bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcription of a yeast telomere alleviates telomere position effect without affecting chromosome stability.
Authors: Sandell L L , Gottschling D E , Zakian V A
Published: May 2006

Telomeres are required for the stable maintenance of chromosomes in the yeast Saccharomyces cerevisiae. Telomeres also repress the expression of genes in their vicinity, a phenomenon known as telomere position effect. In an attempt to construct a conditional telomere, an inducible promoter was introduced adjacent to a single telomere of a chromosome such that transcription could be induced toward the end of the chromosome. Transcription toward two other essential chromosomal elements, centromeres and origins of replication, eliminates their function. In contrast, transcription toward a telomere did not affect the stability function of the telomere as measured by the loss rate of the transcribed chromosome. Transcription proceeded through the entire length of the telomeric tract and caused a modest reduction in the average length of the transcribed telomere. Transcription of the telomere substantially reduced the frequency of cells in which an adjacent URA3 gene was subject to telomere position effect. These results indicate that telomere position effect can be alleviated without compromising chromosome stability.
Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior.
Authors: Schroeder R , London E , Brown D
Published: May 2006

Proteins anchored by GPI are poorly solubilized from cell membranes by cold nonionic detergents because they associate with detergent-resistant membranes rich in cholesterol and sphingolipids. In this study, we demonstrated that cholesterol and sphingolipid-rich liposomes were incompletely solubilized by Triton X-100. GPI-anchored placental alkaline phosphatase incorporated in these liposomes was also not solubilized by cold Triton X-100. As sphingolipids have much higher melting temperatures (Tm) than cellular phospholipids, a property correlated with Tm might cause detergent inextractability. In support of this idea, we found that the low-Tm lipid dioleoyl phosphatidylcholine (DOPC) was efficiently extracted from detergent-resistant liposomes by Triton X-100, whereas the high-Tm lipid dipalmitoyl phosphatidylcholine (DPPC) was not. The fluorescence polarization of liposome-incorporated diphenylhexatriene was measured to determine the "fluidity" of the detergent-resistant liposomes. We found that these liposomes were about as fluid as DPPC/cholesterol liposomes, which were present in the liquid-ordered phase, and much less fluid than DOPC or DOPC/cholesterol liposomes. These findings may explain the behavior of GPI-anchored proteins, which often have saturated fatty acyl chains and should prefer a less-fluid membrane. Therefore, we propose that acyl chain interactions can influence the association of GPI-anchored proteins with detergent-resistant membrane lipids. The affinity of GPI-anchored proteins for a sphingolipid-rich membrane phase that is not in the liquid crystalline state may be important in determining their cellular localization.
Specific photocrosslinking of DNA-protein complexes: identification of contacts between integration host factor and its target DNA.
Authors: Yang S W , Nash H A
Published: May 2006

Azide moieties have been specifically placed in the backbone of DNA by chemical coupling between azidophenacyl bromide and uniquely positioned phosphorothioate residues. The derivatized DNA forms specific complexes with a DNA-binding protein and, following irradiation with 302-nm light, makes specific crosslinks to the protein. Isolation of this covalent complex, followed by tryptic digestion and Edman degradation of the resulting crosslinked peptide, identifies the portion of the protein that is near the derivatized segment of the target DNA. We use this method to probe the interaction between a specific DNA sequence and integration host factor (IHF) protein. A single IHF heterodimer is known to contact > 25 bp of DNA and thereby introduce a sharp bend. Two segments of a typical IHF site were derivatized with aryl azide. Although the segments were separated by only 5 bp, they crosslinked to different subunits of IHF. The locations of the crosslinks support our current view for the way IHF protein binds to and bends its specific targets.
Terminal protein-primed DNA amplification.
Authors: Blanco L , Lázaro J M , de Vega M , Bonnin A , Salas M
Published: May 2006

By using appropriate amounts of four bacteriophage phi 29 DNA replication proteins--terminal protein, DNA polymerase, protein p6 (double-stranded DNA-binding protein), and protein p5 (single-stranded DNA-binding protein)--it has been possible to amplify limited amounts of the 19,285-bp-long phi 29 DNA molecule by three orders of magnitude after 1 hr of incubation at 30 degrees C. Moreover, the quality of the amplified material was demonstrated by transfection experiments, in which infectivity of the synthetic (amplified) phi 29 DNA, measured as the ability to produce phage particles, was identical to that of the natural phi 29 DNA obtained from virions. The results presented in this paper establish some of the requisites for the development of isothermal DNA amplification strategies based on the bacteriophage phi 29 DNA replication machinery that are suitable for the amplification of very large (> 70 kb) segments of DNA.
Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells.
Authors: Sauvageau G , Lansdorp P M , Eaves C J , Hogge D E , Dragowska W H , Reid D S , Largman C , Lawrence H J , Humphries R K
Published: May 2006

