bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoregulation of the Escherichia coli heat shock response by the DnaK and DnaJ heat shock proteins.
Authors: Liberek K , Georgopoulos C
Published: May 2006

All organisms respond to various forms of stress, including heat shock. The heat shock response has been universally conserved from bacteria to humans. In Escherichia coli the heat shock response is under the positive transcriptional control of the sigma 32 polypeptide and involves transient acceleration in the rate of synthesis of a few dozen genes. Three of the heat shock genes--dnaK, dnaJ, and grpE--are special because mutations in any one of these lead to constitutive levels of heat shock gene expression, implying that their products negatively autoregulate their own synthesis. The DnaK, DnaJ, and GrpE proteins have been known to function in various biological situations, including bacteriophage lambda replication. Here, we report the formation of an ATP hydrolysis-dependent complex of DnaJ, sigma 32, and DnaK proteins in vitro. This DnaJ-sigma 32-DnaK complex has been seen under different conditions, including glycerol gradient sedimentation and co-immunoprecipitation. The DnaK and DnaJ proteins in the presence of ATP can interfere with the efficient binding of sigma 32 to the RNA polymerase core, and are capable of disrupting a preexisting sigma 32-RNA polymerase complex. Our results suggest a possible mechanism for the autoregulation of the heat shock response.
Estrogen modulates expression of the glycosyltransferases that synthesize sulfated oligosaccharides on lutropin.
Authors: Dharmesh S M , Baenziger J U
Published: May 2006

The glycoprotein hormone lutropin (LH) bears oligosaccharides terminating with the sequence SO4-4-GalNAc beta 1,4GlcNAc beta 1,2 Man alpha. We have determined that estrogen actively modulates expression of the GalNAc- and sulfotransferases responsible for synthesis of sulfated oligosaccharides on LH alpha and beta subunits. Consequently, terminal glycosylation of LH oligosaccharides with GalNAc-4-SO4 is maintained when LH synthesis and secretion are markedly increased, as occurs during the midcycle surge and following ovariectomy. Maintenance of sulfated oligosaccharides on LH has important biologic consequences because LH circulatory half-life as well as biologic activity at the hormone receptor level are dramatically affected by glycosylation. To our knowledge, regulation of glycosyltransferase levels in response to specific stimuli has not been observed previously, further emphasizing the biologic significance of glycosylation for expression of LH bioactivity in vivo.
Evidence for myosin motors on organelles in squid axoplasm.
Authors: Bearer E L , DeGiorgis J A , Bodner R A , Kao A W , Reese T S
Published: May 2006

Squid axoplasm has proved a rich source for the identification of motors involved in organelle transport. Recently, squid axoplasmic organelles have been shown to move on invisible tracks that are sensitive to cytochalasin, suggesting that these tracks are actin filaments. Here, an assay is described that permits observation of organelles moving on unipolar actin bundles. This assay is used to demonstrate that axoplasmic organelles move on actin filaments in the barbed-end direction, suggesting the presence of a myosin motor on axoplasmic organelles. Indeed, axoplasm contains actin-dependent ATPase activity, and a pan-myosin antibody recognized at least four bands in Western blots of axoplasm. An approximately 235-kDa band copurified in sucrose gradients with KI-extracted axoplasmic organelles, and the myosin antibody stained the organelle surfaces by immunogold electron microscopy. The myosin is present on the surface of at least some axoplasmic organelles and thus may be involved in their transport through the axoplasm, their movement through the cortical actin in the synapse, or some other aspect of axonal function.
Direct gene transfer with DNA-liposome complexes in melanoma: expression, biologic activity, and lack of toxicity in humans.
Authors: Nabel G J , Nabel E G , Yang Z Y , Fox B A , Plautz G E , Gao X , Huang L , Shu S , Gordon D , Chang A E
Published: May 2006

Direct gene transfer offers the potential to introduce DNA encoding therapeutic proteins to treat human disease. Previously, gene transfer in humans has been achieved by a cell-mediated ex vivo approach in which cells from the blood or tissue of patients are genetically modified in the laboratory and subsequently returned to the patient. To determine the feasibility and safety of directly transferring genes into humans, a clinical study was performed. The gene encoding a foreign major histocompatibility complex protein, HLA-B7, was introduced into HLA-B7-negative patients with advanced melanoma by injection of DNA-liposome complexes in an effort to demonstrate gene transfer, document recombinant gene expression, and determine the safety and potential toxicity of this therapy. Six courses of treatment were completed without complications in five HLA-B7-negative patients with stage IV melanoma. Plasmid DNA was detected within biopsies of treated tumor nodules 3-7 days after injection but was not found in the serum at any time by using the polymerase chain reaction. Recombinant HLA-B7 protein was demonstrated in tumor biopsy tissue in all five patients by immunochemistry, and immune responses to HLA-B7 and autologous tumors could be detected. No antibodies to DNA were detected in any patient. One patient demonstrated regression of injected nodules on two independent treatments, which was accompanied by regression at distant sites. These studies demonstrate the feasibility, safety, and therapeutic potential of direct gene transfer in humans.
The ubiquitous subunit of erythroid transcription factor NF-E2 is a small basic-leucine zipper protein related to the v-maf oncogene.
Authors: Andrews N C , Kotkow K J , Ney P A , Erdjument-Bromage H , Tempst P , Orkin S H
Published: May 2006

