bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacteriophage T4 MotA and AsiA proteins suffice to direct Escherichia coli RNA polymerase to initiate transcription at T4 middle promoters.
Authors: Ouhammouch M , Adelman K , Harvey S R , Orsini G , Brody E N
Published: May 2006

Development of bacteriophage T4 in Escherichia coli requires the sequential recognition of three classes of promoters: early, middle, and late. Recognition of middle promoters is known to require the motA gene product, a protein that binds specifically to the "Mot box" located at the -30 region of these promoters. In vivo, the asiA gene product is as critical for middle mode RNA synthesis as is that of the motA gene. In vitro, AsiA protein is known to loosen the sigma 70-core RNA polymerase interactions and to inhibit some sigma 70-dependent transcription, presumably through binding to the sigma 70 subunit. Here we show that, in vitro, purified MotA and AsiA proteins are both necessary and sufficient to activate transcription initiation at T4 middle promoters by the E. coli RNA polymerase in a sigma 70-dependent manner. AsiA is also shown to inhibit recognition of T4 early promoters and may play a pivotal role in the recognition of all three classes of phage promoters.
Expression of a human alpha-1,3/4-fucosyltransferase in the pit cell lineage of FVB/N mouse stomach results in production of Leb-containing glycoconjugates: a potential transgenic mouse model for studying Helicobacter pylori infection.
Authors: Falk P G , Bry L , Holgersson J , Gordon J I
Published: May 2006

Helicobacter pylori is a human pathogen associated with the development of gastric and duodenal ulcers and gastric adenocarcinoma. To test the hypothesis that the human Lewis(b) blood group antigen (Le(b)) functions as a receptor for the bacteria's adhesins and mediates its attachment to gastric pit and surface mucous cells, a human alpha-1,3/4-fucosyltransferase was expressed in these cell lineages in FVB/N transgenic mice. The fucosyltransferase directed production of the Leb epitope without any apparent effect on the proliferation and differentiation programs of this lineage. Moreover, clinical isolates of H. pylori bound to these cells in transgenic mice but not in their normal littermates. Binding was blocked by pretreatment of the bacteria with soluble Le(b). This mouse model could be useful for examining the molecular pathogenesis of diseases caused by H. pylori infection. Creating novel pathways for production of specific oligosaccharides in selected cell lineages of transgenic animals represents an approach for examining the role of complex carbohydrates in regulating cellular differentiation and host-microbe interactions.
Segregation of cardiac and skeletal muscle-specific regulatory elements of the beta-myosin heavy chain gene.
Authors: Rindt H , Knotts S , Robbins J
Published: May 2006

The beta-myosin heavy chain (beta-MyHC) gene is expressed in cardiac and slow skeletal muscles. To examine the regulatory sequences that are required for the gene's expression in the two compartments in vivo, we analyzed the expression pattern of a transgene consisting of the beta-MyHC gene 5' upstream region linked to the chloramphenicol acetyltransferase reporter gene. By using 5600 bp of 5' upstream region, the transgene was expressed at high levels in the slow skeletal muscles. Decreased levels of thyroid hormone led to the up-regulation of the transgene in both cardiac and skeletal muscles, mimicking the behavior of the endogenous beta-MyHC gene. After deleting the distal 5000 bp, the level of reporter gene expression was strongly reduced. However, decreased levels of thyroid hormone led to an 80-fold skeletal muscle-specific increase in transgene expression, even upon the ablation of a conserved cis-regulatory element termed MCAT, which under normal (euthyroid) conditions abolishes muscle-specific expression. In contrast, cardiac-specific induction was not detected with the deletion construct. These observations indicate that the cardiac and skeletal muscle regulatory elements can be functionally segregated on the beta-MyHC gene promoter.
Differential expression of mRNAs for protein kinase inhibitor isoforms in mouse brain.
Authors: Seasholtz A F , Gamm D M , Ballestero R P , Scarpetta M A , Uhler M D
Published: May 2006

Many neurotransmitters are known to regulate neuronal cell function by means of activation of cAMP-dependent protein kinase (PKA) and phosphorylation of neuronal substrate proteins, including transcription factors and ion channels. Here, we have characterized the gene expression of two isoforms of a protein kinase inhibitor (PKI) specific for PKA in mouse brain by RNase protection and in situ hybridization histochemistry. The studies demonstrate that the PKI alpha isoform is abundant in many regions of the adult mouse brain but particularly in cerebellum, hypothalamus, hippocampus, and cortex. In contrast, PKI beta is present at much lower levels in most brain regions but is found in significant amounts in the cerebellum, as well as in distinct nuclei within the pons, medulla, and hypothalamus. These results are consistent with a regulatory role of endogenous PKI in PKA-mediated signal transduction in brain and suggest differential functions for the two isoforms of PKI within the central nervous system.
Immunological and biological evidence for a stanniocalcin-like hormone in human kidney.
Authors: Wagner G F , Guiraudon C C , Milliken C , Copp D H
Published: May 2006

