bluuebunny/crossref_metadata_2025 huggingface dataset:
The DNA-activated protein kinase is required for the phosphorylation of replication protein A during simian virus 40 DNA replication.
Authors: Brush G S , Anderson C W , Kelly T J
Published: May 2006

The 32-kDa subunit of replication protein A (RPA) is phosphorylated during the S phase of the cell cycle in vivo and during simian virus 40 DNA replication in vitro. To explore the functional significance of this modification, we purified a HeLa cell protein kinase that phosphorylates RPA in the presence of single-stranded DNA. By several criteria we identified the purified enzyme as a form of the DNA-activated protein kinase (DNA-PK), a previously described high molecular weight protein kinase that is capable of phosphorylating a number of nuclear DNA binding proteins. Phosphorylation of RPA by DNA-PK is stimulated by natural single-stranded DNAs but not by homopolymers lacking secondary structure. Studies with the simian virus 40 model system indicate that DNA-PK is required for DNA-replication-dependent RPA phosphorylation. Depletion of the kinase activity, however, has no effect on the extent of DNA replication in vitro. Our data support a model in which phosphorylation of RPA by DNA-PK is activated by formation of replication intermediates containing single- and double-stranded regions. This event may be involved in a signaling mechanism that coordinates DNA replication with the cell cycle.
The outer surface protein A of the spirochete Borrelia burgdorferi is a plasmin(ogen) receptor.
Authors: Fuchs H , Wallich R , Simon M M , Kramer M D
Published: May 2006

The spirochete Borrelia burgdorferi is the causative agent of Lyme borreliosis (Lyme disease) and is transmitted to mammalian hosts by tick vectors. In humans, the bacteria induce a complex disease, which involves the skin, joints, heart, and nervous system. However, the pathogenic principles of this multisystem illness are far from being understood. To disseminate from the site of the tick bite and invade multiple organ sites, spirochetes have to penetrate normal tissue barriers, such as vascular basement membranes and other organized extracellular matrices. Substantial evidence from other invasive bacterial infections suggest that spirochetes may use endogenous or host-derived enzymes--in particular, proteinases--for this purpose. Here we show that B. burgdorferi binds human plasmin(ogen)--mainly via its outer cell surface lipoprotein A. Binding of plasminogen to spirochetal receptor leads to an accelerated formation of active plasmin in the presence of host-derived plasminogen activator. The cell-surface-associated plasmin cannot be regulated by the serum inhibitor alpha 2-antiplasmin and degrades high-molecular-weight glycoproteins, such as fibronectin. It is suggested that the acquisition of host-derived proteinase plasmin(ogen) contributes to the pathogenicity of B. burgdorferi.
Tagged mutations at the Tox1 locus of Cochliobolus heterostrophus by restriction enzyme-mediated integration.
Authors: Lu S , Lyngholm L , Yang G , Bronson C , Yoder O C , Turgeon B G
Published: May 2006

We have used the restriction enzyme-mediated integration insertional mutagenesis procedure to tag the Tox1 locus in the filamentous Ascomycete Cochliobolus heterostrophus. Mutations at other, unselected, loci were also identified and a high proportion (30-50%) of them were tagged. This procedure may be of general utility for simultaneously mutating and tagging genes in fungi and in other eukaryotes. The Tox1 locus of C. heterostrophus has been defined by Mendelian analysis as a single genetic element that controls production of T toxin, a linear polyketide involved in virulence of the fungus to its host plant, corn. To tag Tox1, protoplasts of a Tox1+ (T-toxin producing) strain were transformed with a linearized, nonhomologous plasmid along with an excess of the restriction enzyme used to linearize the plasmid. Of 1310 transformants recovered, two produced no detectable T toxin in culture or on corn plants. In each of these transformants, the Tox- mutation mapped at Tox1, was tagged with the selectable marker (hygB) on the transforming plasmid, and was tightly linked to the other tagged Tox- mutation. The two mutations, however, represent two different points of plasmid insertion at the Tox1 locus.
Coherent nuclear dynamics at room temperature in bacterial reaction centers.
Authors: Vos M H , Jones M R , Hunter C N , Breton J , Martin J L
Published: May 2006

A room-temperature study is reported of the femtosecond spectral evolution of the stimulated emission band of the primary electron-transfer precursor P* in bacterial photosynthesis. The study was performed with membranes of the antenna-deficient RCO1 mutant of Rhodobacter sphaeroides. A time-dependent red shift, reflecting nuclear motion out of the Franck-Condon region of the excited state, is resolved. Analysis of oscillatory features persisting for > 1 ps in the kinetics revealed main frequencies of the activated motions at 30, 84, 145, and 192 cm-1. The oscillations occur on the time scale of primary electron transfer. Our results set a lower limit for the vibrational dephasing time in P* that is not compatible with the usual assumption in theoretical treatments of complete vibrational relaxation prior to electron transfer, even at room temperature.
Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras.
Authors: Koch W J , Hawes B E , Allen L F , Lefkowitz R J
Published: May 2006

Stimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.
Oncogenic capacity of the E2F1 gene.
Authors: Johnson D G , Cress W D , Jakoi L , Nevins J R
Published: May 2006