Class I homeobox (Hox) genes encode a major group of transcription factors controlling embryonic development and have been implicated in the continuing process of hematopoietic cell differentiation. They are clustered on four chromosomes and, in early development, exhibit spatially restricted expression with respect to their 3'-->5' chromosomal position. By using an improved PCR-based method for amplifying total cDNA derived from limited cell numbers, we now describe the expression of class I Hox genes in highly purified CD34+ cell subpopulations isolated from normal human bone marrow that represent functionally distinct stem and progenitor cell compartments. Our data indicate that at least 16 different Hox genes, mainly from the A and the B clusters, are expressed in one or more of these subpopulations of human hematopoietic cells. Moreover, markedly elevated expression of some of the Hox genes found at the 3' end of the A and B clusters (e.g., HoxB3) was a unique feature of the subpopulations that contained the most primitive functionally defined cells, whereas genes located in the 5' region of each cluster (e.g., HoxA10) were found to be expressed at nearly equal levels in the CD34+ subpopulations analyzed. In contrast to the findings for CD34+ cells, expression of two selected Hox genes, HoxB3 and HoxA10, was virtually extinguished in the CD34- fraction of bone marrow cells. These results demonstrate the expression of a broad range of Hox genes in primitive hematopoietic cells and point to the existence of a regulated program of Hox gene expression during their normal development.
Critical role of the group A streptococcal capsule in pharyngeal colonization and infection in mice.
Authors: Wessels M R , Bronze M S
Published: May 2006

To study the role of the group A streptococcal capsule in pharyngeal colonization, we used two acapsular mutants derived from a type 24 strain of group A Streptococcus by transposon mutagenesis. One mutant had a stable acapsular phenotype due to a transposon-associated chromosomal deletion of essential capsule synthetic genes, while the second mutant could revert to the encapsulated phenotype at a low frequency (< 10(-4)) upon spontaneous excision of the transposon from the capsule-synthesis region of the chromosome. Both acapsular mutants were sensitive to phagocytic killing in vitro and had reduced virulence in mice after intraperitoneal challenge. Mice inoculated intranasally with the stable acapsular mutant rapidly cleared the organisms from the pharynx, and no mice died. In contrast, throat cultures of animals challenged with the revertible mutant yielded many encapsulated revertants, and mortality was similar to that of animals challenged with the parent strain. The rapid emergence of a population of encapsulated revertants in the pharynx implies that the capsule conferred a powerful selective advantage in this environmental niche. Together with the complete avirulence of the stable acapsular mutant, these observations indicate that the hyaluronic acid capsule plays a critical role in colonization and infection of the pharynx by group A streptococci.
Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids.
Authors: Sigal C T , Zhou W , Buser C A , McLaughlin S , Resh M D
Published: May 2006

Membrane targeting of pp60src (Src) is mediated by its myristoylated amino terminus. We demonstrate that, in addition to myristate, six basic residues in the amino terminus are essential for high-affinity binding to the lipid bilayer via electrostatic interaction with acidic phospholipids. Specifically, c-Src was shown to bind 2500-fold more strongly to vesicles composed of the physiological ratio of 2:1 phosphatidylcholine (PC)/phosphatidylserine (PS) than to neutral PC bilayer vesicles. The apparent Kd for binding of c-Src to the PC/PS bilayer was 6 x 10(-7) M. This interaction is sufficiently strong to account for c-Src membrane targeting. Mutants of c-Src in which the amino-terminal basic residues were replaced by neutral asparagine residues exhibited binding isotherms approaching that of wild-type binding to neutral bilayers (apparent Kd of 2 x 10(-3) M). The transforming v-Src and activated c-Src (Y527F) proteins also bound more strongly to PC/PS bilayers (apparent Kd of approximately 1 x 10(-5) M) than to neutral PC bilayers. In vivo experiments with Src mutants confirmed the role of positive charge in mediating membrane binding and cellular transformation.
Modulation of brain Na+ channels by a G-protein-coupled pathway.
Authors: Ma J Y , Li M , Catterall W A , Scheuer T
Published: May 2006

Na+ channels in acutely dissociated rat hippocampal neurons and in Chinese hamster ovary (CHO) cells transfected with a cDNA encoding the alpha subunit of rat brain type IIA Na+ channel (CNaIIA-1 cells) are modulated by guanine nucleotide binding protein (G protein)-coupled pathways under conditions of whole-cell voltage clamp. Activation of G proteins by 0.2-0.5 mM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable GTP analog, increased Na+ currents recorded in both cell types. The increase in current amplitude was caused by an 8- to 10-mV negative shift in the voltage dependence of both activation and inactivation. The effects of G-protein activators were blocked by treatment with pertussis toxin or guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), a nonhydrolyzable GDP analog, but not by cholera toxin. GDP[beta S] (2 mM) alone had effects opposite those of GTP[gamma S], shifting Na(+)-channel gating 8-10 mV toward more-positive membrane potentials and suggesting that basal activation of G proteins in the absence of stimulation is sufficient to modulate Na+ channels. In CNaIIA-1 cells, thrombin, which activates pertussis toxin-sensitive G proteins in CHO cells, caused a further negative shift in the voltage dependence of Na(+)-channel activation and inactivation beyond that observed with GTP alone. The results in CNaIIA-1 cells indicate that the alpha subunit of the Na+ channel alone is sufficient to mediate G protein effects on gating. The modulation of Na+ channels via a G-protein-coupled pathway acting on Na(+)-channel alpha subunits may regulate electrical excitability through integration of different G-protein-coupled synaptic inputs.
Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction.
Authors: Roskelley C D , Desprez P Y , Bissell M J
Published: May 2006