The corpuscles of Stannius are responsible for the synthesis and secretion of stanniocalcin (STC), a glycoprotein hormone that regulates calcium and phosphate homeostasis in fishes through its actions on the gills and kidneys. The corpuscles of Stannius and STC are considered to be an endocrine system that is unique to fishes. In this report, we provide evidence for the existence of STC-like proteins in vertebrates other than fishes, in particular, humans. By using a well-characterized RIA for salmon STC, sera from vertebrates as diverse as sharks and humans contained measurable levels of STC-like immunoreactivity in the concentration range commonly observed in fishes, and all of these sera exhibited parallelism in the assay. By using Western blot analysis, proteins were also identified in human kidney extracts that shared several properties with the fish hormone in addition to their cross-reactivity with salmon STC antiserum. The same antiserum was used to identify a discrete population of cells in human kidney tubules that could be the source of serum immunoreactivity. Human kidney extracts containing the STC-immunoreactive proteins also had STC-related effects when injected into fishes. Collectively, the data suggest that STC may be more widespread among the vertebrates than is currently accepted.
Rat kidney papilla contains abundant synaptobrevin protein that participates in the fusion of antidiuretic hormone-regulated water channel-containing endosomes in vitro.
Authors: Jo I , Harris H W , Amendt-Raduege A M , Majewski R R , Hammond T G
Published: May 2006

Antidiuretic hormone (ADH) regulates renal water excretion by altering the permeability of the collecting duct to water. ADH-responsive epithelial cells are the major cell type lining kidney tubules in the inner medulla and papilla. ADH modulates apical membrane water permeability by the insertion and removal of vesicles containing aquaporin collecting duct water channel protein (now termed AQP-2). To identify and characterize proteins responsible for trafficking of AQP-2-containing vesicles, we utilized antibody and cDNA probes to synaptobrevin b (also termed VAMP-2, for vesicle-associated membrane protein 2), a protein that mediates synaptic vesicle exocytosis in the brain and whose structural homologs are now considered to be components of a complex responsible for intracellular vesicle fusion in all cells. We now report that rat kidney inner medulla and papilla contain abundant synaptobrevin protein. Only light endosomes, one of two types of purified papillary AQP-2-containing endosomes, possess synaptobrevin. Light endosomes fuse in vitro by means of an ATP-dependent process that is significantly inhibited when endosomes are preincubated with either anti-synaptobrevin antibody or tetanus toxin. These data define a functional role for a synaptobrevin protein in the fusion of endosomes in vitro. The presence of abundant synaptobrevin proteins in endosomes containing AQP-2 water channels, as well as insulin-sensitive glucose transporters [Cain, C. C., Trimble, W. S. & Lienhard, G. E. (1992) J. Biol. Chem. 267, 11681-11684], and in cells of Malpighian tubules responsible for urine formation in insects [Chin, A. S., Burgess, R. W., Wong, B. R., Schwartz, T. L. & Scheller, R. H. (1993) Gene 131, 175-181] suggests a specialized role for synaptobrevin in vesicle-mediated membrane transport modulated by peptide hormones.
opaque-15, a maize mutation with properties of a defective opaque-2 modifier.
Authors: Dannenhoffer J M , Bostwick D E , Or E , Larkins B A
Published: May 2006

An opaque mutation was identified that reduces gamma-zein synthesis in maize endosperm. The mutation, opaque-15, causes a 2- to 3-fold reduction in gamma-zein mRNA and protein synthesis and reduces the proportion of the 27-kDa gamma-zein A gene transcript. Although the protein bodies in opaque-15 are similar in size and morphology compared to wild type, there are fewer of them in developing endosperm cells. The opaque-15 mutation maps near the telomere of chromosome 7L, coincident with an opaque-2 modifier locus. Based on its phenotype, opaque-15 appears to be a mutation of an opaque-2 modifier gene.
A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli.
Authors: Sledjeski D , Gottesman S
Published: May 2006

The regulation of capsular polysaccharide synthesis in Escherichia coli K-12 depends on the level of an unstable positive regulator, RcsA. The amount of RcsA protein is limited both by its rapid degradation by Lon, an ATP-dependent protease, and by its low level of synthesis. We have found that the low level of expression from the rcsA promoter is due to transcriptional silencing by the histone-like protein H-NS; this silencing is sensitive to both sequence and context in a region upstream of the -35 region of the promoter. A small (85-nt) RNA, DsrA, when overproduced, activates transcription of rcsA::lacZ fusions by counteracting H-NS silencing. DsrA RNA does not show any extended homology with the rcsA promoter or other sequenced regions of E. coli. Since the stimulation of rcsA transcription by this small RNA does not depend on any sequences from within the rcsA transcript, DsrA acts, either directly or indirectly, on rcsA transcription initiation.
Myosin-actin interaction plays an important role in human immunodeficiency virus type 1 release from host cells.
Authors: Sasaki H , Nakamura M , Ohno T , Matsuda Y , Yuda Y , Nonomura Y
Published: May 2006

We examined the potential role of myosin and actin in the release of human immunodeficiency virus type 1 (HIV-1) from infected cells. Wortmannin (100 nM to 5 microM), an effective inhibitor of myosin light chain kinase, blocked the release of HIV-1 from infected T-lymphoblastoid and monocytoid cells in a concentration-dependent manner. Cytochalasin D, a reagent that disrupts the equilibrium between monomeric and polymeric actin, also partially inhibited the release of HIV-1 from the infected cells. At the budding stage, myosin and HIV-1 protein were detected in the same areas on the plasma membrane by using dual-label immunofluorescence microscopy and immunoelectron microscopy. In the presence of 5 microM wortmannin, viral components were observed on the plasma membrane by using immunofluorescence microscopy and electron microscopy, implying that wortmannin did not disturb the transport of viral proteins to the plasma membrane but rather inhibited budding.
OR-1, a member of the nuclear receptor superfamily that interacts with the 9-cis-retinoic acid receptor.
Authors: Teboul M , Enmark E , Li Q , Wikström A C , Pelto-Huikko M , Gustafsson J A
Published: May 2006