Previous experiments have identified the E2F transcription factor as a potential downstream target for the action of cellular regulatory activities, such as the Rb tumor suppressor protein, that control cell growth and that, when altered, contribute to the development of human tumors. In light of these findings, we have assayed the ability of the E2F1 and DP1 genes, which encode heterodimeric partners that together create E2F activity, to act in an oncogenic fashion. We find that E2F1, particularly in combination with the DP1 product, cooperates with an activated ras oncogene to induce the formation of morphologically transformed foci in primary rat embryo fibroblast cultures. In addition, an E2F1 chimeric protein, in which sequences involved in Rb binding have been replaced with the herpesvirus VP16 activation domain, exhibits increased transformation activity. Cells transfected with E2F1 and DP1 or the E2F1-VP16 chimera form colonies in soft agar and induce tumor formation in nude mice. We conclude that deregulated E2F1 expression and function can have oncogenic consequences.
Transporters from H-2b, H-2d, H-2s, H-2k, and H-2g7 (NOD/Lt) haplotype translocate similar sets of peptides.
Authors: Schumacher T N , Kantesaria D V , Serreze D V , Roopenian D C , Ploegh H L
Published: May 2006

The TAP complex transports peptides from the cytosol into the lumen of the endoplasmic reticulum for presentation by major histocompatibility complex class I molecules. A limited degree of sequence polymorphism has been observed for the mouse TAP1 and TAP2 genes by restriction fragment length polymorphism and sequence analysis. However, functional polymorphism of the TAP transporter has thus far been observed for the rat only. Here we examine the effect of TAP polymorphism on ATP dependency and peptide specificity of TAP-mediated peptide transport and show that, in the mouse, polymorphism in TAP genes does not measurably alter the function of their gene products. We conclude that TAP polymorphism is unlikely to contribute to the development of autoimmune diseases and that, in the mouse, the specificity of the TAP transporter is matched to that of the F pocket of the class I molecules for which it provides the peptide substrates.
Stable expression of a functional GluR6 homomeric glutamate receptor channel in mammalian cells.
Authors: Tygesen C K , Rasmussen J S , Jones S V , Hansen A , Hansen K , Andersen P H
Published: May 2006

This study demonstrates the stable expression of a functional ionotropic glutamate receptor in a mammalian cell line of non-neuronal origin. The kainate-selective glutamate receptor GluR6 was constitutively expressed under the control of a metallothionein promoter. Clones were isolated expressing approximately 3 pmol of receptor per mg of protein. Functionality of the recombinant GluR6 was demonstrated both by electrophysiology and by Ca2+ imaging. Application of kainate to the GluR6-transfected cells activated an inward current response at a holding potential of -60 mV. The kainate concentration needed to evoke 50% of the maximal response (EC50) was calculated to be 0.82 +/- 0.39 microM. The current-voltage relationship was found to be almost linear, with a reversal potential of -2.5 +/- 4.8 mV. Application of kainate also resulted in an increase in the intracellular Ca2+ concentration measured by Ca2+ imaging. The pharmacological profile of [3H]kainate binding to the recombinant GluR6 resembled the high-affinity [3H]kainate binding sites in rat brain, showing high affinity for domoate (Ki = 5.1 +/- 3.0 nM) and kainate (Kd = 12.9 +/- 2.4 nM). No decrease in GluR6 expression level was observed over > 75 passages of the transfected cells. When domoate, a slowly desensitizing GluR6 agonist, was included in the growth medium for 3 weeks, the number of GluR6 binding sites decreased by 30%, indicating the importance of complete channel closure for stable expression.
Critical roles of the S3 segment and S3-S4 linker of repeat I in activation of L-type calcium channels.
Authors: Nakai J , Adams B A , Imoto K , Beam K G
Published: May 2006

Each of the four repeats (or motifs) of voltage-gated ion channels is thought to contain six transmembrane segments (S1-S6). Mutational analyses indicate that S4 functions as a voltage sensor and that the S5, S6, and S5-S6 linker contribute to formation of the ion pore. However, little information exists regarding the functional role(s) of the amino-terminal portion (S1-S3-S4 linker) of the repeats. Here we report that the amino acid composition of the S3 segment of repeat I and the linker connecting S3 and S4 segments of repeat I is critical for the difference in activation kinetics between cardiac and skeletal muscle L-type calcium channels. Mutant dihydropyridine receptors that have the skeletal muscle dihydropyridine receptor sequence in this region activated relatively slowly with the time constant of current activation (tau act) > 5 ms, whereas mutants that have the cardiac counterpart there activated relatively rapidly with tau act < 5 ms. Comparison of these two mutant groups indicates that a total of 11 conservative and 10 nonconservative amino acid changes from skeletal muscle to cardiac dihydropyridine receptor sequence are sufficient to convert activation from slow to fast. These data demonstrate a functional role for this region of voltage-gated ion channels.
Effect of ethanol on cytochrome P450 in the rat brain.
Authors: Warner M , Gustafsson J A
Published: May 2006

