bluuebunny/crossref_metadata_2025 huggingface dataset:
A membrane form of brain L-glutamate decarboxylase: identification, isolation, and its relation to insulin-dependent mellitus.
Authors: Nathan B , Bao J , Hsu C C , Aguilar P , Wu R , Yarom M , Kuo C Y , Wu J Y
Published: May 2006

A membrane form of L-glutamate decarboxylase (GAD) was identified and purified to apparent homogeneity from hog brain. The purified GAD was established as an integral membrane protein by phase-partitioning assay, charge-shift electrophoresis, and chromatography on a hydrophobic interaction column. This membrane GAD has a native molecular mass of 96 +/- 5 kDa and is a homodimer of 48 +/- 3-kDa subunits. Immunoprecipitation and immunoblotting tests revealed the presence of antibodies against this membrane GAD in sera from patients with insulin-dependent diabetes mellitus. Since this form of GAD appears to be an integral membrane protein and is presumed to have extracellular domains exposed, it seems reasonable to suggest that membrane GAD is more likely than soluble GAD to be involved in the pathogenesis of insulin-dependent diabetes and related autoimmune disorders such as stiff-man syndrome.
Functional communication in the recognition of tRNA by Escherichia coli glutaminyl-tRNA synthetase.
Authors: Rogers M J , Adachi T , Inokuchi H , Söll D
Published: May 2006

Wild-type Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) poorly aminoacylates opal suppressors (GLN) derived from tRNA(Gln). Mutations in glnS (the gene encoding GlnRS) that compensate for impaired aminoacylation were isolated by genetic selection. Two glnS mutants were obtained by using opal suppressors differing in the nucleotides composing the base pair at 3.70: glnS113 with an Asp-235-->Asn change selected with GLNA3U70 (GLN carrying G3-->A and C70-->U changes), and glnS114 with a Gln-318-->Arg change selected with GLNU70 (GLN carrying a C70-->U change). The Asp-235-->Asn change was identified previously by genetic selection. Additional mutants were isolated by site-directed mutagenesis followed by genetic selection; the mutant enzymes have single amino acid changes (Lys-317-->Arg and Gln-318-->Lys). A number of mutants with no phenotype also were obtained randomly. In vitro aminoacylation of a tRNA(Gln) transcript by GlnRS enzymes with Lys-317-->Arg, Gln-318-->Lys, or Gln-318-->Arg changes shows that the enzyme's kinetic parameters are not greatly affected by the mutations. However, aminoacylation of a tRNA(Gln) transcript with an opal (UCA) anticodon shows that the specificity constants (kcat/Km) for the mutant enzymes were 5-10 times above that of the wild-type GlnRS. Interactions between Lys-317 and Gln-318 with the inside of the L-shaped tRNA and with the side chain of Gln-234 provide a connection between the acceptor end-binding and anticodon-binding domains of GlnRS. The GlnRS mutants isolated suggest that perturbation of the interactions with the inside of the tRNA L shape results in relaxed anticodon recognition.
Specific ablation of human immunodeficiency virus Tat-expressing cells by conditionally toxic retroviruses.
Authors: Brady H J , Miles C G , Pennington D J , Dzierzak E A
Published: May 2006

The identification of human immunodeficiency virus (HIV) as the etiologic agent of AIDS has led to the proposal of novel intervention strategies to block HIV infection and viral replication or eliminate HIV-infected cells. We have produced recombinant retroviruses for a molecular ablation system, whereby a toxin gene can be delivered to hematopoietic cells for the specific elimination of HIV Tat-expressing cells. For this cell-specific ablation, we have coupled the conditional toxin herpes simplex virus type 1 thymidine kinase (tk) gene to the HIV-2 promoter and Tat responsive region (TAR) in order that transcriptional activity be under the absolute control of HIV and simian immunodeficiency virus Tat trans-activator proteins. Since the HIV-2 promoter has a considerable level of basal expression in the absence of Tat, we constructed a number of modifications in the HIV-2 promoter to minimize the risk of cytotoxicity to cells not containing HIV Tat. We demonstrate that certain promoter modifications reduce basal transcription while maintaining high trans-activated levels of expression when transfected or transduced by retroviral vectors into several different cell lines. In mouse and human cells infected with HIV-2 tk retroviruses, we show that Tat-induced expression from the HIV-2 promoter results in differential ablation and a massive reduction in Tat-positive cells after ganciclovir treatment. Thus, the retroviruses produced in these studies may be applicable to HIV ablative therapy.
The retinoblastoma gene product is a cell cycle-dependent, nuclear matrix-associated protein.
Authors: Mancini M A , Shan B , Nickerson J A , Penman S , Lee W H
Published: May 2006

The retinoblastoma gene product (Rb) has been established as a tumor suppressor and cell cycle regulator, although its mechanism of action remains obscure. The observations that several Rb-binding viral oncoproteins all associate with the nuclear matrix suggest that these interactions may occur on this structure. To determine whether Rb itself is a component of the matrix, we extracted synchronized cultured cells to isolate matrix proteins while preserving nuclear architecture. Immunoblot and immunolabeling data show that a significant portion of hypophosphorylated Rb associates with the matrix only during early G1. Mutant Rb in tumor cells did not associate with the matrix, whereas Rb-reconstituted cells contained abundant matrix-bound Rb. Rb is distributed widely throughout the matrix, particularly concentrated at the nuclear periphery and in nucleolar remnants. Core filaments of the matrix contained no detectable Rb. Our screening of expression libraries for potential Rb-associated proteins has identified several that are part of the matrix. Specifically, the peripheral matrix proteins lamin A and C bound Rb in vitro. We therefore suggest that Rb interactions with the nuclear matrix may be important for its ability to regulate cell cycle progression.
Isolation and sequencing of cDNAs for proteins with multiple domains of Gly-Xaa-Yaa repeats identify a distinct family of collagenous proteins.
Authors: Oh S P , Kamagata Y , Muragaki Y , Timmons S , Ooshima A , Olsen B R
Published: May 2006