Erythroid transcription factor NF-E2 is a tissue-restricted heterodimeric protein which recognizes an extended AP-1 motif [(T/C)TGCTGA(C/G)TCA(T/C)] found in the upstream locus control regions of the alpha- and beta-globin gene clusters. A cDNA clone encoding a cell-type-specific subunit of NF-E2, designated p45 NF-E2, has previously been characterized and shown to encode a basic-leucine zipper DNA-binding protein. Here we describe protein purification and cloning of cDNA that encodes the second basic-leucine zipper subunit of the native NF-E2 heterodimer. This polypeptide, designated p18, is widely expressed. It displays extensive homology to the v-maf oncogene product and a human retinal-specific protein, NRL. Unusual features in the basic region shared by v-Maf, NRL, and p18 place them in a distinct subfamily of AP-1-like proteins.
Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo.
Authors: Kass-Eisler A , Falck-Pedersen E , Alvira M , Rivera J , Buttrick P M , Wittenberg B A , Cipriani L , Leinwand L A
Published: May 2006

To optimize the use of modified adenoviruses as vectors for gene delivery to the myocardium, we have characterized infection of cultured fetal and adult rat cardiac myocytes in vitro and of adult cardiac myocytes in vivo by using a replication-defective adenovirus carrying the chloramphenicol acetyltransferase (CAT) reporter gene driven by the cytomegalovirus promoter (AdCMVCATgD). In vitro, virtually all fetal or adult cardiocytes express the CAT gene when infected with 1 plaque-forming unit of virus per cell. CAT enzymatic activity can be detected in these cells as early as 4 hr after infection, reaching near-maximal levels at 48 hr. In fetal cells, CAT expression was maintained without a loss in activity for at least 1 week. Using in vitro studies as a guide, we introduced the AdCMVCATgD virus directly into adult rat myocardium and compared the expression results obtained from virus injection with those obtained by direct injection of pAdCMVCATgD plasmid DNA. The amount of CAT activity resulting from adenovirus infection of the myocardium was orders of magnitude higher than that seen from DNA injection and was proportional to the amount of input virus. Immunostaining for CAT protein in cardiac tissue sections following adenovirus injection demonstrated large numbers of positive cells, reaching nearly 100% of the myocytes in many regions of the heart. Expression of genes introduced by adenovirus peaked at 5 days but was still detectable 55 days following infection. Adenoviruses are therefore a very useful tool for high-efficiency gene transfer into the cardiovascular system.
Human cytochrome P450 3A4: enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase.
Authors: Shet M S , Fisher C W , Holmans P L , Estabrook R W
Published: May 2006

Human cytochrome P450 3A4 is recognized as the catalyst for the oxygen-dependent metabolism of a diverse group of medically important chemicals, including the immunosuppressive agent cyclosporin; macrolide antibiotics, such as erythromycin; drugs such as benzphetamine, nifedipine, and cocaine; and steroids; such as cortisol and testosterone to name but a few. We have engineered the cDNA for human cytochrome P450 3A4 by linkage to the cDNA for the rat or human flavoprotein, NADPH-P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 1.6.2.4). An enzymatically active fusion protein (rF450[mHum3A4/mRatOR]L1) has been expressed at high levels in Escherichia coli and purified to homogeneity. Enzymatic studies show a requirement for lipid, detergent, and cytochrome b5 for the 6 beta-hydroxylation of steroids and the N-oxidation of nifedipine. In contrast, these additions are not required for the N-demethylation of erythromycin or benzphetamine. A spectrophotometrically detectable metabolite complex of P450 3A4 is formed during the metabolism of triacetyloleandomycin, and this has a pronounced inhibitory effect on the metabolism of both testosterone and erythromycin. These results relate to the interpretation of current methods used to assess the in vivo activity of P450 3A4.
Human T-cell receptor (TCR) alpha/beta + CD4-CD8- T cells express oligoclonal TCRs, share junctional motifs across TCR V beta-gene families, and phenotypically resemble memory T cells.
Authors: Brooks E G , Balk S P , Aupeix K , Colonna M , Strominger J L , Groh-Spies V
Published: May 2006

Most human T cells express the TCR alpha/beta and either CD4 or CD8 molecules (single positive, SP); however, small numbers lack CD4 and CD8. In inbred mice, alpha/beta CD4-CD8- (double negative, DN) T cells preferentially express certain beta variable region (V beta) families and may arise via unique developmental pathways. Increased percentages of alpha/beta DN T cells have been identified in some human and murine autoimmune and immunodeficiency diseases. However, their contribution to disease pathology or normal immunity is unknown. To study the cell surface phenotype and TCR diversity of human alpha/beta DN T cells, these cells were isolated from the peripheral blood of healthy adults. The proportion of alpha/beta DN T cells expressing molecules associated with activation (HLA-DR), previous exposure to antigen (CD45RO), and cytotoxic function (CD56, CD57, and CD11b) was increased relative to SP T cells. The TCR V beta repertoire of alpha/beta DN T cells was different from that of alpha/beta SP T cells, although most major gene families were present. For example, higher proportions of V beta 11, a minor gene family in peripheral blood leukocytes, were found in most alpha/beta DN T-cell samples. In contrast to mice, no dominant V beta family was used consistently in different human individuals. Within an individual alpha/beta DN T cells possessed an oligoclonal TCR beta repertoire with conservation of several distinct junctional amino acid motifs with one joined to three different V beta genes in two individuals, suggesting that these cells have undergone a selection process driven by a limited set of ligands. The possibility that they may represent, at least in part, originally SP T cells anergized by down-modulation of CD4 or CD8 must also be entertained. Overall, this study demonstrates that human peripheral blood alpha/beta DN T cells possess unique phenotypic and TCR beta repertoire characteristics when compared with the major alpha/beta SP T cell populations and thus may serve specialized immunologic functions and/or have an unusual origin.
Zinc modulation of a transient potassium current and histochemical localization of the metal in neurons of the suprachiasmatic nucleus.
Authors: Huang R C , Peng Y W , Yau K W
Published: May 2006