We have cloned a member of the nuclear receptor superfamily. The cDNA was isolated from a rat liver library and encodes a protein of 446 aa with a predicted mass of 50 kDa. This clone (OR-1) shows no striking homology to any known member of the steroid/thyroid hormone receptor superfamily. The most related receptor is the ecdysone receptor and the highest homologies represent < 10% in the amino-terminal domain, between 15-37% in the carboxyl-terminal domain and 50-62% in the DNA binding domain. The expression of OR-1 appears to be widespread in both fetal and adult rat tissues. Potential DNA response elements composed of a direct repeat of the hexameric motif AGGTCA spaced by 0-6 nt were tested in gel shift experiments. OR-1 was shown to interact with the 9-cis-retinoic acid receptor (retinoid X receptor, RXR) and the OR-1/RXR complex to bind to a direct repeat spaced by 4 nt (DR4). In transfection experiments, OR-1 appears to activate RXR-mediated function through the DR4. Therefore OR-1 might modulate 9-cis-retinoic acid signaling by interacting with RXR.
Subunit structure of the nonactivated human estrogen receptor.
Authors: Segnitz B , Gehring U
Published: May 2006

The nonactivated estrogen receptor of human MCF-7 mammary carcinoma cells was investigated with respect to stoichiometry of protein subunits. The native receptor complex stabilized by molybdate had a molecular mass of approximately 300 kDa. Chemical cross-linking with several bifunctional reagents resulted in complete stabilization of the same receptor form of approximately 300 kDa and was achieved both in cell extracts and in intact cells. Incubation of the cross-linked receptor with a receptor-specific monoclonal IgG1 antibody increased the molecular mass by approximately 135 kDa--i.e., no more than one immunoglobulin molecule bound to the complex. Partial and progressive cross-linking of affinity-labeled receptors revealed patterns of labeled bands upon denaturing gel electrophoresis indicative of a heteromeric structure. The completely cross-linked receptor was purified to homogeneity and analyzed for protein components. In addition to the receptor polypeptide of approximately 65 kDa, we detected the heat shock proteins hsp90 and p59; the hsp90 band was roughly twice as intense as the p59 band. The heat shock protein hsp70 and the 40-kDa cyclophilin were not detected as components of the highly purified cross-linked receptor of approximately 300 kDa. We suggest a heterotetrameric structure consisting of one receptor polypeptide, two hsp90 molecules, and one p59 subunit, for which the molecular mass adds up to approximately 300 kDa. Thus, the nonactivated estrogen receptor has a molecular architecture homologous to those of glucocorticoid and progesterone receptors, even though phylogenetically the estrogen receptor gene forms a distinct subgroup within the gene family of nuclear hormone receptors.
Enhanced activity of the bacteriophage lambda PL promoter at low temperature.
Authors: Giladi H , Goldenberg D , Koby S , Oppenheim A B
Published: May 2006

The response of the early phage lambda PL promoter to temperature was investigated. Experiments with lacZ reporter gene fusions demonstrated that the activity of the phage lambda PL promoter is inversely dependent on temperature. The bacterial DNA-binding protein integration host factor (IHF) further enhances lambda PL promoter activity at low temperature, although no apparent changes in the cellular level of IHF protein were observed at the different temperatures. IHF protein binds DNA in vitro more avidly at low temperatures. In vitro transcription assays further revealed that the temperature response of PL is the result of an intrinsic property of the promoter as well as its activation by IHF.
Glycine reductase selenoprotein A is not a glycoprotein: the positive periodic acid-Schiff reagent test is the result of peptide bond cleavage and carbonyl group generation.
Authors: Kimura Y , Stadtman T C
Published: May 2006

The complete amino acid sequence of Clostridium sticklandii selenoprotein A, a selenocysteine-containing protein component of the glycine reductase complex, has been established. Both the intact protein and peptide fragments produced by Staphylococcus aureus V8 protease or trypsin were purified by reversed-phase high-performance liquid chromatography and subjected to electrospray ionization mass spectrometric analysis and standard Edman degradation. Selenoprotein A consists of 157 amino acids with a chemical molecular weight of 17,011, in reasonable agreement with the observed molecular weight (17,022.7) determined from its ionization mass spectrum. The sequence of the amino-terminal region of the isolated native protein is Ser-Arg-Phe-Thr-Gly-Lys- Lys-Ile-Val-Ile-Ile-Gly-Asp-Arg-Asp-. An N-terminal methionine residue deduced from the gene sequence was not present. Although selenoprotein A reacted positively in a glycoprotein stain when using either the periodic acid-Schiff reagent procedure or a commercial glycan detection kit, no saccharide was detected by carbohydrate analyses after acid hydrolysis or methanolysis. Identity of the amino acid sequence determined by analysis with that deduced from the gene sequence is further evidence of the absence of bound carbohydrate.
Site-specific rates of excision repair of benzo[a]pyrene diol epoxide adducts in the hypoxanthine phosphoribosyltransferase gene of human fibroblasts: correlation with mutation spectra.
Authors: Wei D , Maher V M , McCormick J J
Published: May 2006