Extracellular matrix (ECM) profoundly influences the growth and differentiation of the mammary gland epithelium, both in culture and in vivo. Utilizing a clonal population of mouse mammary epithelial cells that absolutely requires an exogenous ECM for function, we developed a rapid assay to study signal transduction by ECM. Two components of the cellular response to a basement membrane overlay that result in the expression of the milk protein beta-casein were defined. The first component of this response involves a rounding and clustering of the cells that can be physically mimicked by plating the cells on a nonadhesive substratum. The second component is biochemical in nature, and it is associated with beta 1 integrin clustering and increased tyrosine phosphorylation. The second component is initiated in a morphology-independent manner, but the proper translation of this biochemical signal into a functional response requires cell rounding and cell clustering. Thus, physical and biochemical signal transduction events contribute to the ECM-dependent regulation of tissue-specific gene expression in mouse mammary epithelial cells.
Time course of Ca2+ concentration triggering exocytosis in neuroendocrine cells.
Authors: Chow R H , Klingauf J , Neher E
Published: May 2006

We have used the secretory response of chromaffin cells to estimate the submembrane intracellular Ca2+ concentration ([Ca2+]i) "seen" by secretory granules during short depolarizations. The rate of secretion during a depolarization was assessed by combining the electrochemical method of amperometry and electrical capacitance measurements. The rate was then related to [Ca2+]i based on a previous characterization of how Ca2+ affects the dynamics of vesicle priming and fusion in chromaffin cells [Heinemann, C., Chow, R. H., Neher, E. & Zucker, R. S. (1994) Biophys. J. 67, in press]. Calculated [Ca2+]i rose during the depolarization to a peak of < 10 microM, then decayed over tens of milliseconds. In synapses, vesicles are presumed to be located within nanometers of Ca2+ channels where [Ca2+]i is believed to rise in only microseconds to near steady-state levels of hundreds of micromolar. Channel closure should lead to a decrease in [Ca2+]i also in microseconds. Our findings of the slower time course and the lower peak [Ca2+]i suggest that in chromaffin cells, unlike synapses, Ca2+ channels and vesicles are not strictly colocalized. This idea is consistent with previously published data on dense-core vesicle secretion from diverse cell types.
Keratinocyte expression of B7-1 in transgenic mice amplifies the primary immune response to cutaneous antigens.
Authors: Williams I R , Ort R J , Kupper T S
Published: May 2006

Resting epidermal keratinocytes do not express B7-1 and other known CD28 counterligands with costimulatory activity. The absence of these costimulators on keratinocytes correlates with their ability to preferentially induce T-cell anergy instead of T-cell activation. To test the hypothesis that keratinocytes expressing a CD28 counterligand would be more effective inducers of T-cell-mediated immune responses in skin, we prepared transgenic mice in which expression of the B7-1 costimulator was targeted to basal keratinocytes by using the human K14 promoter. Keratinocytes from the K14/B7-1 transgenic line expressed high levels of surface B7-1. No spontaneous inflammatory changes were seen in transgenic skin, but epicutaneous application of contact sensitizers to these mice elicited a stronger primary ear swelling response than in controls. Sites of initial hapten application in transgenic mice also responded much more strongly to reapplication of hapten to a remote cutaneous site. Epidermal cell suspensions from transgenic mice contained normal numbers of Langerhans cells and dendritic epidermal T cells when analyzed by flow cytometry. Systemic treatment of the transgenic mice with interferon gamma induced high levels of class II major histocompatibility complex expression on keratinocytes but was not sufficient to initiate an inflammatory response. We conclude that the constitutive expression of the B7-1 molecule in vivo on a nonprofessional antigen-presenting cell is not by itself sufficient to trigger inflammatory changes, but B7-1 expression amplifies the host immune responses after exposure to nonself antigens presented by B7-1-expressing cells.
Comparisons of eukaryotic genomic sequences.
Authors: Karlin S , Ladunga I
Published: May 2006

A method for assessing genomic similarity based on relative abundances of short oligonucleotides in large DNA samples is introduced. The method requires neither homologous sequences nor prior sequence alignments. The analysis centers on (i) dinucleotide (and tri- and tetra-) relative abundance extremes in genomic sequences, (ii) distances between sequences based on all dinucleotide relative abundance values, and (iii) a multidimensional partial ordering protocol. The emphasis in this paper is on assessments of general relatedness of genomes as distinguished from phylogenetic reconstructions. Our methods demonstrate that the relative abundance distances almost always differ more for genomic interspecific sequence comparisons than for genomic intraspecific sequence comparisons, indicating congruence over different genome sequence samples. The genomic comparisons are generally concordant with accepted phylogenies among vertebrate and among fungal species sequences. Several unexpected relationships between the major groups of metazoa, fungal, and protist DNA emerge, including the following. (i) Schizosaccharomyces pombe and Saccharomyces cerevisiae in dinucleotide relative abundance distances are as similar to each other as human is to bovine. (ii) S. cerevisiae, although substantially far from, is significantly closer to the vertebrates than are the invertebrates (Drosophila melanogaster, Bombyx mori, and Caenorhabditis elegans). This phenomenon may suggest variable evolutionary rates during the metazoan radiations and slower changes in the fungal divergences, and/or a polyphyletic origin of metazoa. (iii) The genomic sequences of D. melanogaster and Trypanosoma brucei are strikingly similar. This DNA similarity might be explained by some molecular adaptation of the parasite to its dipteran (tsetse fly) host, a host-parasite gene transfer hypothesis. Robustness of the methods may be due to a genomic signature of dinucleotide relative abundance values reflecting DNA structures related to dinucleotide stacking energies, constraints of DNA curvature, and mechanisms attendant to replication, repair, and recombination.
Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins.
Authors: Hoffmann H M , Catron K M , van Wijnen A J , McCabe L R , Lian J B , Stein G S , Stein J L
Published: May 2006