The effect of Zn2+ on a voltage-dependent, transient potassium current (IA) in acutely dissociated neurons from the suprachiasmatic nucleus was studied with the whole-cell patch-clamp technique. At micromolar concentrations, Zn2+ markedly potentiated IA activated from a holding potential of -60 mV, which is the resting potential of these neurons. This potentiation occurred at a Zn2+ concentration as low as 2 microM and increased with higher Zn2+ concentrations. The Zn2+ action appears to arise from a shift in the steady-state inactivation of IA to more positive voltages. At 30 microM, Zn2+ shifted the half-inactivation voltage by +20 mV (from -80 mV to -60 mV), and 200 microM Zn2+ shifted this voltage by +45 mV (from -80 mV to -35 mV). Histochemically, we have also observed Zn2+ staining throughout the suprachiasmatic nucleus; the staining is particularly intense in the ventrolateral region of the nucleus, which receives the major fiber inputs. Our findings suggest that Zn2+, presumably synaptically released, may modulate the electrical activity of suprachiasmatic nucleus neurons through IA. Because vesicular Zn2+ is fairly widespread in the central nervous system, it is conceivable that this kind of Zn2+ modulation on IA, and possibly on other voltage-activated currents, exists elsewhere in the brain.
Inherited amplification of an active gene in the cytochrome P450 CYP2D locus as a cause of ultrarapid metabolism of debrisoquine.
Authors: Johansson I , Lundqvist E , Bertilsson L , Dahl M L , Sjöqvist F , Ingelman-Sundberg M
Published: May 2006

Deficient hydroxylation of debrisoquine is an autosomal recessive trait that affects approximately 7% of the Caucasian population. These individuals (poor metabolizers) carry deficient CYP2D6 gene variants and have an impaired metabolism of severely commonly used drugs. The opposite phenomenon also exists, and certain individuals metabolize the drugs very rapidly, resulting in subtherapeutic plasma concentrations at normal doses. In the present study, we have investigated the molecular genetic basis for ultrarapid metabolism of debrisoquine. Restriction fragment length polymorphism analysis of the CYP2D locus in two families with very rapid metabolism of debrisoquine [metabolic ratio (MR) for debrisoquine = 0.01-0.1] revealed the variant CYP2D6 gene CYP2D6L. Eco RI RFLP and Xba I pulsed-field gel electrophoresis analyses showed that this gene had been amplified 12-fold in three members (father and his two children) of one of the families, and two copies were present among members of the other family. The CYP2D6L gene had an open reading frame and carried two mutations causing amino acid substitutions: one in exon 6, yielding an Arg-296-->Cys exchange and one in exon 9 causing Ser-486-->Thr. The MR of subjects carrying one copy of the CYP2D6L gene did not significantly differ from that of those with the wild-type gene, indicating that the structural alterations were not of importance of the catalytic properties of the gene product. Examination of the MR among subjects carrying wild-type CYP2D6, CYP2D6L, or deficient alleles revealed a relationship between the number of active genes and MR. The data show the principle of inherited amplification of an active gene. Furthermore, the finding of a specific haplotype with two or more active CYP2D6 genes allows genotyping for ultrarapid drug metabolizers. This genotyping could be of predictive value for individualized and more efficient drug therapy.
The presequence of Euglena LHCPII, a cytoplasmically synthesized chloroplast protein, contains a functional endoplasmic reticulum-targeting domain.
Authors: Kishore R , Muchhal U S , Schwartzbach S D
Published: May 2006

The precursor to the Euglena light-harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) is unique; it is a polyprotein, synthesized on membrane-bound ribosomes and transported to the Golgi apparatus prior to chloroplast localization. A cDNA corresponding to the 5' end of LHCPII mRNA has been isolated and sequenced. The deduced amino acid sequence of this cDNA indicates that Euglena pLHCPII contains a 141-amino acid N-terminal extension. The N-terminal extension contains three hydrophobic domains and a potential signal peptidase cleavage site at amino acid 35. Cotranslational processing by canine microsomes removed approximately 35 amino acids from an in vitro synthesized 33-kDa pLHCPII composed of a 141-amino acid N-terminal extension and a 180-amino acid partial LHCPII unit truncated at the beginning of the third membrane-spanning hydrophobic domain. Processed pLHCPII was degraded by exogenous protease, indicating that it had not been translocated to the microsomal lumen. Extraction with 0.1 M Na2CO3, pH 11.5, did not remove the processed pLHCPII from the microsomal membrane. A stop-transfer membrane anchor sequence appears to anchor the nascent protein within the membrane, preventing translocation into the lumen. Taken together, these results provide biochemical evidence for a functional cleaved signal sequence within the N-terminal extension of a Euglena cytoplasmically synthesized chloroplast-localized protein.
Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21.
Authors: McCormick M K , Campbell E , Deaven L , Moyzis R
Published: May 2006