When populations of repair-proficient diploid human fibroblasts were treated with (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) during early S phase, just as the hypoxanthine phosphoribosyltransferase gene (HPRT) was being replicated, 5% of the induced base substitutions were found at nt 212, and 5% of the substitutions were found at nt 229 in exon 3. However, when the population was treated in early G1 phase to allow at least 12 hr for repair before the onset of S phase, 21% of the substitutions were found at nt 212, and 10% were found at nt 229. No such cell-cycle-dependent difference in distribution of base substitutions occurred in excision-repair-deficient cells. To test whether the increase in the relative frequency of mutations resulted from inefficient repair at these sites, we adapted ligation-mediated PCR to measure the rates of removal of BPDE adducts from individual sites in exon 3 of the HPRT gene. Cells were treated with 0.5 microM BPDE in early G1 phase and harvested immediately or after 10, 20, and 30 hr for repair. the nontranscribed strand of exon 3 was analyzed for the original distribution of adducts and those remaining after repair, using Escherichia coli UvrABC excinuclease to excise the adducts and annealing a 5' biotinylated gene-specific primer to the DNA and extending it with Sequenase 2.0 to generate a blunt end at the site of each cut. A linker was ligated to the blunt end, and the desired fragments were isolated from the rest of the genomic DNA by using magnetic beads, amplified by PCR, and analyzed on a sequencing gel. The distribution of fragments of particular lengths indicated the relative number of BPDE adducts initially formed or remaining at specific sites. The rates of repair at individual sites varied widely along exon 3 of the HPRT gene and were very slow at nt 212 and 229, strongly supporting the hypothesis that inefficient DNA repair plays an important role in the formation of mutation hotspots.
A 7-base-pair sequence protects DNA from exonucleolytic degradation in Lactococcus lactis.
Authors: Biswas I , Maguin E , Ehrlich S D , Gruss A
Published: May 2006

Linear DNA molecules are subject to degradation by various exonucleases in vivo unless their ends are protected. It has been demonstrated that a specific 8-bp sequence, 5'-GCTGGTGG-3', named Chi, can protect linear double-stranded DNA from the major Escherichia coli exonuclease RecBCD. Chi protects linear replication products of rolling-circle plasmids from RecBCD degradation in vivo, in agreement with observations in vitro. A unique 7-bp sequence, 5'-GCGCGTG-3', is shown to protect similar replication products from degradation in Lactococcus lactis strains but not in more distantly related Gram-positive bacteria. The properties of this sequence in L. lactis correspond to those of a Chi site. Linear plasmid replication products have been detected in numerous prokaryotes, suggesting the widespread existence of short species-specific sequences that preserve linear DNA from extensive degradation by host cell exonucleases.
Activation-dependent carboxyl methylation of neutrophil G-protein gamma subunit.
Authors: Philips M R , Staud R , Pillinger M , Feoktistov A , Volker C , Stock J B , Weissmann G
Published: May 2006

The gamma subunits of heterotrimeric guanine nucleotide-binding regulatory (G) proteins (G gamma) are post-translationally processed at their C termini by prenylation, proteolysis, and carboxyl methylation. Whereas prenylation of G gamma is required for membrane association of G proteins, the role of carboxyl methylation is unknown. Here we show that human neutrophils express G gamma 2 but not G gamma 3 or G gamma 7 and that carboxyl methylation of G gamma 2 is associated with signal transduction. In a reconstituted cell-free system, neutrophil G gamma 2 was labeled by the methyl donor S-[methyl-3H]adenosyl-L-methionine. Carboxyl methylation was confirmed by alkaline hydrolysis and quantitation of volatile [3H]methanol. Neutrophil G gamma 2 methylation was stimulated by activation of G protein with guanosine 5'-[beta, gamma-thio]triphosphate. We estimate that after 1 hr of G-protein activation at least 6% of the total pool of G gamma 2 was carboxyl-methylated. The inflammatory agonist fMet-Leu-Phe stimulated guanosine 5'-[beta,gamma-thio]triphosphate-dependent carboxyl methylation. Methylation of G gamma 2 was inhibited by the carboxyl methyltransferase inhibitor N-acetyl-S-trans,trans-farnesylcysteine at concentrations that affected signal transduction in neutrophils. These results demonstrate that activation of neutrophil Gi is associated with alpha-carboxyl methyl esterification of G gamma 2 and suggest that carboxyl methylation of G gamma may play a role in signal transduction.
A role for ultraviolet A in solar mutagenesis.
Authors: Drobetsky E A , Turcotte J , Châteauneuf A
Published: May 2006

It is well established that exposure to solar UVB (290-320 nm) gives rise to mutations in oncogenes and tumor suppressor genes that initiate the molecular cascade toward skin cancer. Although UVA (320-400 nm) has also been implicated in multistage photocarcinogenesis, its potential contribution to sunlight mutagenesis remains poorly characterized. We have determined the DNA sequence specificity of mutations induced by UVB (lambda > 290 nm), and by UVA (lambda > 350 nm), at the adenine phosphoribosyltransferase locus of Chinese hamster ovary cells. This has been compared to results previously obtained for stimulated sunlight (lambda > or = 310 nm) and 254-nm UVC in the same gene. We demonstrate that T-->G transversions, a generally rare class of mutation, are induced at high frequency (up to 50%) in UVA-exposed cells. Furthermore, this event comprises a substantial proportion of the simulated sunlight-induced mutant collection (25%) but is significantly less frequent (P < 0.05) in cells irradiated with either UVB (9%) or UVC (5%). We conclude that the mutagenic specificity of broad-spectrum solar light in rodent cells is not determined entirely by the UVB component and that UVA also plays an important role.
Alloantigen priming induces a state of unresponsiveness in human umbilical cord blood T cells.
Authors: Risdon G , Gaddy J , Horie M , Broxmeyer H E
Published: May 2006