After a single dose of ethanol (0.8 ml/kg) administered intraperitoneally, the P450 content of the rat brain increased from 62 +/- 19 to 230 +/- 97 pmol/g (wet weight) of tissue (mean +/- SD, n = 5). Most of this increase could be accounted for by a 10- to 20-fold increase in the olfactory lobes and hypothalamic preoptic area. The P450s were identified by Western blot analysis and by microsequencing of the N-terminal ends after resolution of the proteins on SDS gels. They were identified as P450 2C7, 2C11, 2E1, 4A3, 4A8, and a member of the P450 2D family. In P450 extracted from the brains of control rats, P450 2C and 4A were also detectable but at a much lower concentration. P450 1A1, 2A1, 2B1, or 3A was not detected in the brains of either control or ethanol-treated rats. Oral administration of the same dose of ethanol resulted in a similar increase in the whole brain but smaller effects in the olfactory lobes. This effect of ethanol on the P450 in the brain has implications for the mechanism of toxicity and the development of tolerance to ethanol and for the effects of other drugs and environmental pollutants that act on the central nervous system.
Crystal structure of a disulfide-linked "trefoil" motif found in a large family of putative growth factors.
Authors: De A , Brown D G , Gorman M A , Carr M , Sanderson M R , Freemont P S
Published: May 2006

Porcine pancreatic spasmolytic polypeptide (PSP) belongs to a large family of homologous growth factor-like polypeptides characterized by a disulfide-linked "trefoil motif," duplicated and conserved in various family members. PSP contains two trefoil motifs, has several pharmacological actions on the gut, and has growth factor properties on epithelial cells in vitro. The human PSP analogue, human spasmolytic polypeptide, appears to be involved in many regenerative situations and, especially, in healing gastrointestinal ulcers. One member of the trefoil family, pS2, is secreted in approximately 50% of estrogen-dependent human breast carcinomas, which has led to its use as a tumor prognostic marker. Both pS2 and human spasmolytic polypeptide are also widely expressed in chronic gastrointestinal ulcerative conditions such as Crohn disease. Here we report the three-dimensional structure at 2.6-A resolution of a trefoil-containing protein, namely PSP, purified from porcine pancreas. The structure shows two homologous domains that share a supersecondary structure and disulfide bond pattern. The two domains pack asymmetrically giving rise to a number of protruding loops, exposed clefts, and an unusual electrostatic surface potential. Knowledge of the structure of PSP should allow the design of mutants to investigate further the function of PSP and other trefoil-containing peptides.
Bound water molecules and conformational stabilization help mediate an antigen-antibody association.
Authors: Bhat T N , Bentley G A , Boulot G , Greene M I , Tello D , Dall'Acqua W , Souchon H , Schwarz F P , Mariuzza R A , Poljak R J
Published: May 2006

We report the three-dimensional structures, at 1.8-A resolution, of the Fv fragment of the anti-hen egg white lysozyme antibody D1.3 in its free and antigen-bound forms. These structures reveal a role for solvent molecules in stabilizing the complex and provide a molecular basis for understanding the thermodynamic forces which drive the association reaction. Four water molecules are buried and others form a hydrogen-bonded network around the interface, bridging antigen and antibody. Comparison of the structures of free and bound Fv fragment of D1.3 reveals that several of the ordered water molecules in the free antibody combining site are retained and that additional water molecules link antigen and antibody upon complex formation. This solvation of the complex should weaken the hydrophobic effect, and the resulting large number of solvent-mediated hydrogen bonds, in conjunction with direct protein-protein interactions, should generate a significant enthalpic component. Furthermore, a stabilization of the relative mobilities of the antibody heavy- and light-chain variable domains and of that of the third complementarity-determining loop of the heavy chain seen in the complex should generate a negative entropic contribution opposing the enthalpic and the hydrophobic (solvent entropy) effects. This structural analysis is consistent with measurements of enthalpy and entropy changes by titration calorimetry, which show that enthalpy drives the antigen-antibody reaction. Thus, the main forces stabilizing the complex arise from antigen-antibody hydrogen bonding, van der Waals interactions, enthalpy of hydration, and conformational stabilization rather than solvent entropy (hydrophobic) effects.
CD40 ligand expression is defective in a subset of patients with common variable immunodeficiency.
Authors: Farrington M , Grosmaire L S , Nonoyama S , Fischer S H , Hollenbaugh D , Ledbetter J A , Noelle R J , Aruffo A , Ochs H D
Published: May 2006

Common variable immunodeficiency (CVI) is characterized by hypogammaglobulinemia and recurrent bacterial infections due to failure of CVI B cells to differentiate in vivo into immunoglobulin-secreting plasma cells. We hypothesized that T-cell dysfunction resulting in abnormal contact-mediated B-cell activation may play a prominent role in the failure of CVI B cells to produce specific antibody. We have previously shown that B-cell proliferation and IgE production after stimulation with anti-CD40 and interleukin (IL) 4 were normal in 22 CVI patients evaluated, indicating that CVI B cells respond to signals delivered via CD40. Here we report that CD40 ligand (gp39) mRNA expression by activated lymphocytes from CVI patients (n = 31) as a group was significantly depressed (P < 0.0001) compared with normal controls (n = 32). gp39 mRNA expression by activated lymphocytes from 13 CVI patients fell below the normal control range. T-cell surface expression of functional gp39 protein was correspondingly low in those patients with gp39 mRNA levels below normal control range and normal in patients with gp39 mRNA levels within normal control range. In CVI patients as a group, gp39 mRNA levels correlated with IL-2 mRNA levels (P < 0.002, r = 0.6) and production (P < 0.001, r = 0.7) but not with gene expression or production of other lymphokines evaluated, suggesting an as-yet-undetermined association between gp39 and IL-2 gene regulation. Of the 13 patients whose activated T cells exhibited gp39 mRNA expression below the normal control range, 2 had normal T-cell-derived lymphokine production, whereas the remaining 11 exhibited broader T-cell dysfunction, resulting in IL-2 deficiency, and in some patients deficient production of other lymphokines as well, reflecting a heterogeneity in the underlying mechanisms leading to depressed gp39 expression in these patients. The observation that both gene and surface expression of gp39 by activated T cells is depressed in a subgroup of CVI patients suggests that inefficient signaling via CD40 may be responsible, in part, for failure of B-cell differentiation in these patients.
Selection for mefloquine resistance in Plasmodium falciparum is linked to amplification of the pfmdr1 gene and cross-resistance to halofantrine and quinine.
Authors: Cowman A F , Galatis D , Thompson J K
Published: May 2006

