaccination must correspond in length to naturally processed peptides.
Ligand occupancy mimicked by single residue substitutions in a receptor: transmembrane signaling induced by mutation.
Authors: Yaghmai R , Hazelbauer G L
Published: May 2006

We used mixed, mutagenic oligonucleotides to create single amino acid substitutions in the bacterial chemoreceptor Trg. Mutagenesis was directed at a 20-residue segment of the periplasmic domain implicated in ligand recognition. Transmembrane signaling by the mutant receptors was assayed in vivo by monitoring adaptational covalent modification. Among 20 functionally altered but stable receptors there were two distinct signaling phenotypes. Insensitive receptors did not signal upon stimulation and thus appeared defective in productive ligand interaction. Mimicked-occupancy receptors exhibited transmembrane signaling without ligand. Many mimicked-occupancy receptors produced additional signaling upon ligand binding and in appropriate conditions mediated effective chemotaxis; most insensitive receptors did not. Like normal receptors with one binding site occupied, mimicked-occupancy proteins adapted to persistent transmembrane signaling by increased methylation and thus could respond to other stimuli. Signaling phenotypes were strikingly segregated by residue position. Substitutions mimicking ligand occupancy occurred in half the segment, and those creating insensitive phenotypes occurred in the other half. These observations could be related to the three-dimensional structure of the periplasmic domain of the Tar(s) chemoreceptor. Insensitive substitutions occurred near the distal end of helix 1, where bulky protein ligands could interact; occupancy-mimicking substitutions were on the same helix at positions buried in the subunit interface between helices 1 and 1'. Thus perturbation of the interface induced transmembrane signaling, implicating changes at that interface in signal transduction, a conclusion consistent with differences in crystal structures of unoccupied and ligand-occupied Tar(s).
Redox state of a one-electron component controls the rate of photoinhibition of photosystem II.
Authors: Nedbal L , Samson G , Whitmarsh J
Published: May 2006

Photosystem II reaction centers in plants, algae, and cyanobacteria are susceptible to damage by excess light that irreversibly impairs activity and eventually results in the proteolytic degradation of at least one of the core proteins. The sequence of events and underlying molecular mechanisms that lead to photoinhibition are poorly understood. Here we present evidence for a one-electron redox component that exerts strong control over the rate of photosystem II photoinhibition in isolated thylakoid membranes. Monitoring the impact of various doses of visible light on the rate of water oxidation and on the variable chlorophyll fluorescence, we found that reduction of the redox component increased the rate of photoinhibition >15-fold. Anaerobic potentiometric titrations of the rate of photoinhibition revealed a redox component with a midpoint potential near 20 mV at pH 7.5. The titrations fit a Nernst equation for a one-electron reaction and were nearly pH independent. Although we have not yet identified the chemical species being titrated, a likely candidate is lowpotential cytochrome b-559. We believe this observation reveals an electron transfer pathway in photosystem II that functions to protect the reaction center against excess light energy.
Intranuclear Ca2+ transients during neurite regeneration of an adult mammalian neuron.
Authors: Birch B D , Eng D L , Kocsis J D
Published: May 2006

Depolarization-induced increases in cytoplasmic and intranuclear Ca2+ were visualized in adult mammalian dorsal root ganglion (DRG) neurons during different stages of neurite extension by using confocal laser scanning microscopy and the long-wavelength Ca2+ indicator dye fluo 3-AM (acetoxymethyl ester of fluo 3). In neurons beginning to extend neurites, depolarization led to pronounced increases in nuclear and nucleolar Ca2+ levels severalfold greater than corresponding increases in the cytoplasm. The nucleolar Ca2+ signal often exceeded that of the nucleus, indicating regional heterogeneity of the nucleus. The subcellular calcium transients were dependent on extracellular Ca2+ and the level of depolarization, indicating the importance of transmembrane Ca2+ fluxes in triggering the nuclear events. After neurite extension, the nuclear Ca2+ signals were attenuated and never exceeded cytoplasmic levels. These results indicate that activity-dependent modulation of intranuclear Ca2+ levels is greater in DRG neurons during early neurite extension. Given the importance of Ca2+ in gene expression, the results may be relevant to Ca(2+)-dependent nuclear events responsible for axonal regeneration.
Primary structure, import, and assembly of the yeast homolog of succinate dehydrogenase flavoprotein.
Authors: Schülke N , Blobel G , Pain D
Published: May 2006