The OC box of the rat osteocalcin promoter (nt -99 to -76) is the principal proximal regulatory element contributing to both tissue-specific and developmental control of osteocalcin gene expression. The central motif of the OC box includes a perfect consensus DNA binding site for certain homeodomain proteins. Homeodomain proteins are transcription factors that direct proper development by regulating specific temporal and spatial patterns of gene expression. We therefore addressed the role of the homeodomain binding motif in the activity of the OC promoter. In this study, by the combined application of mutagenesis and site-specific protein recognition analysis, we examined interactions of ROS 17/2.8 osteosarcoma cell nuclear proteins and purified Msx-1 homeodomain protein with the OC box. We detected a series of related specific protein-DNA interactions, a subset of which were inhibited by antibodies directed against the Msx-1 homeodomain but which also recognize the Msx-2 homeodomain. Our results show that the sequence requirements for binding the Msx-1 or Msx-2 homeodomain closely parallel those necessary for osteocalcin gene promoter activity in vivo. This functional relationship was demonstrated by transient expression in ROS 17/2.8 osteosarcoma cells of a series of osteocalcin promoter (nt -1097 to +24)-reporter gene constructs containing mutations within and flanking the homeodomain binding site of the OC box. Northern blot analysis of several bone-related cell types showed that all of the cells expressed msx-1, whereas msx-2 expression was restricted to cells transcribing osteocalcin. Taken together, our results suggest a role for Msx-1 and -2 or related homeodomain proteins in transcription of the osteocalcin gene.
Discovery of endogenous catecholamines in lymphocytes and evidence for catecholamine regulation of lymphocyte function via an autocrine loop.
Authors: Bergquist J , Tarkowski A , Ekman R , Ewing A
Published: May 2006

Evidence has been obtained that catecholamines and their metabolites are present in single lymphocytes and extracts of T- and B-cell clones by use of capillary electrophoresis with electrochemical detection. Pharmacological inhibition of tyrosine hydroxylase reduces observed catecholamine levels, suggesting catecholamine synthesis by lymphocytes. Intracellular dopamine levels are shown to be increased by extra-cellular dopamine, suggesting a cellular-uptake mechanism. Furthermore, incubation with either dopamine or L-dihydroxyphenylalanine, a precursor of dopamine, results in a dose-dependent inhibition of lymphocyte proliferation and differentiation. Together, these results suggest the presence of an autocrine loop whereby lymphocytes down-regulate their own activity.
The beetle Tribolium castaneum has a fushi tarazu homolog expressed in stripes during segmentation.
Authors: Brown S J , Hilgenfeld R B , Denell R E
Published: May 2006

The genetic control of embryonic organization is far better understood for the fruit fly Drosophila melanogaster than for any other metazoan. A gene hierarchy acts during oogenesis and embryogenesis to regulate the establishment of segmentation along the anterior-posterior axis, and homeotic selector genes define developmental commitments within each parasegmental unit delineated. One of the most intensively studied Drosophila segmentation genes is fushi tarazu (ftz), a pair-rule gene expressed in stripes that is important for the establishment of the parasegmental boundaries. Although ftz is flanked by homeotic selector genes conserved throughout the metazoa, there is no evidence that it was part of the ancestral homeotic complex, and it has been unclear when the gene arose and acquired a role in segmentation. We show here that the beetle Tribolium castaneum has a ftz homolog located in its Homeotic complex and expressed in a pair-rule fashion, albeit in a register differing from that of the fly gene. These and other observations demonstrate that a ftz gene preexisted the radiation of holometabolous insects and suggest that it has a role in beetle embryogenesis which differs somewhat from that described in flies.
Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators.
Authors: Finley R L , Brent R
Published: May 2006

We characterized interactions between Drosophila melanogaster cell cycle regulatory proteins by a yeast interaction-mating technique. The results were displayed as two-dimensional matrices that revealed individual binary interactions between proteins. Each protein (Cdi, cyclin-dependent kinase interactor) interacted with a distinct spectrum of cyclin-dependent kinases (Cdk) from Drosophila and other organisms. Some Cdis interacted with other Cdis, indicating that these proteins may form trimeric complexes that include Cdks. Similar analysis of interaction matrices may be generally useful in detecting other multiprotein complexes and in establishing connectivity between individual complex members. Moreover, such analysis may also help assign function to newly identified proteins, identify domains involved in protein-protein interactions, and aid the dissection of genetic regulatory networks.
Enrichment for RNA molecules that bind a Diels-Alder transition state analog.
Authors: Morris K N , Tarasow T M , Julin C M , Simons S L , Hilvert D , Gold L
Published: May 2006