Two chloroquine-resistant cloned isolates of Plasmodium falciparum were subjected to mefloquine selection to test if this resulted in alterations in chloroquine sensitivity and amplification of the pfmdr1 gene. The mefloquine-resistant lines derived by this selection were shown to have amplified and overexpressed the pfmdr1 gene and its protein product (Pgh1). Macrorestriction maps of chromosome 5, where pfmdr1 is encoded, showed that this chromosome has increased in size in response to mefloquine selection, indicating the presence of a gene(s) in this area of the genome that confers a selective advantage in the presence of mefloquine. Concomitant with the increase in mefloquine resistance was a corresponding increase in the level of resistance to halofantrine and quinine, suggesting a true multidrug-resistance phenotype. The mefloquine-selected parasite lines also showed an inverse relationship between the level of chloroquine resistance and increased pfmdr1 gene copy number. These results have important implications for the derivation of amplified copies of the pfmdr1 gene in field isolates, as they suggest that quinine pressure may be involved.
A human amphotropic retrovirus receptor is a second member of the gibbon ape leukemia virus receptor family.
Authors: van Zeijl M , Johann S V , Closs E , Cunningham J , Eddy R , Shows T B , O'Hara B
Published: May 2006

Retrovirus infection is initiated by binding of the viral envelope glycoprotein to a cell-surface receptor. The envelope proteins of type C retroviruses of mammals demonstrate similarities in structural organization and protein sequence. These similarities suggest the possibility that retroviruses from different interference groups might use related proteins as receptors, despite the absence of any relationship between retrovirus receptors isolated to date. To investigate this possibility, we have identified a human cDNA clone encoding a protein closely related to the receptor for gibbon ape leukemia virus and have found that it functions as the receptor for the amphotropic group of murine retroviruses. Expression of this protein (GLVR-2) is likely to be a requirement for infection of human cells by amphotropic retroviral vectors for purposes of gene therapy.
PLZF-RAR alpha fusion proteins generated from the variant t(11;17)(q23;q21) translocation in acute promyelocytic leukemia inhibit ligand-dependent transactivation of wild-type retinoic acid receptors.
Authors: Chen Z , Guidez F , Rousselot P , Agadir A , Chen S J , Wang Z Y , Degos L , Zelent A , Waxman S , Chomienne C
Published: May 2006

Recently, we described a recurrent variant translocation, t(11;17)(q23;q21), in acute promyelocytic leukemia (APL) which juxtaposes PLZF, a gene encoding a zinc finger protein, to RARA, encoding retinoic acid receptor alpha (RAR alpha). We have now cloned cDNAs encoding PLZF-RAR alpha chimeric proteins and studied their transactivating activities. In transient-expression assays, both the PLZF(A)-RAR alpha and PLZF(B)-RAR alpha fusion proteins like the PML-RAR alpha protein resulting from the well-known t(15;17) translocation in APL, antagonized endogenous and transfected wild-type RAR alpha in the presence of retinoic acid. Cotransfection assays showed that a significant repression of RAR alpha transactivation activity was obtained even with a very low PLZF-RAR alpha-expressing plasmid concentration. A "dominant negative" effect was observed when PLZF-RAR alpha fusion proteins were cotransfected with vectors expressing RAR alpha and retinoid X receptor alpha (RXR alpha). These abnormal transactivation properties observed in retinoic acid-sensitive myeloid cells strongly implicate the PLZF-RAR alpha fusion proteins in the molecular pathogenesis of APL.
A cocaine-sensitive Drosophila serotonin transporter: cloning, expression, and electrophysiological characterization.
Authors: Corey J L , Quick M W , Davidson N , Lester H A , Guastella J
Published: May 2006

A cocaine-sensitive, high-affinity Drosophila serotonin (5-hydroxytryptamine; 5HT) transporter cDNA, denoted dSERT1, was isolated and characterized in oocytes. dSERT1 shows little transport of other monoamines and is Na+ and Cl- dependent. Sequence analysis indicates 12 putative transmembrane domains and strong homologies (approximately 50%) among dSERT1 and mammalian 5HT, norepinephrine, and dopamine transporters. Interestingly, the pharmacological properties of dSERT1, including sensitivity to antidepressants, are more similar to those of mammalian catecholamine transporters than to mammalian 5HT transporters. Two-electrode voltage-clamp analysis demonstrated 5HT-induced, voltage-dependent currents. Cloning and characterization of dSERT1 adds significantly to our knowledge of the diversity of 5HT transporters with regard to primary sequence, pharmacological profile, and permeation properties.
Role of N-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control.
Authors: Hammond C , Braakman I , Helenius A
Published: May 2006