Construction of chromosome-specific yeast artificial chromosome (YAC) libraries from sorted chromosomes was undertaken (i) to eliminate drawbacks associated with first-generation total genomic YAC libraries, such as the high frequency of chimeric YACs, and (ii) to provide an alternative method for generating chromosome-specific YAC libraries in addition to isolating such collections from a total genomic library. Chromosome-specific YAC libraries highly enriched for human chromosomes 16 and 21 were constructed. By maximizing the percentage of fragments with two ligatable ends and performing yeast transformations with less than saturating amounts of DNA in the presence of carrier DNA, YAC libraries with a low percentage of chimeric clones were obtained. The smaller number of YAC clones in these chromosome-specific libraries reduces the effort involved in PCR-based screening and allows hybridization methods to be a manageable screening approach.
Developmental control of glucocorticoid receptor transcriptional activity in embryonic retina.
Authors: Ben-Dror I , Havazelet N , Vardimon L
Published: May 2006

In chicken embryo retina, competence for induction of the glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] gene by glucocorticoid hormones increases progressively with development; this competence is minimal in 6-day retina (E6) and high by day 10 (E10). Because the level of glucocorticoid receptors (GRs) in the retina does not increase during that time, we investigated whether the transcriptional activity of GR increased between days 6 and 10 of development. The glucocorticoid-inducible chloramphenicol acetyltransferase (CAT) constructs 2GRE-37TK and p delta G46TCO, which contain glucocorticoid-responsive elements attached to a TATA box and to the thymidine kinase promoter, respectively, were transfected into E6 and E10 retinas, and their inducibility was examined. CAT expression could be induced in the transfected E10 retina but was not induced in the transfected E6 retina. However, induction was obtained also in E6 retina after cotransfection with a GR expression vector. Noninducible CAT constructs (pRSV-CAT, pSV2CAT, and pBLCAT2) were expressed at both ages at similar levels. The CAT construct pGS2.1CAT, which is controlled by the upstream sequence of the chicken glutamine synthetase gene, could be induced in E10 retina but was not induced in E6 retina; however, cotransfection with the GR expression vector resulted in induction of pGS2.1CAT also in E6 retina. We interpret these results as showing that the transcriptional activity of GR in embryonic retina is developmentally controlled and suggest that its increase is causally implicated in the development of competence for glutamine synthetase induction.
Recombinant human sickle hemoglobin expressed in yeast.
Authors: Martin de Llano J J , Schneewind O , Stetler G , Manning J M
Published: May 2006

Sickle hemoglobin has been expressed in the yeast Saccharomyces cerevisiae after site-directed mutagenesis of a plasmid containing normal human alpha- and beta-globin genes. Cassette mutagenesis of this plasmid was achieved by inserting a DNA fragment containing the beta-globin gene in the replicative form of M13mp18 to make a point mutation and then reconstituting the original plasmid containing the mutated beta-globin gene. Pure recombinant hemoglobin S was shown to be identical to natural sickle hemoglobin in its ultraviolet and visible absorption bands and by gel electrophoresis, isoelectric focusing, amino acid analysis, mass spectrometry, partial N-terminal sequencing, and functional properties (P50, cooperativity, and response to 2,3-bisphosphoglycerate). In yeast and in mammalian cells, cotranslational processing yields the same N-terminal valine residues of hemoglobin alpha- and beta-chains, but in bacterial expression systems the N terminus is extended by an additional amino acid because the initiator methionine residue is retained. Since the N-terminal valine residues of both chains of hemoglobin S participate in important physiological functions, such as oxygen affinity, interaction with anions, and the Bohr coefficient, the yeast expression system is preferable to the bacterial system for recombinant DNA studies. Hence, mutagenesis employing this expression system should permit definitive assignments of the role of any amino acid side chain in hemoglobin S aggregation and could suggest additional approaches to therapeutic intervention. The engineering of this system for the synthesis of sickle hemoglobin and its purification to homogeneity in a single column procedure are described.
Molecular recognition analyzed by docking simulations: the aspartate receptor and isocitrate dehydrogenase from Escherichia coli.
Authors: Stoddard B L , Koshland D E
Published: May 2006

Protein docking protocols are used for the prediction of both small molecule binding to DNA and protein macromolecules and of complexes between macromolecules. These protocols are becoming increasingly automated and powerful tools for computer-aided drug design. We review the basic methodologies and strategies used for analyzing molecular recognition by computer docking algorithms and discuss recent experiments in which (i) substrate and substrate analogues are docked to the active site of isocitrate dehydrogenase and (ii) maltose binding protein is docked to the extracellular domain of the receptor, which signals maltose chemotaxis.
Futile cycles in Saccharomyces cerevisiae strains expressing the gluconeogenic enzymes during growth on glucose.
Authors: Navas M A , Cerdán S , Gancedo J M
Published: May 2006