RNA molecules that bind a transition state analog for a Diels-Alder reaction (Kd = 0.35 +/- 0.05 mM) were isolated from a starting pool of approximately 10(14) sequences by affinity chromatography. After the initial rise and plateau of the amount of RNA that eluted with soluble analog, a step gradient elution was used to further enrich the pool for sequences with higher affinities for the target. To our knowledge, the isolation of RNA molecules that bind either a nonplanar or a hydrophobic ligand has not been reported previously. A conserved nucleotide sequence and secondary structure present in many of the RNA molecules are necessary but not sufficient for binding the analog. No catalysts of the targeted Diels-Alder reaction were found among the binders. The absence of catalysis contrasts with previous successful experiments with antibodies and suggests that other strategies may be needed to identify oligonucleotides with diverse catalytic activities.
Oxymetry deep in tissues with low-frequency electron paramagnetic resonance.
Authors: Halpern H J , Yu C , Peric M , Barth E , Grdina D J , Teicher B A
Published: May 2006

We have measured the oxygen concentration in the body water of murine FSa and NFSa fibrosarcomas using a new method for quantitative oxygen concentration determination deep in the tissues of a living animal. The measurement uses unusually low-frequency electron paramagnetic spectroscopy sensitive to substrate 7 cm deep in tissue, partially deuterated spin probes (spin labels of molecular mass 195, approximating that of glucose) whose distribution compartment can be targeted with facile adduct substitution, and novel analytic techniques. We show that the water-compartment oxygen concentration of the tumors decreases as the tumor size increases and also shows a trend to decrease as radiobiologic hypoxia increases. An oxymetric spectral image of the tumor is presented. The technique will improve with larger human tissue samples. It provides the potential to quantitatively assess tissue hypoxia in ischemic or preischemic states in stroke and myocardial infarction. It will allow direct assessment of tumor hypoxia to determine the usefulness of radiation and chemotherapy adjuvants directed to hypoxic cell compartments.
Spectral hole burning and selection of conformational substates in chromoproteins.
Authors: Friedrich J , Gafert J , Zollfrank J , Vanderkooi J , Fidy J
Published: May 2006

We investigated spectral holes burnt at 1.5 K into the origins of several tautomeric forms of mesoporphyrin IX-substituted horseradish peroxidase at pH 8 under pressures up to 2 MPa. From the pressure-induced lineshift the compressibility of the apoprotein could be determined. We found that the compressibility changed significantly when measured at different tautomer origins. It was concluded that there must be a correlation between the tautomer configurations of the chromophore and the actual structures of the apoprotein. As a consequence, specific conformational substates of the protein can be selected by optical selection of the associated tautomers.
Lysophosphatidylcholine upregulates the level of heparin-binding epidermal growth factor-like growth factor mRNA in human monocytes.
Authors: Nakano T , Raines E W , Abraham J A , Klagsbrun M , Ross R
Published: May 2006

Lysophosphatidylcholine is increased in the plasma of hypercholesterolemic patients, is a component of oxidatively modified low-density lipoprotein, and, as such, may play an important role in atherosclerosis. Here we demonstrate that in human monocytes, lysophosphatidylcholine increases the level of mRNA encoding the heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent smooth muscle mitogen. Lysophosphatidylcholine treatment also enhances the release of heparin-binding mitogenic activity by these cells in culture. The anti-inflammatory glucocorticoid dexamethasone inhibits the upregulation of HB-EGF mRNA induced by either lysophosphatidylcholine or bacterial lipopolysaccharide in cultured monocytes. However, the responses induced by lysophosphatidylcholine and by lipopolysaccharide differ in their kinetics. In addition, the response to lysophosphatidylcholine is resistant to the action of cycloheximide, whereas the response to lipopolysaccharide is not, suggesting that the activation mechanisms induced by these two stimuli are different. Since a nuclear run-on assay showed no effect of lysophosphatidylcholine on the transcription of the HB-EGF gene, we speculate that lysophosphatidylcholine may increase the level of HB-EGF mRNA by altering the processing or degradation of primary or mature transcripts. Lysophosphatidylcholine enhancement of monocyte production of HB-EGF may represent an important result of the interactions among oxidized low-density lipoprotein and monocyte-derived macrophages and may play a role in initiation of smooth muscle proliferation in atherogenesis.
Atomic force microscopy produces faithful high-resolution images of protein surfaces in an aqueous environment.
Authors: Karrasch S , Hegerl R , Hoh J H , Baumeister W , Engel A
Published: May 2006

The atomic force microscope has the potential to monitor structural changes of a biological system in its native environment. To correlate them with the biological function at a molecular level, high lateral and vertical resolution are required. Here we demonstrate that the atomic force microscope is capable of imaging the surface of the hexagonally packed intermediate layer of Deinococcus radiodurans in buffer solution with a lateral resolution of 1 nm and a vertical resolution of 0.1 nm. On average, these topographs differ from those determined by electron microscopy by <0.5 nm.
Oncogene activation of human keratin 18 transcription via the Ras signal transduction pathway.
Authors: Pankov R , Umezawa A , Maki R , Der C J , Hauser C A , Oshima R G
Published: May 2006