Using a pulse-chase approach combined with immunoprecipitation, we showed that newly synthesized influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein associate transiently during their folding with calnexin, a membrane-bound endoplasmic reticulum (ER) chaperone. Inhibitors of N-linked glycosylation (tunicamycin) and glucosidases I and II (castanospermine and 1-deoxynojirimycin) prevented the association, whereas inhibitors of ER alpha-mannosidases did not. Our results indicated that binding of these viral glycoproteins to calnexin correlated closely with the composition of their N-linked oligosaccharide side chains. Proteins with monoglucosylated oligosaccharides were the most likely binding species. On the basis of our data and existing information concerning the role of monoglucosylated oligosaccharides on glycoproteins, we propose that the ER contains a unique folding and quality control machinery in which calnexin acts as a chaperone that binds proteins with partially glucose-trimmed carbohydrate side chains. In this model glucosidases I and II serve as signal modifiers and UDP-glucose:glycoprotein glucosyltransferase, as a folding sensor.
Determination of spin-label orientation within the myosin head.
Authors: Fajer P G
Published: May 2006

Current methods of analyzing EPR spectra of spin-labeled muscle fibers allow the determination of spin-label orientation within the fiber, rather than the orientation of the myosin head itself. In order to describe the orientational distribution of spin labeled myosin heads within the muscle fibers, the orientation of the spin label within the myosin head must be known. The iodoacetamide label orientation in the myosin head was determined to be (16.8 degrees, 28.3 degrees, 4.2 degrees) or (16.6 degrees, 72.0 degrees, 4.3 degrees). These Eulerian angles were obtained from the analysis of EPR spectra of fibers decorated with labeled myosin heads in the absence of ATP, with the assumption that the head's tilt angle is 40 degrees, as observed in a recent EM study [Pollard, T., Bhandari, D., Maupin, P., Wachsstock, D., Weeds, A. & Zot, H. (1993) Biophys. J. 64, 454-471]. Knowledge of spin-label orientation will allow for quantitative determination of myosin head orientation in the various states of the contractile cycle.
PANG, a gene encoding a neuronal glycoprotein, isectopically activated by intracisternal A-type particle long terminal repeats inmurine plasmacytomas.
Authors: Connelly M A , Grady R C , Mushinski J F , Marcu K B
Published: May 2006

Plasmacytomagenesis provides a murine model to
decipher progressive genetic events culminating in a B-cell neoplasia.
Activation of the c-myc protooncogene by chromosomal translocation is considered
an initiating event. Intracisternal A-type particles (IAPs) are defective
retroviral-like structures present in the endoplasmic reticulum of plasmacytomas
(PCTs). IAP proviral insertions have been documented to engender negative or
positive effects on the expression of nearby cellular genes. We have isolated a
gene, PANG (plasmacytoma-associated neuronal glycoprotein), that is ectopically
transcribed in a number of PCTs due to IAP long terminal repeat (LTR)
activation. A full-length PANG cDNA was isolated from an MPC-11 plasma cell
tumor cDNA library and encodes a polypeptide of about 113 kDa with six
immunoglobulin C2-like and four type III fibronectin-like domains. PANG bears a
striking resemblance to axonal glycoproteins TAG-1 and F11 known to function in
neuronal outgrowth. An extensive survey revealed a predominant 3.6-kb PANG
transcript in 60% (30 of 50) of PCTs as well as unique smaller and larger
species. All other normal and transformed lymphoid and nonlymphoid cell lines
and normal tissues were negative for PANG expression except for the brain,
wherein unique 4.0- and 6.1-kb transcripts were detected. Reverse transcriptase
PCR analysis revealed IAP LTR fusion to PANG mRNAs in five PCTs and in a
neuroblastoma line. The 5' end of a mouse brain PANG cDNA was identical to
the MPC-11 PANG transcript except for the precise replacement of its 5' LTR
sequence.
Direct functional assay for tobacco mosaic viruscell-to-cell movement protein and identification of a domain involved inincreasing plasmodesmal permeability.
Authors: Waigmann E , Lucas W J , Citovsky V , Zambryski P
Published: May 2006

Plasmodesmata are cytoplasmic bridges between
plant cells thought to generally allow only the passage of small molecules and
metabolites. However, large structures such as plant viruses also move from cell
to cell via plasmodesmata. In tobacco mosaic virus (TMV) infection a viral
movement protein (TMV-MP) mediates viral spread. Here, a microinjection assay is
used to monitor the dynamics of TMV-MP function directly in wild-type plants.
The results indicate that TMV-MP interacts with an endogenous plant pathway
increasing plasmodesmal size exclusion limit to permit passage of 20-kDa
dextrans. Furthermore, TMV-MP influences plasmodesmal size exclusion limit
several cells distant from the injection site, indicating either that TMV-MP
itself crosses plasmodesmata or that TMV-MP induces a diffusable signal capable
of dilating microchannels of plasmodesmata. The region of TMV-MP responsible for
increasing plasmodesmal size exclusion limit was mapped to the carboxyl-terminal
part of the 268-amino acid residue protein between amino acid residues 126 and
224.
Reconstructing evolutionary trees from DNA and proteinsequences: paralinear distances.
Authors: Lake J A
Published: May 2006