The systems which control the levels of the gluconeogenic enzymes in Saccharomyces cerevisiae have been bypassed to ascertain their physiological significance. The coding regions of the genes FBP1 and PCK1, which encode fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, have been put under the control of the promoter of ADC1 (alcohol dehydrogenase I), a gene not repressed by glucose, and introduced into yeast in multicopy plasmids. The transformed yeast cells show high levels of the gluconeogenic enzymes during growth on glucose. Generation time and growth yield of yeast expressing either fructose-1,6-bisphosphatase or phosphoenolpyruvate carboxykinase are not significantly different from those of the wild-type strain. For a strain expressing both enzymes the increase in generation time is about 20% and the decrease in growth yield around 30%. The concentration of ATP is about 1.5 mM in the growing cells of the different strains. The extent of in vivo cycling was measured by 13C NMR in cell-free extracts from yeast growing on [6-13C]glucose. Cycling between fructose-6-phosphate and fructose-1,6-bisphosphate is < 2%, most likely due to the very strong inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate. Cycling between phosphoenolpyruvate and pyruvate is low, but a precise figure could not be obtained due to poor equilibration of label between carbons 2 and 3 of oxaloacetate.
Telomere-like DNA polymorphisms associated with genetic predisposition to acute myeloid leukemia in irradiated CBA mice.
Authors: Silver A , Cox R
Published: May 2006

There is evidence that interstitial telomere (TTAGGG)n-like sequences at chromosome 2 fragile sites play an important role in the somatic events that characterize the earliest phases of radiation-induced acute myeloid leukemia in the CBA/H mouse. Here we show that the highly inbred CBA/H colony unexpectedly contains four genotypic variants for telomere-like sequence arrays and that almost all induced myeloid leukemias derive from one of the variant subpopulations that constitutes approximately 20% of the colony. Preliminary evidence on the irregular inheritance patterns for these variant sequences is discussed together with the proposal that one form of these telomere sequence arrays either represents or is closely linked to a locus that influences chromosome 2 breakage patterns in hemopoietic cells following irradiation and, through this, susceptibility to induced myeloid leukemia.
Guanine nucleotide binding protein involvement in early steps of phytochrome-regulated gene expression.
Authors: Romero L C , Lam E
Published: May 2006

The transmission process of light signals from plant photoreceptors to target cellular events is largely unknown. In the present work, we show that treatment of dark-adapted soybean cells (SB-P) with cholera toxin or pertussis toxin uncouples phytochrome-dependent gene expression. Addition of as little as 10 ng of toxin per ml is sufficient to activate expression of genes encoding the major chlorophyll a/b-binding protein (cab) in the dark. Significant levels of cab transcript accumulation are detected within 0.5 h after addition of the toxins and expression of these genes is desensitized to further light treatments. Treatment of SB-P cells with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphtha-lenesulfonamide hydrochloride (W-7) prevents induction of the photoregulated gene by phytochrome or bacterial toxins. These results indicate the involvement of guanine nucleotide binding protein(s) in phytochrome-mediated cab gene activation. A likely site of action for this step is between the photoreceptor and a downstream W-7-sensitive effector.
cDNA cloning of a human mRNA preferentially expressed in hematopoietic cells and with homology to a GDP-dissociation inhibitor for the rho GTP-binding proteins.
Authors: Lelias J M , Adra C N , Wulf G M , Guillemot J C , Khagad M , Caput D , Lim B
Published: May 2006

We have identified the mRNA for a human gene, denoted D4, which is expressed at very high levels in hematopoietic cell lines and in normal cells of lymphoid and myeloid origin. The 1.5-kb transcript is absent or detectable only at low levels in nonhematopoietic tissues. D4 encodes a 201-amino acid protein with homology to rhoGDI, an inhibitor of GDP dissociation for the ras-homologous protein rho. D4 might function also as a regulator of guanine nucleotide exchange for small GTP-binding proteins. A homologous transcript of similar size is also preferentially expressed in murine hematopoietic tissues. When totipotent murine embryonic stem cells develop in vitro into hematopoietic cells, the gene is activated with the onset of hematopoiesis. When hematopoietic cell lines are induced to differentiate, the expression of D4 is modulated. Thus, D4 appears to be a developmentally regulated gene. Its preferential expression in hematopoietic cells indicates that D4 likely plays some significant role in the growth and differentiation processes of hematopoietic cells. This significance is underscored by increasing evidence for the involvement of regulators of G proteins in clinical diseases.
5' sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts.
Authors: Salvador M L , Klein U , Bogorad L
Published: May 2006

We have found that sequences in the 5' leader of the Chlamydomonas chloroplast rbcL gene, when fused 5' to foreign genes, destabilize transcripts of these chimeric genes in the chloroplast of transgenic Chlamydomonas but that 5' sequences of the rbcL structural gene prevent this destabilization. Transcripts of the chloroplast rbcL gene are about equally abundant at all times in Chlamydomonas reinhardtii growing on an alternating 12-h light/12-h dark cycle. However, Chlamydomonas chloroplast transformants, harboring chimeric genes containing the same rbcL promoter with 63 or 92 bp of the rbcL 5' leader sequence fused upstream of the Escherichia coli uidA (beta-glucuronidase, GUS) gene, accumulated GUS transcripts only in the dark. Transcripts disappeared rapidly upon illumination of the cells. The same phenomenon was exhibited by transcripts of chimeric genes in which the GUS gene coding sequence was replaced by other unrelated genes. The precipitous light-induced drop in GUS transcript abundance was found to be due to an approximately 16-fold increase in the rate of degradation of GUS transcripts in light rather than to a decrease in the rate of transcription of the GUS gene. Transcripts of a chimeric rbcL-GUS construct in which the leader sequence of the rbcL gene was replaced by 103 bp of the leader sequence of the atpB gene were stable in illuminated cells. The destabilizing effect of the rbcL 5' leader sequence was reversed by adding 257 bp of the 5' coding region of the rbcL gene. The results show that chloroplast transcript levels in illuminated Chlamydomonas cells--and perhaps in other cases--can be determined, at least to some extent, by sequences and interactions of sequences transcribed from the 5' ends of genes.
The activation domain of transcription factor PU.1 binds the retinoblastoma (RB) protein and the transcription factor TFIID in vitro: RB shows sequence similarity to TFIID and TFIIB.
Authors: Hagemeier C , Bannister A J , Cook A , Kouzarides T
Published: May 2006