Induction of alloreactivity in human adult and umbilical cord blood T cells was evaluated in mixed leukocyte culture by exposure to an allogeneic lymphoblastoid line that expresses known costimulatory molecules. Initial exposure to alloantigen-presenting cells (allo-APC) induced strong proliferative responses in both adult and cord blood T cells. However, in contrast to adult T cells, cord blood T cells exhibited little proliferation after restimulation with donor APC. Primed cord blood T cells could respond to interleukin 2 (IL-2), but unresponsiveness to alloantigen was not overcome by addition of exogenous IL-2. Unresponsiveness was long-lasting and appeared to be maintained by a combination of induction of anergy and activity of CD8+ suppressor cells. This information may contribute to use of human cord blood as an allogeneic source of transplantable stem cells.
Total chemical synthesis of a ribozyme derived from a group I intron.
Authors: Whoriskey S K , Usman N , Szostak J W
Published: May 2006

We describe the complete chemical synthesis of a ribozyme that catalyzes template-directed oligonucleotide ligation. The specific activity of the synthetic ribozyme is nearly identical to that of the same enzyme generated by in vitro transcription with T7 RNA polymerase. The ribozyme is derived from a group I intron and consists of three RNA fragments of 36, 43, and 59 nt that self-assemble to form a catalytically active complex. We have site-specifically substituted ribonucleotide analogs into this enzyme and have identified two 2'-hydroxyl groups that are required for full catalytic activity. In contrast, neither the 2'-hydroxyl nor the exocyclic amino group of the conserved guanosine in the guanosine binding site is necessary for catalysis. By allowing the ribozyme to be modified as easily as its substrates, this synthetic ribozyme system should be useful for testing specific hypotheses concerning ribozyme-substrate interactions and tertiary interactions within the ribozyme.
Colocalization of calcium entry and exocytotic release sites in adrenal chromaffin cells.
Authors: Robinson I M , Finnegan J M , Monck J R , Wightman R M , Fernandez J M
Published: May 2006

"Snapshot" images of localized Ca2+ influx into patch-clamped chromaffin cells were captured by using a recently developed pulsed-laser imaging system. Transient opening of voltage-sensitive Ca2+ channels gave rise to localized elevations of Ca2+ that had the appearance of either "hotspots" or partial rings found immediately beneath the plasma membrane. When the Ca2+ imaging technique was employed in conjunction with flame-etched carbon-fiber electrodes to spatially map the release sites of catecholamines, it was observed that the sites of Ca2+ entry and catecholamine release were colocalized. These results provide functional support for the idea that secretion occurs from "active zone"-like structures in neuroendocrine cells.
Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.
Authors: Sanderson N , Factor V , Nagy P , Kopp J , Kondaiah P , Wakefield L , Roberts A B , Sporn M B , Thorgeirsson S S
Published: May 2006

Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine.
Mutation of juxtamembrane tyrosine residue 1001 suppresses loss-of-function mutations of the met receptor in epithelial cells.
Authors: Weidner K M , Sachs M , Riethmacher D , Birchmeier W
Published: May 2006

Signals transduced by the met tyrosine kinase, which is the receptor for scatter factor/hepatocyte growth factor, are of major importance for the regulation of epithelial cell motility, morphogenesis, and proliferation. We report here that different sets of tyrosine residues in the cytoplasmic domain of the met receptor affect signal transduction in epithelial cells in a positive or negative fashion: mutation of the C-terminal tyrosine residues 13-16 (Y1311, Y1347, Y1354, and Y1363) reduced or abolished ligand-induced cell motility and branching morphogenesis. In contrast, mutation of the juxtamembrane tyrosine residue 2 (Y1001) produced constitutively mobile, fibroblastoid cells. Furthermore, the gain-of-function mutation of tyrosine residue 2 suppressed the loss-of-function mutations of tyrosine residue 15 or 16. The opposite roles of the juxtamembrane and C-terminal tyrosine residues may explain the suggested dual function of the met receptor in both epithelial-mesenchymal interactions and tumor progression.
A random-walk/giant-loop model for interphase chromosomes.
Authors: Sachs R K , van den Engh G , Trask B , Yokota H , Hearst J E
Published: May 2006

Fluorescence in situ hybridization data on distances between defined genomic sequences are used to construct a quantitative model for the overall geometric structure of a human chromosome. We suggest that the large-scale geometry during the G0/G1 part of the cell cycle may consist of flexible chromatin loops, averaging approximately 3 million bp, with a random-walk backbone. A fully explicit, three-parametric polymer model of this random-walk/giant-loop structure can account well for the data. More general models consistent with the data are briefly discussed.
A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects.
Authors: Macreadie I G , Castelli L A , Hewish D R , Kirkpatrick A , Ward A C , Azad A A
Published: May 2006