The reconstruction of phylogenetic trees from DNA
and protein sequences is confounded by unequal rate effects. These effects can
group rapidly evolving taxa with other rapidly evolving taxa, whether or not
they are genealogically related. All algorithms are sensitive to these effects
whenever the assumptions on which they are based are not met. The algorithm
presented here, called paralinear distances, is valid for a much broader class
of substitution processes than previous algorithms and is accordingly less
affected by unequal rate effects. It may be used with all nucleic acid, protein,
or other sequences, provided that their evolution may be modeled as a succession
of Markov processes. The properties of the method have been proven both
analytically and by computer simulations. Like all other methods, paralinear
distances can fail when sequences are misaligned or when site-to-site sequence
variation of rates is extensive. To examine the usefulness of paralinear
distances, the "origin of the eukaryotes" has been investigated by the
analysis of elongation factor Tu sequences with a variety of sequence
alignments. It has been found that the order in which sequences are pairwise
aligned strongly determines the topology which is reconstructed by paralinear
distances (as it does for all other reconstruction methods tested). When the
parts of the alignment that are unaffected by alignment order are analyzed,
paralinear distances strongly select the eocyte topology. This provides evidence
that the eocyte prokaryotes are the closest prokaryotic relatives of the
eukaryotes.
Detoxication of base propenals and other alpha,beta-unsaturated aldehyde products of radical reactions and lipid peroxidationby human glutathione transferases.
Authors: Berhane K , Widersten M , Engström A , Kozarich J W , Mannervik B
Published: May 2006

Radiation and chemical reactions that give rise to
free radicals cause the formation of highly cytotoxic base propenals,
degradation products of DNA. Human glutathione transferases (GSTs;
RX:glutathione R-transferase, EC 2.5.1.18) of classes Alpha, Mu, and Pi were
shown to promote the conjugation of glutathione with base propenals and related
alkenes. GST P1-1 was particularly active in catalyzing the reactions with the
propenal derivatives, and adenine propenal was the substrate giving the highest
activity. The catalytic efficiency of GST P1-1 with adenine propenal (kcat/Km =
7.7 x 10(5) M-1.s-1) is the highest so far reported with any substrate for this
enzyme. In general, GST A1-1 and GST M1-1, in contrast to GST P1-1, were more
active with 4-hydroxyalkenals (products of lipid peroxidation) than with base
propenals. The adduct resulting from the Michael addition of glutathione to the
alkene function of one of the base propenals (adenine propenal) was identified
by mass spectrometry. At the cellular level, GST P1-1 was shown to provide
protection against alpha, beta-unsaturated aldehydes. GST P1-1 added to the
culture medium of HeLa cells augmented the protective effect of glutathione
against the toxicity of adenine propenal and thymine propenal. No protective
effect of the enzyme was observed in the presence of the competitive inhibitor
S-hexylglutathione. GST P1-1 introduced into Hep G2 cells by electroporation was
similarly found to increase their resistance to acrolein. The results show that
glutathione transferases may play an important role in cellular detoxication of
electrophilic alpha, beta-unsaturated carbonyl compounds produced by radical
reactions, lipid peroxidation, ionizing radiation, and drug
metabolism.
Diffusible factors regulate hair cell regeneration inthe avian inner ear.
Authors: Tsue T T , Oesterle E C , Rubel E W
Published: May 2006

Damage to the avian inner ear results in
up-regulation of mitotic activity resulting in regeneration of hair cells. The
objective of this investigation was to determine whether the damaged inner ear
epithelium releases a soluble mitogen that is responsible for the up-regulation
of proliferation. The sensory epithelium from normal and drug-damaged avian
inner ears was cultured alone or in the presence of other cultures. As
previously shown in vivo and in vitro, damaged organs displayed increased
supporting cell proliferation compared with undamaged organs, leading to
eventual morphologic and functional recovery. When damaged organs were
cocultured with an undamaged organ, proliferation was increased in the undamaged
tissue. When undamaged organs were cultured together, proliferation was
decreased. These results indicate that a soluble factor released from the
damaged inner ear epithelium stimulates proliferation and suggest the release of
a factor from normal tissue that suppressed mitotic activity. Thus, reparative
hair cell regeneration in the inner ear appears to be regulated by a balance
between proliferative and antiproliferative paracrine factors.
A collision gradient method to determine the immersiondepth of nitroxides in lipid bilayers: application to spin-labeled mutants ofbacteriorhodopsin.
Authors: Altenbach C , Greenhalgh D A , Khorana H G , Hubbell W L
Published: May 2006