The retinoblastoma (RB) tumor suppressor protein and the TATA-box-binding protein TFIID form contacts with a number of viral transactivator proteins. One of these, the adenovirus E1A protein, can bind to both proteins. Here we present evidence that the cellular transcription factor PU.1 can bind to both RB and TFIID. Like E1A, PU.1 binds to the conserved C-terminal domain of TFIID and to the RB "pocket" domain. The PU.1 sequences required to bind either protein lie within a 75-amino acid region which functions as an independent activation domain in vivo. The ability of PU.1 to contact directly both RB and TFIID through the same 75-residue domain prompted us to look for sequence similarity between these two proteins. We find that the previously defined domain A of the RB pocket shows sequence similarity to the conserved C terminus of TFIID, whereas domain B shows sequence similarity to a second general transcription factor, TFIIB. The potential for RB to influence transcription by using TFIID- and TFIIB-related functions is discussed.
Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase.
Authors: Gupta A S , Heinen J L , Holaday A S , Burke J J , Allen R D
Published: May 2006

Transgenic tobacco plants that express a chimeric gene that encodes chloroplast-localized Cu/Zn superoxide dismutase (SOD) from pea have been developed. To investigate whether increased expression of chloroplast-targeted SOD could alter the resistance of photosynthesis to environmental stress, these plants were subjected to chilling temperatures and moderate (500 mumol of quanta per m2 per s) or high (1500 mumol of quanta per m2 per s) light intensity. During exposure to moderate stress, transgenic SOD plants retained rates of photosynthesis approximately 20% higher than untransformed tobacco plants, implicating active oxygen species in the reduction of photosynthesis during chilling. Unlike untransformed plants, transgenic SOD plants were capable of maintaining nearly 90% of their photosynthetic capacity (determined by their photosynthetic rates at 25 degrees C) following exposure to chilling at high light intensity for 4 hr. These plants also showed reduced levels of light-mediated cellular damage from the superoxide-generating herbicide methyl viologen. These results demonstrate that SOD is a critical component of the active-oxygen-scavenging system of plant chloroplasts and indicate that modification of SOD expression in transgenic plants can improve plant stress tolerance.
A selective transcriptional induction system for mammalian cells based on Gal4-estrogen receptor fusion proteins.
Authors: Braselmann S , Graninger P , Busslinger M
Published: May 2006

Most mammalian cells neither express any Gal4-like activity nor endogenous estrogen receptor, thus rendering estrogen an inert signal for them. For these two reasons we have developed a selective induction system based on the estrogen-regulable transcription factor Gal-ER. Gal-ER consists of the DNA-binding domain of the yeast Gal4 protein fused to the hormone-binding domain of the human estrogen receptor and hence should exclusively regulate a transfected gene under the control of a Gal4-responsive promoter in mammalian cells. Two major improvements of this induction system were made. First, a synthetic Gal4-responsive promoter was constructed which consisted of four Gal4-binding sites, an inverted CCAAT element, a TATA box, and the adenovirus major late initiation region. This promoter showed extremely low basal activity in the absence and high inducibility in the presence of ligand-activated Gal-ER. Second, the transcription factor Gal-ER was rendered more potent and less susceptible to cell type-specific variation by fusing the strong activating domain of the herpesvirus protein VP16 onto its C terminus. In response to estrogen, Gal-ER-VP16 induced the Gal4-responsive promoter at least 100-fold in transiently transfected NIH 3T3 and P19 cells. Rat fibroblast cell lines expressing integrated Gal-ER and Gal4-responsive fos genes were transformed in a strictly estrogen-dependent manner. The exogenous fos gene was rapidly induced to maximal levels within 1-2 hr of estrogen addition. Elevated Fos activity in turn stimulated transcription of the endogenous fra-1 gene. These data demonstrate the utility of the Gal-ER induction system as a powerful genetic switch for regulating heterologous genes and, in particular, for identifying Fos targets in mammalian cells.
Tumor-suppressor function of muscarinic acetylcholine receptors is associated with activation of receptor-operated calcium influx.
Authors: Felder C C , MacArthur L , Ma A L , Gusovsky F , Kohn E C
Published: May 2006