Keratin 8 (K8) and keratin 18 (K18) are intermediate filament proteins normally expressed in simple epithelial tissues and persistently expressed in a wide variety of carcinomas. Ectopic expression of K8 and K18 occurs in some epidermal and murine skin carcinomas induced by chemical carcinogenesis or oncogenic ras expression. We show here that K18 is a direct target of the Ras signal transduction pathway, by demonstrating that activated Ha-Ras, as well as activated Src, Lck, or Raf, stimulates the transcription of K18. This activation is mediated by an enhancer element containing essential and closely spaced Ets and AP-1 transcription factor binding sites. Oncogene activation of K18 transcription provides a molecular explanation for the persistent and sometimes unexpected expression of K18 in such a wide variety of tumors.
Splicing function of mammalian U6 small nuclear RNA: conserved positions in central domain and helix I are essential during the first and second step of pre-mRNA splicing.
Authors: Wolff T , Menssen R , Hammel J , Bindereif A
Published: May 2006

On the basis of mutational analyses in yeast, the highly conserved ACAGAGA sequence of U6 small nuclear RNA (snRNA) and the adjacent U6-U2 helix I have been proposed to be part of the active center of the spliceosome. We report here a detailed analysis of the human U6 snRNA sequence requirements during the first and second step of splicing, using a mammalian in vitro splicing-complementation system and a mutational approach. Positions A53G54C55 (helix Ib) were identified as important specifically for the first step, but not for spliceosome assembly. A45 of the ACAGAGA sequence and U52 of helix Ia function during the second step; in addition, the bulge separating helices Ia and Ib appears critical for the second step. In contrast, no splicing-essential sequences could be identified in the central domain upstream of the ACAGAGA sequence. In sum, our data demonstrate for the mammalian splicing system that discrete positions within the ACAGAGA sequence and helix I of U6 snRNA function during the first and second step of splicing, suggesting that these two sequence elements are closely associated with the catalytic center of the spliceosome. Comparison with previous results in yeast indicates a fundamental conservation of the U6 snRNA function in the pre-mRNA splicing mechanism.
Candida albicans estrogen-binding protein gene encodes an oxidoreductase that is inhibited by estradiol.
Authors: Madani N D , Malloy P J , Rodriguez-Pombo P , Krishnan A V , Feldman D
Published: May 2006

Candida albicans, the most common fungal pathogen of humans, possesses an estrogen-binding protein (EBP) that binds mammalian estrogens with high affinity. We report here the cloning and complete nucleotide sequence of a gene encoding a C. albicans EBP. Amino acid sequences obtained from cyanogen bromide fragments of purified EBP were used to design oligonucleotide primers for PCR. An 800-bp product was amplified and used to screen a C. albicans genomic library. A clone was isolated containing an insert with an open reading frame of 1221 nt capable of encoding a protein with 407 amino acids and having a calculated molecular mass of 46,073 Da, the estimated size of EBP. The cloned gene, expressed in Escherichia coli as a lacZ fusion protein, demonstrated high-affinity binding for estradiol and a competition profile comparable to C. albicans wild-type EBP. Northern blots of C. albicans RNA revealed a single transcript of approximately 1600 nt, whereas Southern blots identified three hybridizing fragments. Computer searches of data bases showed that EBP shares a 46% amino acid identity with the old yellow enzyme, an oxidoreductase from Saccharomyces cerevisiae, but was unrelated to the human estrogen receptor as previously speculated. In addition, a 51-amino acid region of EBP is highly conserved among a group of flavoproteins including old yellow enzyme. Expressed EBP was shown to exhibit oxidoreductase activity that could be inhibited by 17 beta-estradiol in vitro. In conclusion, the EBP from C. albicans has no evident homology to the mammalian steroid receptor superfamily but appears to be a member of a recently identified family of flavoproteins.
Social factors and health: the causation-selectionissue revisited.
Authors: Goldman N
Published: May 2006

Since social scientists rarely have access to
experimental data, they rely heavily upon observational studies. As a
consequence, their attempts to make causal inferences about the effects of
social factors--such as occupation or marital status--on health are plagued by
potential selection problems. Some researchers have addressed this
selection-causation problem on the basis of the presence or absence of a
particular aggregate pattern of health status. The rationale underlying this
approach derives from the investigators' hypotheses that the presence of
selection would lead to a particular type of pattern that is distinct from the
pattern that would result in the absence of selection. Although intuitively
appealing, this strategy appears to be seriously flawed. The essential weakness
is that the range of patterns that can result from selection is often much
broader than researchers have speculated.
Positioned nucleosomes inhibit Dam methylation invivo.
Authors: Kladde M P , Simpson R T
Published: May 2006

Escherichia coli Dam DNA methyltransferase can
methylate genomic GATC sites when expressed in Saccharomyces cerevisiae. Others
have observed changes in the level of methylation at specific sites and
suggested that these changes are related to transcriptional state or chromosomal
context. To test directly the influence of nucleosome location on the ability of
Dam methyltransferase to modify GATC sites in chromatin, we analyzed
minichromosomes containing precisely positioned nucleosomes in dam-expressing
yeast strains. Levels of methylation at individual GATC sites were rigorously
quantified by an oligonucleotide-probing procedure. Within the linker and
adjacent 21 bp of nucleosome-associated DNA, GATC sites were highly methylated,
whereas methylation was severely inhibited by histone-DNA contacts nearer to the
nucleosomal pseudodyad. Other DNA-protein complexes also interfere with Dam
methylation. These data are consistent with a model in which nucleosomes exert a
repressive influence on the biological functions of DNA by restricting access of
trans-acting factors to DNA.
Remarkable archaeal diversity detected in aYellowstone National Park hot spring environment.
Authors: Barns S M , Fundyga R E , Jeffries M W , Pace N R
Published: May 2006