Vpr is a virion-associated protein of human immunodeficiency type 1 (HIV-1) whose function in acquired immunodeficiency syndrome (AIDS) has been uncertain. Employing the yeast Saccharomyces cerevisiae as a model to examine the effects of HIV-1 auxiliary proteins on basic cellular functions, we found that the vpr gene caused cell growth arrest and structural defects indicated by osmotic sensitivity and gross cell enlargement. Production of various domains by gene expression showed that this effect arose from within the carboxyl-terminal third of the Vpr protein and implicated the sequence HFRIGCRHSRIG, containing two H(S/F)RIG motifs. Electroporation with a series of peptides containing these motifs caused structural defects in yeast that resulted in osmotic sensitivity. A protein with functions relating to the yeast cytoskeleton, Sac1p [Cleves, A. E., Novick, P.J. & Bankaitis, V.A. (1989) J. Cell Biol. 109, 2939-2950], shows sequence similarity to Vpr, and Vpr's effect in yeast may be to disrupt normal Sac1p functions. The Sac1p equivalent has not yet been described in mammalian cells, but in rhabdomyosarcoma and osteosarcoma cell lines Vpr also caused gross cell enlargement and replication arrest [Levy, D.N., Fernandes, L.S., Williams, W.V. & Weiner, D.B. (1993) Cell 72, 541-550]. We note that there is a correlation between the region containing the H(S/F)RIG motifs and the pathogenicity of primate lentiviruses and we suggest that the function of Vpr may be to bring about cell growth arrest and/or cytoskeletal changes as an early step in HIV-1 infection.
Intraspecific diversity and ecological zonation in coral-algal symbiosis.
Authors: Rowan R , Knowlton N
Published: May 2006

All reef-building corals are obligately associated with photosynthetic microalgal endosymbionts called zooxanthellae. Zooxanthella taxonomy has emphasized differences between species of hosts, but the possibility of ecologically significant zooxanthella diversity within hosts has been the subject of speculation for decades. Analysis of two dominant Caribbean corals showed that each associates with three taxa of zooxanthellae that exhibit zonation with depth--the primary environmental gradient for light-dependent marine organisms. Some colonies apparently host two taxa of symbionts in proportions that can vary across the colony. This common occurrence of polymorphic, habitat-specific symbioses challenges conventional understanding of the units of biodiversity but also illuminates many distinctive aspects of marine animal-algal associations. Habitat specificity provides ecological explanations for the previously documented poor concordance between host and symbiont phylogenies and the otherwise surprising lack of direct, maternal transmission of symbionts in many species of hosts. Polymorphic symbioses may underlie the conspicuous and enigmatic variability characteristic of responses to environmental stress (e.g., coral "bleaching") and contribute importantly to the phenomenon of photoadaptation.
Erythropoietin-induced cellular differentiation requires prolongation of the G1 phase of the cell cycle.
Authors: Carroll M , Zhu Y , D'Andrea A D
Published: May 2006

Erythropoietin (EPO), like many other hematopoietic growth factors, can induce either growth or differentiation of hematopoietic cells. Little is known about the molecular basis of this cellular decision, in part because of a paucity of cell lines in which these two phenomena can be dissociated. Ectopic expression of the EPO receptor (EPO-R) in Ba/F3, a murine interleukin 3 (IL-3)-dependent progenitor cell line, confers EPO-dependent cell growth. In these cells (Ba/F3-EPO-R), EPO also induces beta-globin mRNA, a specific marker of erythroid differentiation. Here we show that the induction of erythroid differentiation by EPO requires a delay in cell growth and a prolongation of the (G1) phase of the cell cycle. Interestingly, this effect on G1 prolongation was concentration dependent. At low EPO concentrations (0.05-0.1 unit of EPO per ml; 1 pM EPO = 0.01 unit of EPO per ml), EPO prolonged G1 and induced differentiation; at high concentrations (0.5-10.0 units per ml), EPO shortened G1 and preferentially stimulated growth. IL-3 stimulated Ba/F3 growth but not differentiation at all growth factor concentrations ranging from 0.1 to 500 pM. Moreover, IL-3 suppressed EPO-induced beta-globin induction in a dose-dependent manner. This suppression correlated with the shortening of G1 by IL-3. Taken together, these data demonstrate distinct effects of EPO and IL-3 and a balance between erythroid growth and differentiation that is cell cycle dependent.
Macrophage-inducible expression of a model antigen in Salmonella typhimurium enhances immunogenicity.
Authors: Hohmann E L , Oletta C A , Loomis W P , Miller S I
Published: May 2006