Several members of the family of guanine nucleotide-binding protein (G protein)-coupled receptors have recently been shown to induce agonist-dependent foci development in NIH 3T3 cells and tumors in nude mice. We selected the five subtypes of the muscarinic acetylcholine receptor family to investigate their role in tumor suppression. When transfected and expressed in CHO-K1 Chinese hamster ovary cells, m1, m3, and m5 muscarinic acetylcholine receptor activation resulted in a morphology change. Receptor activation did not slow or inhibit monolayer growth of CHOm5 cells in culture but markedly inhibited density-independent growth in soft agar and suppressed tumor formation in nude mice. Receptor-mediated tumor suppression was found to be agonist-dependent and reversible and was blocked with a muscarinic receptor antagonist. Of the five signaling pathways associated with the m1, m3, and m5 receptors, only receptor-operated, and inositol trisphosphate-independent, calcium influx was found to correlate with inhibition of tumorigenicity. These data suggest a pivotal role for inositol trisphosphate-independent receptor-regulated calcium homeostasis in CHO-K1 tumor suppression.
Aberrant transcription caused by the insertion of an early transposable element in an intron of the Fas antigen gene of lpr mice.
Authors: Adachi M , Watanabe-Fukunaga R , Nagata S
Published: May 2006

The mouse lpr (lymphoproliferation) mutation carries a rearrangement in the chromosomal gene for the Fas antigen, which mediates apoptosis. Isolation and characterization of mouse Fas antigen chromosomal gene from wild-type and lpr mice indicated an insertion of an early transposable element (ETn) in intron 2 of the Fas antigen gene of lpr mice. Hybrid transcripts carrying the Fas antigen and ETn sequences were expressed in the thymus and liver of the mutant. This indicated that premature termination and aberrant splicing of the Fas antigen transcript caused by the insertion of the ETn in the intron are responsible for the lymphoproliferation and autoimmune phenotype of the mutant mouse. On the other hand, an insertion of the ETn into an intron of a mammalian expression vector dramatically but not completely reduced the expression efficiency. These findings suggest that lpr mice are able to express a very low level of the Fas antigen.
Development of potent gastrin-releasing peptide antagonists having a D-Pro-psi(CH2NH)-Phe-NH2 C terminus.
Authors: Leban J J , Kull F C , Landavazo A , Stockstill B , McDermed J D
Published: May 2006

Gastrin-releasing peptide (GRP) is a 27-amino acid neuroendocrine hormone that may play a role in the pathophysiology of small cell lung carcinoma. GRP and bombesin, a structurally related peptide, stimulate the growth of some cultured cell types. C-terminal GRP peptide analogs were developed that inhibited 6 nM bombesin-induced [3H]thymidine incorporation into quiescent murine Swiss 3T3 cells, which routinely produced a 6-fold stimulation over the basal extent of incorporation. The peptides were also analyzed for their capacity to inhibit the binding of 50 pM 125I-labeled GRP to Swiss 3T3 cells. The combination of two chemical modifications, each antagonistic in itself, led to the creation of antagonists with orders of magnitude greater potency than either modification alone. (i) Antagonist analogs of the form -Leu26-psi(CH2NH)-Xaa27-NH2 [where Xaa is Leu, norleucine (Nle), or Phe; residues numbered after GRP], similar to those introduced by Coy and coworkers [for review, see Jensen, R. T. & Coy, D. H. (1991) Trends Pharmacol. Sci. 12, 13-19], were found to have nanomolar potencies. (ii) We found that an octapeptide C-terminal GRP analog having D-Pro adjacent to the C-terminal amino acid amide was antagonistic, with a potency of 40 nM. By combining both modifications, specific analogs were found with potencies > 1000-fold greater than our lead structure--[(4'-hydroxy)-3-phenylpropanoyl]-Pro-Arg-Gly-Asn-His-Tr p-Ala-Val - Gly-His-Leu-psi(CH2NH)-Nle-NH2--and greater than any antagonist previously reported. The analogs [(4'-hydroxy)-3-phenylpropanoyl]-His-Trp-Ala-Val-D-Ala-His-D-Pro- psi(CH2NH)-Phe-NH2 and 1-naphthoyl-His-Trp-Ala-Val-D-Ala-His-D-Pro-psi(CH2NH)-Phe-NH2 antagonized [3H]thymidine incorporation with IC50 values of approximately 0.3 nM and inhibited the binding of 125I-labeled GRP with IC50 values of approximately 1 pM. These peptides may be of use in the study of the physiology of GRP.
Developmental expression of the platelet-derived growth factor alpha-receptor gene in mammalian central nervous system.
Authors: Yeh H J , Silos-Santiago I , Wang Y X , George R J , Snider W D , Deuel T F
Published: May 2006

We recently reported that the platelet-derived growth factor (PDGF) A-chain gene is highly expressed in neurons of embryonic and adult mouse central nervous system and suggested that its secretion by neurons may support development and maintenance of glia. We have now analyzed the levels and sites of expression of the cognate PDGF alpha-receptor gene in brain and spinal cord of embryonic and adult mice by in situ hybridization. The predominant cell populations in both gray and white matter expressing transcripts of the PDGF alpha-receptor gene are glial cells or their precursors. Transcripts consistently were not detected in neurons. Expression of the PDGF alpha-receptor gene was first observed at embryonic day 15, increased through postnatal day 14, and fell to lower levels in adults. Expression of the alpha-receptor gene corresponds in temporal sequence to the developmental period of glial migration and proliferation and to the expression of PDGF A by neurons. The results indicate that glia but not neurons have the potential to respond to PDGF A and suggest that neurons influence glial cell development through paracrine regulation.
Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers.
Authors: Keller H , Dreyer C , Medin J , Mahfoudi A , Ozato K , Wahli W
Published: May 2006