Ten mutants of bacteriorhodopsin, each containing
a single cysteine residue regularly spaced along helix D and facing the lipid
bilayer, were derivatized with a nitroxide spin label. Collision rates of the
nitroxide with apolar oxygen increased with distance from the membrane/solution
interface. Collision rates with polar metal ion complexes decreased over the
same distance. Although the collision rates depend on steric constraints imposed
by the local protein structure and on the depth in the membrane, the ratio of
the collision rate of oxygen to those of a polar metal ion complex is
independent of structural features of the protein. The logarithm of the ratio is
a linear function of depth within the membrane. Calibration of this ratio
parameter with spin-labeled phospholipids allows localization of the individual
nitroxides, and hence the bacteriorhodopsin molecule, relative to the plane of
the phosphate groups of the bilayer. The spacing between residues is consistent
with the pitch of an alpha-helix. These results provide a general strategy for
determining the immersion depth of nitroxides in bilayers.
Cre recombinase-mediated site-specific recombinationbetween plant chromosomes.
Authors: Qin M , Bayley C , Stockton T , Ow D W
Published: May 2006

We report the use of the bacteriophage P1 Cre-lox
system for generating conservative site-specific recombination between tobacco
chromosomes. Two constructs, one containing a promoterless hygromycin-resistance
gene preceded by a lox site (lox-hpt) and the other containing a cauliflower
mosaic virus 35S promoter linked to a lox sequence and the cre coding region
(35S-lox-cre), were introduced separately into tobacco plants. Crosses between
plants harboring either construct produced plants with the two constructs
situated on different chromosomes. Plants with recombination events were
identified by selecting for hygromycin resistance, a phenotype expressed upon
recombination. Molecular analysis showed that these recombination events
occurred specifically at the lox sites and resulted in the reciprocal exchange
of flanking host DNA. Progenies of these plants showed 67-100% cotransmission of
the new transgenes, 35S-lox-hpt and lox-cre, consistent with the preferential
cosegregation of translocated chromosomes. These results illustrate that
site-specific recombination systems can be useful tools for the large-scale
manipulation of eukaryotic chromosomes in vivo.
Deactivation of the sporulation transcription factorSpo0A by the Spo0E protein phosphatase.
Authors: Ohlsen K L , Grimsley J K , Hoch J A
Published: May 2006

The spo0E locus of Bacillus subtilis codes for a
negative regulator of sporulation that, when overproduced, represses sporulation
and, if deleted, results in inappropriate timing of sporulation. The product of
this locus, Spo0E, was purified and found to be a protein phosphatase, which
specifically dephosphorylated the sporulation transcription factor Spo0A-P,
converting it to an inactive form. Spo0E was not significantly active as a
phosphatase on other components of the phosphorelay signal-transduction pathway
producing Spo0A-P. A mutant Spo0E protein that results in sporulation deficiency
was purified and found to be hyperactive as a phosphatase. The Spo0E phosphatase
may provide an additional control point for environmental, metabolic, or
cell-cycle regulation of phosphate flow in the phosphorelay. These results
reinforce the concept that the phosphorelay is subject to a host of positive and
negative signals for sporulation that are recognized and interpreted as signal
integration circuit that has the role of regulating the cellular level of active
phosphorylated Spo0A sporulation transcription factor.
Insulin-like growth factors act synergistically withbasic fibroblast growth factor and nerve growth factor to promote chromaffincell proliferation.
Authors: Frödin M , Gammeltoft S
Published: May 2006

We have investigated the effects of insulin-like
growth factors (IGFs), basic fibroblast growth factor (bFGF), and nerve growth
factor (NGF) on DNA synthesis in cultured chromaffin cells from fetal, neonatal,
and adult rats by using 5-bromo-2'-deoxyuridine (BrdUrd) pulse labeling for
24 or 48 h and immunocytochemical staining of cell nuclei. After 6 days in
culture in the absence of growth factors, nuclear BrdUrd incorporation was
detected in 30% of fetal chromaffin cells, 1.5% of neonatal cells, and 0.1% of
adult cells. Addition of 10 nM IGF-I or IGF-II increased the fraction of
BrdUrd-labeled nuclei to 50% of fetal, 20% of neonatal, and 2% of adult
chromaffin cells. The ED50 value of IGF-I- and IGF-II-stimulated BrdUrd labeling
in neonatal chromaffin cells was 0.3 nM and 0.8 nM, respectively. In neonatal
and adult chromaffin cells, addition of 1 nM bFGF or 2 nM NGF stimulated nuclear
BrdUrd incorporation to approximately the same level as 10 nM IGF-I or IGF-II.
However, the response to bFGF or NGF in combination with either IGF-I or IGF-II
was more than additive, indicating that the combined effect of the IGFs and bFGF
or NGF is synergistic. The degree of synergism was 2- to 4-fold in neonatal
chromaffin cells and 10- to 20-fold in adult chromaffin cells compared with the
effect of each growth factor alone. In contrast, the action of bFGF and NGF
added together in the absence of IGFs was not synergistic or additive. IGF-II
acted also as a survival factor on neonatal chromaffin cells and the cell
survival was further improved when bFGF or NGF was added together with IGF-II.
In conclusion, we propose that IGF-I and IGF-II act in synergy with bFGF and NGF
to stimulate proliferation and survival of chromaffin cells during neonatal
growth and adult maintenance of the adrenal medulla. Our findings may have
implications for improving the survival of chromaffin cell implants in diseased
human brain.
Thermophilic, anaerobic bacteria isolated from a deepborehole in granite in Sweden.
Authors: Szewzyk U , Szewzyk R , Stenström T A
Published: May 2006