Of the three primary phylogenetic domains--Archaea
(archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes)--Archaea is
the least understood in terms of its diversity, physiologies, and ecological
panorama. Although many species of Crenarchaeota (one of the two recognized
archaeal kingdoms sensu Woese [Woese, C. R., Kandler, O. & Wheelis, M. L.
(1990) Proc. Natl. Acad. Sci. USA 87, 4576-4579]) have been isolated, they
constitute a relatively tight-knit cluster of lineages in phylogenetic analyses
of rRNA sequences. It seemed possible that this limited diversity is merely
apparent and reflects only a failure to culture organisms, not their absence. We
report here phylogenetic characterization of many archaeal small subunit rRNA
gene sequences obtained by polymerase chain reaction amplification of mixed
population DNA extracted directly from sediment of a hot spring in Yellowstone
National Park. This approach obviates the need for cultivation to identify
organisms. The analyses document the existence not only of species belonging to
well-characterized crenarchaeal genera or families but also of crenarchaeal
species for which no close relatives have so far been found. The large number of
distinct archaeal sequence types retrieved from this single hot spring was
unexpected and demonstrates that Crenarchaeota is a much more diverse group than
was previously suspected. The results have impact on our concepts of the
phylogenetic organization of Archaea.
Delineation of a small region within the majortransactivation domain of the human glucocorticoid receptor that mediatestransactivation of gene expression.
Authors: Dahlman-Wright K , Almlöf T , McEwan I J , Gustafsson J A , Wright A P
Published: May 2006

Previous deletion analysis localized the major
transactivation function of the human glucocorticoid receptor to a 185-amino
acid segment close to the N terminus of the receptor protein. This region was
named tau 1 [Hollenberg, S. M. & Evans, R. M. (1988) Cell 55, 899-906]. To
delineate the smallest active region within tau 1, we have systematically tested
the transactivation capacity of deletion derivatives of the tau 1 domain, fused
to the glucocorticoid receptor DNA-binding domain, in yeast cells. Internal
scanning deletions suggested that residues near the C terminus of tau 1 are most
important for activity. Deletions of N-terminal and C-terminal sequences
identified a 41-amino acid "core" region near the C terminus of tau 1
that is crucial for tau 1 function. Small peptide fragments containing the tau 1
core region are competent for transactivation, while regions outside the tau 1
core are not active. We have previously demonstrated that the intact tau 1
domain squelches the activity of a minimal promoter in vivo and in vitro,
suggesting involvement of interactions with a component/components of the basal
transcription machinery in the mechanism of transactivation. This activity was
maintained in the tau 1 core-containing segments.
Fibroblast growth factor, but not activin, is a potentactivator of mitogen-activated protein kinase in Xenopusexplants.
Authors: Graves L M , Northrop J L , Potts B C , Krebs E G , Kimelman D
Published: May 2006

Isolated explants from the animal hemisphere of
Xenopus embryos were incubated with Xenopus basic fibroblast growth factor
(XbFGF) or human activin A. XbFGF incubation resulted in the rapid activation of
mitogen-activated protein kinase (MAPK) and ribosomal S6 protein kinase
(pp90rsk) in a dose-dependent manner with the highest levels of activation
occurring at 50 ng/ml. Maximal activation occurred within 6-10 min after the
addition of growth factor, and the activity of both kinases declined to
unstimulated levels after 30 min. Activin was unable to activate either MAPK or
pp90rsk in the Xenopus explants to a substantial level, although it induced
dorsal mesoderm better than XbFGF under the same experimental conditions. The
regulatory protein Xwnt-8 did not activate MAPK, nor did it enhance the
activation of MAPK by XbFGF. XbFGF was able to activate MAPK through at least
the midgastrula stage, suggesting that this family of growth factors may have a
role in gastrula-stage events.
Heat stress induces hsc70/nuclear topoisomerase Icomplex formation in vivo: evidence for hsc70-mediated, ATP-independentreactivation in vitro.
Authors: Ciavarra R P , Goldman C , Wen K K , Tedeschi B , Castora F J
Published: May 2006

We previously demonstrated that in murine T cells
thermotolerance correlated with heat shock protein 70 (hsp70) synthesis and
protection of nuclear type I topoisomerase (topo I). Topo I activity returned to
normal levels following heat stress even in cells not rendered thermotolerant by
a prior heat shock. Recovery of topo I activity was not dependent on de novo
protein synthesis, suggesting that the cell possesses a pathway(s) for refolding
this nuclear protein. In this report we demonstrate that topo I and hsc70, the
constitutively produced member of the hsp70 family, associated in vivo during
heat stress. That this association may play a physiologically important role in
protecting topo I activity from heat stress was suggested by the observation
that hsc70 protected topo I from heat inactivation in vitro. hsc70 but not actin
also reactivated previously heat-denatured topo I in a dose-dependent fashion.
However, refolding of heat-denatured topo I by purified hsc70 was inefficient
relative to a hsc70-containing cell lysate. Protection from heat inactivation as
well as reactivation by hsc70 did not require exogenous ATP. Similarly,
reactivation by the cell lysate was not inhibited by ADP or a nonhydrolyzable
analogue of ATP. Thus, our studies suggest that nuclear topo I complexes with
hsc70 during heat stress, which may explain, at least in part, why hsp70
proteins accumulate in the nucleus, particularly the nucleolus. This interaction
may limit heat-induced protein damage and/or accelerate restoration of protein
function in an ATP-independent reaction.
UV repair and resistance to solar UV-B in amphibianeggs: a link to population declines?
Authors: Blaustein A R , Hoffman P D , Hokit D G , Kiesecker J M , Walls S C , Hays J B
Published: May 2006