The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPAR alpha heterodimerizes with retinoid X receptor beta (RXR beta; RXR is the receptor for 9-cis-retinoic acid) and that the two receptors cooperate for the activation of the acyl-CoA oxidase gene promoter. The strongest stimulation of this promoter was obtained when both receptors were exposed simultaneously to their cognate activators. Furthermore, we show that natural fatty acids, and especially polyunsaturated fatty acids, activate PPARs as potently as does the hypolipidemic drug Wy 14,643, the most effective activator known so far. Moreover, we discovered that the synthetic arachidonic acid analogue 5,8,11,14-eicosatetraynoic acid is 100 times more effective than Wy 14,643 in the activation of PPAR alpha. In conclusion, our data demonstrate a convergence of the PPAR and RXR signaling pathways in the regulation of the peroxisomal beta-oxidation of fatty acids by fatty acids and retinoids.
A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans.
Authors: Kaplan J M , Horvitz H R
Published: May 2006

After light touch to its nose, the nematode Caenorhabditis elegans halts forward locomotion and initiates backing. Here we show that three classes of neurons (ASH, FLP, and OLQ) sense touch to the nose and hence are required for this avoidance response. ASH, FLP, and OLQ have sensory endings that contain axonemal cilia. Mutant animals that have defective ciliated sensory endings as well as laser-operated animals that lack ASH, FLP, and OLQ fail to respond to touch to the nose. Together with the previous work of others, these results demonstrate that C. elegans has at least five morphologically distinct classes of mechanosensory neurons. Interestingly, the ASH neuron also acts as a chemosensory neuron; it mediates the avoidance of noxious chemicals. Since ASH possesses both chemosensory and mechanosensory modalities, this neuron might be functionally analogous to vertebrate nociceptors, which mediate the sensation of pain.
Spinach thioredoxin m inhibits DNA synthesis in fertilized Xenopus eggs.
Authors: Hartman H , Wu M , Buchanan B B , Gerhart J C
Published: May 2006

A role for thioredoxin in metazoan DNA synthesis has been assessed by injecting rapidly dividing Xenopus eggs with purified heterologous thioredoxins, which might act as inhibitors if they were to replace resident thioredoxins in some but not all reaction steps. Of 10 tested proteins, spinach chloroplast thioredoxin m is the most potent inhibitor. Eggs cleave and produce cells lacking nuclei. DNA synthesis is severely reduced. Development arrests before gastrulation. In egg extracts, thioredoxin m inhibits incorporation of radioactive dCTP into DNA of sperm nuclei and M13 phage. Inhibition exceeds 90% when thioredoxin m and M13 DNA are preincubated together. The data support the interpretation that thioredoxins normally participate in initiation of metazoan DNA synthesis.
Transgenic mice expressing human CD14 are hypersensitive to lipopolysaccharide.
Authors: Ferrero E , Jiao D , Tsuberi B Z , Tesio L , Rong G W , Haziot A , Goyert S M
Published: May 2006

In vitro studies have previously shown that the myelomonocytic differentiation antigen CD14 is a receptor for a complex consisting of lipopolysaccharide (LPS) and LPS-binding protein. To investigate the role of CD14 in vivo and its relationship to induction of LPS-induced endotoxin shock, transgenic mice expressing human CD14 were produced. These mice express human CD14 strongly on the surface of their monocytes, neutrophils, and Thy-1(+) lymphocytes and are hypersensitive to LPS, as evidenced by their increased susceptibility to endotoxin shock. These results document the importance of CD14 in vivo as a primary mediator of this lethal syndrome. Furthermore, these mice provide an important model for testing the therapeutic effects of agents directed specifically against the human, as opposed to the murine, CD14 protein in preventing LPS-induced endotoxin shock.
Participation of the transcription factor C/EBP delta in the acute-phase regulation of the human gene for complement component C3.
Authors: Juan T S , Wilson D R , Wilde M D , Darlington G J
Published: May 2006

C3, the third component of complement, is critical in the host immune response in that it is involved in both the classical and alternative pathways of complement activation. We have previously shown that a region (bp -127 to -70) within the C3 promoter is indispensable for conferring interleukin 1 (IL-1) responsiveness to this gene. A sequence comparison reveals two CCAAT/enhancer binding protein (C/EBP) consensus sequences, basic DNA binding region and leucine zippers 1 and 2 (bZIP1 and bZIP2), within this region. Site-directed mutagenesis of the more 3' C/EBP site (bZIP1) in the C3 promoter significantly reduced the basal level of expression and the IL-1 responsiveness of the reporter gene, whereas mutation in the second, more 5', C/EBP consensus sequence (bZIP2) had a minimal effect on basal expression and IL-1 inducibility. Electrophoretic-mobility-shift assays, with and without antibodies to the different C/EBP proteins that "supershift" protein-DNA complexes, demonstrated that proteins binding at the 3' C/EBP site formed several complexes. Antibodies to C/EBP alpha supershifted the majority of complexes formed with extracts from control cells. Antibodies directed against C/EBP delta supershifted the major IL-1-inducible complexes. Western immunoblot analyses showed that the level of C/EBP delta protein was increased dramatically in the nuclei of Hep 3B2 cells after 4 h of IL-1 treatment. When Hep 3B2 cells were cotransfected with a C/EBP delta expression vector and a construct with a C3 promoter and a reporter gene, C/EBP delta was able to trans-activate the C3 promoter in an IL-1-responsive manner. The data strongly suggest that C/EBP delta is the major protein responsible for regulating the acute-phase expression of the human C3 gene.