Attenuated Salmonella are useful oral vaccine vectors capable of carrying multiple heterologous antigen genes, but optimal expression of foreign antigens has not yet been achieved. We hypothesized that Salmonella phoP-activated genes, which are transcriptionally activated within antigen-processing macrophages, could prove useful for delivery of heterologous antigens to the immune system. We have created a suicide vector that allows the stable chromosomal insertion of heterologous antigen genes within the phoP-activated gene C (pagC) of Salmonella and permits the expression of heterologous antigens as fusion proteins between the first 84 amino acids of PagC and the chosen antigen. The Escherichia coli phoA gene encoding alkaline phosphatase was cloned into this vector; the resultant plasmid was used to construct Salmonella typhimurium strains that express PagC-alkaline phosphatase fusion proteins from a single chromosomal gene copy. Such strains were administered orally and i.p. as vaccines to BALB/c mice and compared with control strains expressing alkaline phosphatase constitutively. After 3 weeks, mouse sera were analyzed for IgG responses to S. typhimurium lipopolysaccharide and alkaline phosphatase. Remarkably, though all mice had comparable antibody responses to lipopolysaccharide, only mice immunized with strains bearing phoP-activated fusion genes had antibody responses to the heterologous antigen. We conclude that expression of a heterologous antigen from an S. typhimurium in vivo-induced promoter that is activated within macrophages markedly enhances the immunogenicity of a model antigen expressed from a single chromosomal gene copy.
Induction of an outer surface protein on Borrelia burgdorferi during tick feeding.
Authors: Schwan T G , Piesman J , Golde W T , Dolan M C , Rosa P A
Published: May 2006

Lyme disease spirochetes, Borrelia burgdorferi sensu lato, are maintained in zoonotic cycles involving ticks and small mammals. In unfed ticks, the spirochetes produce one outer surface protein, OspA, but not OspC. During infection in mammals, immunological data suggest that the spirochetes have changed their surface, now expressing OspC but little or no OspA. We find by in vitro growth experiments that this change is regulated in part by temperature; OspC is produced by spirochetes at 32-37 degrees C but not at 24 degrees C. Furthermore, spirochetes in the midgut of ticks that have fully engorged on mice now have OspC on their surface. Thus two environmental cues, an increase in temperature and tick feeding, trigger a major alteration of the spirochetal outer membrane. This rapid synthesis of OspC by spirochetes during tick feeding may play an essential role in the capacity of these bacteria to successfully infect mammalian hosts, including humans, when transmitted by ticks.
Photochemistry and spectroscopy of a five-chlorophyll reaction center of photosystem II isolated by using a Cu affinity column.
Authors: Vacha F , Joseph D M , Durrant J R , Telfer A , Klug D R , Barber J
Published: May 2006

A reaction center of photosystem II was isolated from Pisum sativum by using immobilized metal affinity chromatography. This reaction center is photochemically active and has a room temperature Qgamma chlorophyll (Chl) absorption band peaking at 677.5 nm. From HPLC analysis, the pigment stoichiometry was suggested to be 5 Chls per 1 beta-carotene per 2 pheophytins. Low-temperature absorption measurements at 77 K were consistent with the removal of one of the Chls associated with the usual form of the reaction center isolated by using ion-exchange chromatography. Transient absorption spectroscopy on the picosecond time scale indicated that the Chl removed belongs to a pool of Chl absorbing at approximately 670 nm (C670II) that transfers energy relatively slowly to the primary donor P680 in support of our recently proposed model. The results also support the previous conclusion that radical pair formation is largely associated with a 21-ps time constant when P680 is directly excited and that the identity of C670II is likely to be peripherally bound Chls possibly ligated to conserved His residues at positions 118 on the D1 and D2 proteins.
DNA-dependent protein kinase specifically represses promoter-directed transcription initiation by RNA polymerase I.
Authors: Labhart P
Published: May 2006

DNA-dependent protein kinase (DNA-PK) is a nuclear enzyme that phosphorylates several transcription factors, but its cellular function has not been elucidated. Here I show that DNA-PK strongly inhibits promoter-directed transcription initiation by Xenopus RNA polymerase I in vitro. The repression is due to protein phosphorylation, since it is relieved by 6-dimethylaminopurine, an inhibitor of protein kinases. DNA-PK inhibits transcription from both linear and circular templates, but the repression is more efficient on linear templates. DNA-PK has no effect on promoter-directed transcription by RNA polymerases II and III. Partial fractionation of the in vitro transcription system shows that a protein fraction containing transcription factor Rib1, the Xenopus equivalent of human SL1, mediates the repression of transcription by DNA-PK. The present data suggest a role for DNA-PK in down-regulating ribosomal gene transcription.
Interaction of the anthracycline 4'-iodo-4'-deoxydoxorubicin with amyloid fibrils: inhibition of amyloidogenesis.
Authors: Merlini G , Ascari E , Amboldi N , Bellotti V , Arbustini E , Perfetti V , Ferrari M , Zorzoli I , Marinone M G , Garini P
Published: May 2006

All types of amyloidosis are structurally characterized by the cross beta-pleated sheet conformation of the fibrils, irrespective of their biochemical composition. The clinical observation that the anthracycline 4'-iodo-4'-deoxy-doxorubicin (IDOX) can induce amyloid resorption in patients with immunoglobulin light chain amyloidosis was the starting point for this investigation of its possible mechanism of action. IDOX binds strongly to all five types of natural amyloid fibrils tested: immunoglobulin light chains, amyloid A, transthyretin (methionine-30 variant), beta-protein (Alzheimer), and beta 2-microglobulin. Quantitative binding studies showed that IDOX, but not doxorubicin, binds strongly to amyloid fibrils. This binding is saturable and involves two apparently distinct binding sites with Kd values of 5.9 x 10(-11) M and 3.4 x 10(-9) M. IDOX inhibited in vitro insulin amyloid fibrillogenesis. In vivo studies using the experimental amyloid murine model confirmed the specific targeting of IDOX to amyloid deposits. Preincubation of amyloid enhancing factor with IDOX significantly reduced the formation of amyloid deposits. It is hypothesized that IDOX exerts its beneficial effects through the inhibition of fibril growth, thus increasing the solubility of existing amyloid deposits and facilitating their clearance. IDOX may represent the progenitor of a class of amyloid-binding agents that could have both diagnostic and therapeutic potential in all types of amyloidoses.
Retroviral transfer of a human beta-globin/delta-globin hybrid gene linked to beta locus control region hypersensitive site 2 aimed at the gene therapy of sickle cell disease.
Authors: Takekoshi K J , Oh Y H , Westerman K W , London I M , Leboulch P
Published: May 2006