The populations of many amphibian species, in
widely scattered habitats, appear to be in severe decline; other amphibians show
no such declines. There is no known single cause for the declines, but their
widespread distribution suggests involvement of global agents--increased UV-B
radiation, for example. We addressed the hypothesis that differential
sensitivity among species to UV radiation contributes to these population
declines. We focused on species-specific differences in the abilities of eggs to
repair UV radiation damage to DNA and differential hatching success of embryos
exposed to solar radiation at natural oviposition sites. Quantitative
comparisons of activities of a key UV-damage-specific repair enzyme, photolyase,
among oocytes and eggs from 10 amphibian species were reproducibly
characteristic for a given species but varied > 80-fold among the species.
Levels of photolyase generally correlated with expected exposure of eggs to
sunlight. Among the frog and toad species studied, the highest activity was
shown by the Pacific treefrog (Hyla regilla), whose populations are not known to
be in decline. The Western toad (Bufo boreas) and the Cascades frog (Rana
cascadae), whose populations have declined markedly, showed significantly lower
photolyase levels. In field experiments, the hatching success of embryos exposed
to UV radiation was significantly greater in H. regilla than in R. cascadae and
B. boreas. Moreover, in R. cascadae and B. boreas, hatching success was greater
in regimes shielded from UV radiation compared with regimes that allowed UV
radiation. These observations are thus consistent with the UV-sensitivity
hypothesis.
Mammalian class IV alcohol dehydrogenase (stomachalcohol dehydrogenase): structure, origin, and correlation withenzymology.
Authors: Parés X , Cederlund E , Moreno A , Hjelmqvist L , Farrés J , Jörnvall H
Published: May 2006

The structure of a mammalian class IV alcohol
dehydrogenase has been determined by peptide analysis of the protein isolated
from rat stomach. The structure indicates that the enzyme constitutes a separate
alcohol dehydrogenase class, in agreement with the distinct enzymatic
properties; the class IV enzyme is somewhat closer to class I (the
"classical" liver alcohol dehydrogenase; approximately 68% residue
identities) than to the other classes (II, III, and V; approximately 60% residue
identities), suggesting that class IV might have originated through duplication
of an early vertebrate class I gene. The activity of the class IV protein toward
ethanol is even higher than that of the classical liver enzyme. Both Km and kcat
values are high, the latter being the highest of any class characterized so far.
Structurally, these properties are correlated with replacements at the active
site, affecting both substrate and coenzyme binding. In particular, Ala-294
(instead of valine) results in increased space in the middle section of the
substrate cleft, Gly-47 (instead of a basic residue) results in decreased charge
interactions with the coenzyme pyrophosphate, and Tyr-363 (instead of a basic
residue) may also affect coenzyme binding. In combination, these exchanges are
compatible with a promotion of the off dissociation and an increased turnover
rate. In contrast, residues at the inner part of the substrate cleft are bulky,
accounting for low activity toward secondary alcohols and cyclohexanol.
Exchanges at positions 259-261 involve minor shifts in glycine residues at a
reverse turn in the coenzyme-binding fold. Clearly, class IV is distinct in
structure, ethanol turnover, stomach expression, and possible emergence from
class I.
The non-B-DNA structure of d(CA/TG)n differs from thatof Z-DNA.
Authors: Kladde M P , Kohwi Y , Kohwi-Shigematsu T , Gorski J
Published: May 2006

Chemical probing of two predominantly alternating
purine-pyrimidine d(CA/TG)n repeats led us to propose previously that in
supercoiled plasmids these elements adopt a non-B-DNA structure distinct from
that of Z-DNA formed by d(CG)n sequences. Here, we present further evidence
supporting this contention. Reactivity with the conformation-sensitive reagent
chloroacetaldehyde, which reacts with unpaired adenines and cytosines, was
confined strictly to adenines in the d(CA/TG)n repeat. In contrast, only bases
outside the d(CG)n repeat exhibited chloroacetaldehyde reactivity.
Two-dimensional gel analysis of topoisomers containing d(CA/TG)n tracts with
bases out of strict purine-pyrimidine alteration revealed multiple
superhelical-dependent transitions to an alternative left-handed structure.
Within individual plasmid molecules, these multiple transitions resulted from
the stepwise conversion of contiguous segments of alternating purine-pyrimidine
sequence, which are delimited by bases out of alternation, to the full-length
alternative conformation. When the left-handed helices increased in length to
include more bases out of alternation, the average helical pitch changed
substantially to produce a less tightly wound left-handed helix. Overall, these
data indicate that d(CA/TG)n tracts adopt a left-handed conformation
significantly different from that of the canonical Z-DNA structure of d(CG)n
sequences.