A borehole drilled to a total depth of 6779 m in
granitic rock in Gravberg, Sweden, was sampled and examined for the presence of
anaerobic, thermophilic, fermenting bacteria and sulfate-reducing bacteria.
Growth in enrichment cultures was obtained only from water samples collected
from a specific sampling depth in the borehole (3500 m). The hole was cased down
to a depth of 5278 m and open to the formation below that level. All the water
below 2000 m in depth standing in the borehole at the time of sampling must have
entered at the 5278-m level or below, during a prior pumping operation. A strong
salinity stratification certifies that no major amount of vertical mixing had
taken place. The depth from which bacteria could be enriched was that of a
pronounced local minimum of salinity. Pure cultures of thermophilic, anaerobic,
fermenting bacteria were obtained with the following substrates: glucose,
starch, xylan, ethanol, and lactate. The morphology and physiology of the
glucose- and starch-degrading strains indicate a relationship to
Thermoanaerobacter and Thermoanaerobium species. All but one of the newly
isolated strains differ however from those by lacking acetate as a fermentation
product. The glucose-degrading strain Gluc1 is phylogenetically related to
Clostridium thermohydrosulfuricum, with an evolutionary distance based upon rRNA
sequence comparisons of 3%. No sulfate-reducing or methanogenic bacteria were
found.
Expression and partial purification of enzymaticallyactive recombinant Ty1 integrase in Saccharomyces cerevisiae.
Authors: Moore S P , Garfinkel D J
Published: May 2006

Integration of the Saccharomyces cerevisiae
retrotransposon Ty1 into the genome requires Ty1 integrase (IN). Apparent
functions of Ty1 IN are target-site determination, cleavage, and joining of
donor strands. To further study the mechanism of Ty1 integration, an IN
expression plasmid has been constructed for use in yeast. The recombinant IN
coding sequence differs from mature Ty1 IN associated with Ty1 virus-like
particles only in that it has several additional N-terminal amino acid codons.
Inclusion of a polyhistidine tag facilitates purification of recombinant IN by
metal chelate chromatography. Recombinant Ty1 IN is active in an in vitro assay
with short double-stranded oligonucleotide substrates and has biochemical
properties similar to those observed with Ty1 virus-like particles. The
full-length Ty1 IN produced in yeast should be useful for further biochemical,
genetic, and structural analyses of Ty1 integration and for comparative analyses
with retroviral IN proteins.
RGD sequence of foot-and-mouth disease virus isessential for infecting cells via the natural receptor but can be bypassed by anantibody-dependent enhancement pathway.
Authors: Mason P W , Rieder E , Baxt B
Published: May 2006

Foot-and-mouth disease virus appears to initiate
infection by binding to cells at an Arg-Gly-Asp (RGD) sequence found in the
flexible beta G-beta H loop of the viral capsid protein VP1. The role of the RGD
sequence in attachment of virus to cells was tested by using synthetic
full-length viral RNAs mutated within or near the RGD sequence. Baby hamster
kidney (BHK) cells transfected with three different RNAs carrying mutations
bordering the RGD sequence produced infectious viruses with wild-type plaque
morphology; however, one of these mutant viruses bound to cells less efficiently
than wild type. BHK cells transfected with RNAs containing changes within the
RGD sequence produced noninfectious particles indistinguishable from wild-type
virus in terms of sedimentation coefficient, binding to monoclonal antibodies,
and protein composition. These virus-like particles are defined as ads- viruses,
since they were unable to adsorb to and infect BHK cells. These mutants were
defective only in cell binding, since antibody-complexed ads- viruses were able
to infect Chinese hamster ovary cells expressing an immunoglobulin Fc receptor.
These results confirm the essential role of the RGD sequence in binding of
foot-and-mouth disease virus to susceptible cells and demonstrate that the
natural cellular receptor for the virus serves only to bind virus to the
cell.
External blockade of the major cardiacdelayed-rectifier K+ channel (Kv1.5) by polyunsaturated fattyacids.
Authors: Honoré E , Barhanin J , Attali B , Lesage F , Lazdunski M
Published: May 2006

The present work shows that arachidonic acid and
some other long chain polyunsaturated fatty acids such as docosahexaenoic acid,
which is abundant in fish oil, produce a direct open channel block of the major
voltage-dependent K+ channel (Kv1.5) cloned in cardiac cells. The inhibitory
action of these selected fatty acids is seen when they are applied
extracellularly but not when they are included in the patch pipette. Fatty acids
then appear to bind to an external site on the Kv1.5 channel structure.
Inhibition of Kv1.5 channel activity by polyunsaturated fatty acids
(acceleration of the apparent inactivation and decrease of the peak current) is
similar to that produced by the class III antiarrhythmic tedisamil.
Docosahexaenoic acid and arachidonic acid also inhibit the delayed-rectifier K+
channel currents in cultured mouse and rat cardiomyocytes. These results are
discussed in the light of the reported fatty acids effects on cardiac function
in diseased states. Since Kv1.5 is also present in the brain, the results
reported here could also have a significance in terms of processes such as
long-term potentiation or depression.