Human gamma-globin and delta-globin chains have been previously identified as strong inhibitors of the polymerization of hemoglobin S, in contrast to the beta-globin chain, which exerts only a moderate antisickling effect. However, gamma-globin and delta-globin are normally expressed at very low levels in adult erythroid cells, in contrast to beta-globin. We report the design of a beta-globin/delta-globin hybrid gene, beta/delta-sickle cell inhibitor 1 (beta/delta-SCI1) and its transduction by retrovirus-mediated gene transfer. The beta/delta-SCI1-encoding gene retains the overall structure of the human beta-globin gene, while incorporating specific amino acid residues from the delta chain previously found responsible for its enhanced antisickling properties. To achieve high expression levels of beta/delta-SCI1 in adult erythrocytes, the hybrid gene was placed under the transcriptional control of the human beta-globin promoter and the DNase I hypersensitive site 2 of the human beta locus control region. High-titer retroviruses were generated, and stable proviral transmission was achieved in infected cells. The mRNA expression levels of the beta/delta-SCI1 gene in infected, dimethyl sulfoxide-induced murine erythroleukemia cells approached 85% of the endogenous murine beta maj-globin mRNA, on a per gene basis, evidence that high gene expression levels were achieved in adult erythroid cells. Further evaluation of this strategy in transgenic animal models of sickle cell disease should assess its efficacy for the gene therapy of human patients.
The primary structure of sensory rhodopsin II: a member of an additional retinal protein subgroup is coexpressed with its transducer, the halobacterial transducer of rhodopsin II.
Authors: Seidel R , Scharf B , Gautel M , Kleine K , Oesterhelt D , Engelhard M
Published: May 2006

The blue-light receptor genes (sopII) of sensory rhodopsin (SR) II were cloned from two species, the halophilic bacteria Haloarcula vallismortis (vSR-II) and Natronobacterium pharaonis (pSR-II). Upstream of both sopII gene loci, sequences corresponding to the halobacterial transducer of rhodopsin (Htr) II were recognized. In N. pharaonis, psopII and phtrII are transcribed as a single transcript. Comparison of the amino acid sequences of vHtr-II and pHtr-II with Htr-I and the chemotactic methyl-accepting proteins from Escherichia coli revealed considerable identities in the signal domain and methyl-accepting sites. Similarities with Htr-I in Halobacterium salinarium suggest a common principle in the phototaxis of extreme halophiles. Alignment of all known retinal protein sequences from Archaea identifies both SR-IIs as an additional subgroup of the family. Positions defining the retinal binding site are usually identical with the exception of Met-118 (numbering is according to the bacteriorhodopsin sequence), which might explain the typical blue color shift of SR-II to approximately 490 nm. In archaeal retinal proteins, the function can be deduced from amino acids in positions 85 and 96. Proton pumps are characterized by Asp-85 and Asp-96; chloride pumps by Thr-85 and Ala-96; and sensors by Asp-85 and Tyr-96 or Phe-96.
Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein.
Authors: Munemitsu S , Albert I , Souza B , Rubinfeld B , Polakis P
Published: May 2006

The APC tumor-suppressor protein associates with beta-catenin, a cell adhesion protein that is upregulated by the WNT1 oncogene. We examined the effects of exogenous APC expression on the distribution and amount of beta-catenin in a colorectal cancer cell containing only mutant APC. Expression of wild-type APC caused a pronounced reduction in total beta-catenin levels by eliminating an excessive supply of cytoplasmic beta-catenin indigenous to the SW480 colorectal cancer cell line. This reduction was due to an enhanced rate of beta-catenin protein degradation. Truncated mutant APC proteins, characteristic of those associated with cancer, lacked this activity. Mutational analysis revealed that the central region of the APC protein, which is typically deleted or severely truncated in tumors, was responsible for the down-regulation of beta-catenin. These results suggest that the tumor-suppressor activity of mutant APC may be compromised due to a defect in its ability to regulate beta-catenin.
Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration.
Authors: Colley N J , Cassill J A , Baker E K , Zuker C S
Published: May 2006

Retinitis pigmentosa (RP) is a group of hereditary human diseases that cause retinal degeneration and lead to eventual blindness. More than 25% of all RP cases in humans appear to be caused by dominant mutations in the gene encoding the visual pigment rhodopsin. The mechanism by which the mutant rhodopsin proteins cause dominant retinal degeneration is still unclear. Interestingly, the great majority of these mutants appear to produce misfolded rhodopsin. We now report the isolation and characterization of 13 rhodopsin mutations that act dominantly to cause retinal degeneration in Drosophila; four of these correspond to identical substitutions in human autosomal dominant RP patients. We demonstrate that retinal degeneration results from interference in the maturation of wild-type rhodopsin by the mutant proteins.