We have isolated a homolog for the flavoprotein subunit of succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1] from Saccharomyces cerevisiae and used the obtained peptide sequences to clone and characterize the corresponding gene. It contained an open reading frame of 1923 base pairs and encoded a protein of 640 amino acids (M(r), 70,238) that showed approximately 49% and approximately 28% identity with the Escherichia coli and Bacillus subtilis enzymes, respectively. All features of the FAD cofactor binding site were completely conserved. Comparison of the deduced protein sequence with the N-terminal sequence determined from the isolated protein revealed an N-terminal extension of 28 amino acids that presumably represents a mitochondrial signal sequence. After in vitro transcription and translation, the preprotein was efficiently imported into isolated yeast mitochondria, cleaved to its mature form, and assembled into the membrane-bound succinate dehydrogenase complex.
Single amino acid substitutions in "low-risk" human papillomavirus (HPV) type 6 E7 protein enhance features characteristic of the "high-risk" HPV E7 oncoproteins.
Authors: Sang B C , Barbosa M S
Published: May 2006

HPV types associated with genital disease are termed "high-risk" or "low-risk" viruses according to their prevalence in cancers. Two viral genes, E6 and E7, are invariably expressed in cervical carcinomas. The E7 gene product has been found to bind the retinoblastoma tumor suppressor protein and to be phosphorylated by casein kinase II. Although present in both high- and low-risk E7 proteins, these activities are diminished in the low-risk HPV-6 E7 polypeptide. To better understand the oncogenic potential of the HPV-6 E7 protein, we replaced four of its amino acids with HPV-16 E7 residues present in the analogous region of the N-terminal half of the protein. Replacement of the arginine at position 4 of the HPV-6 E7 protein with an aspartate present in HPV-16 E7 slowed the mobility of the protein when expressed in vivo. Replacement of the glycine at position 22 with an aspartate resulted in higher affinity for retinoblastoma protein binding. Replacement of valine residues at positions 30 and 37 with asparagine and aspartate, respectively, resulted in higher levels of casein kinase II phosphorylation. The substitution at position 22 was the only mutation that exhibited increased transforming activity, suggesting a correlation between the HPV E7 protein affinity for the retinoblastoma tumor suppressor protein and its ability to transform established cells. Our results show that subtle changes in sequence may result in marked differences in biological activity of HPV oncogenes.
Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase.
Authors: Saito K , Miura N , Yamazaki M , Hirano H , Murakoshi I
Published: May 2006

Cysteine synthase (CSase) [O-acetyl-L-serine acetate-lyase (adding hydrogen sulfide), EC 4.2.99.8] catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide. We report here the isolation and characterization of cDNA clones encoding cysteine synthase from spinach (Spinacia oleracea L.). Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase. A lambda gt10 cDNA library was constructed from poly(A)+ RNA of young green leaves of spinach. Screening with two synthetic mixed nucleotides encoding the partial peptide sequences revealed 19 positively hybridized clones among 2 x 10(5) clones. Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34,185 Da. Sequence comparison of the deduced amino acids revealed 53% identity with CSases of Escherichia coli and Salmonella typhimurium. Sequence homology was also observed with other metabolic enzymes for amino acids in bacteria and yeast and with rat hemoprotein H-450. A bacterial expression vector was constructed and could genetically complement an E. coli auxotroph that lacks CSases. The accumulation of functionally active spinach CSase in E. coli was also demonstrated by immunoblotting and assaying enzymatic activity. Southern hybridization analysis showed the presence of two to three copies of the cDNA sequence in the genome of spinach. RNA blot hybridization suggested constitutive expression in leaves and roots of spinach.