We have isolated overlapping mouse cDNAs encoding a collagenous polypeptide that we have designated alpha 1(XVIII) collagen. Nucleotide sequence analysis shows that alpha 1(XVIII) collagen contains 10 triple-helical domains separated and flanked by non-triple-helical regions. Within the non-triple-helical regions, there are several Ser-Gly-containing sequences that conform to consensus sequences for glycosaminoglycan attachment sites in proteoglycan core proteins. Northern blots show that alpha 1(XVIII) transcripts are present in multiple organs, with the highest levels in liver, lung, and kidney. We have also isolated overlapping cDNAs encoding human alpha 1(XV) collagen, and their sequence extends a published partial alpha 1(XV) sequence to the 3' end. Comparison of the alpha 1(XV) and alpha 1(XVIII) sequences reveals a striking similarity in the lengths of the six most carboxyl-terminal triple-helical domains. In addition, within the carboxyl non-triple-helical domain NC1 of the two chains, a region of 177 amino acid residues shows about 60% identity at the amino acid level. We suggest, therefore, that alpha 1(XV) and alpha 1(XVIII) collagens are structurally related. Their structure is different from that of other known collagen types. We conclude that they belong to a subfamily of extracellular matrix proteins and we suggest the designation multiplexins (for protein with multiple triple-helix domains and interruptions) for members of this subfamily.
Effector region of the translation elongation factor EF-Tu.GTP complex stabilizes an orthoester acid intermediate structure of aminoacyl-tRNA in a ternary complex.
Authors: Förster C , Limmer S , Zeidler W , Sprinzl M
Published: May 2006

tRNA(Val) from Escherichia coli was aminoacylated with [1-13C]valine and its complex with Thermus thermophilus elongation factor EF-Tu.GTP was analyzed by 13C NMR spectroscopy. The results suggest that the aminoacyl residue of the valyl-tRNA in ternary complex with bacterial EF-Tu and GTP is not attached to tRNA by a regular ester bond to either a 2'- or 3'-hydroxyl group; instead, an intermediate orthoester acid structure with covalent linkage to both vicinal hydroxyls of the terminal adenosine-76 is formed. Mutation of arginine-59 located in the effector region of EF-Tu, a conserved residue in protein elongation factors and the alpha subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), abolishes the stabilization of the orthoester acid structure of aminoacyl-tRNA.
Spreading of differentiating human monocytes is associated with a major increase in membrane-bound CDC42.
Authors: Aepfelbacher M , Vauti F , Weber P C , Glomset J A
Published: May 2006

In a search for a model cell system that might allow studies of the function of the Rho-related GTPase CDC42Hs in human cells, we measured the content and distribution of CDC42Hs in monocytes that were differentiating into macrophages. The total content of this protein increased 5- to 6-fold in phorbol ester-treated human monocytic THP-1 and U-937 cells and increased 13-fold in normal human blood monocytes. Moreover, membrane-associated CDC42Hs in these cells increased 13-fold and 30-fold, respectively, while cytosolic CDC42Hs increased only 3- and 6-fold. Measurements made specifically in U-937 cells showed that the increase in membrane CDC42Hs correlated closely with an increase in cell spreading. The changes in CDC42Hs in U-937 cells probably depended on increased mRNA translation and/or decreased protein degradation, since no change in CDC42Hs mRNA could be detected. Finally, the changes in CDC42Hs were relatively specific, since contents of the CDC42Hs-binding protein Rho-GDI and the Rho-related protein Rac2 were unaffected and no change in CDC42Hs occurred when the cells were stimulated by agonists that induce monocytes to differentiate into nonadherent cells. These findings show that marked changes in content and distribution of CDC42Hs occur when monocytes differentiate into macrophages, suggesting that membrane CDC42Hs may play a role in cell spreading.
Development of sun compensation by honeybees: how partially experienced bees estimate the sun's course.
Authors: Dyer F C , Dickinson J A
Published: May 2006

Honeybees and some other insects, in learning the sun's course, behave as if they can estimate the sun's position at times of day when they have never seen it, but there are competing ideas about the computational mechanisms underlying this ability. In an approach to this problem, we provided incubator-reared bees with opportunities to fly and see the sun only during the late afternoon. Then, on a cloudy day, we allowed bees to fly for the first time during the morning and early afternoon, and we observed how they oriented their waggle dances to indicate their direction of flight relative to the sun's position. The clouds denied the bees a direct view of celestial orientation cues and thus forced them to estimate the sun's position on the basis of their experience on previous evenings. During the test days, experience-restricted bees behaved during the entire morning as if they expected the sun to be in an approximately stationary position about 180 degrees from the average solar azimuth that they had experienced on previous evenings; then from about local noon onward they used the evening azimuth. This pattern suggests that honeybees are innately informed of the general pattern of solar movement, such that they can generate an internal representation that incorporates spatial and temporal features of the sun's course that they have never directly seen.
Delineation of a human histone H4 cell cycle element in vivo: the master switch for H4 gene transcription.
Authors: Ramsey-Ewing A , Van Wijnen A J , Stein G S , Stein J L
Published: May 2006

Histone gene expression is cell cycle regulated at the transcriptional and the post-transcriptional levels. Upon entry into S phase, histone gene transcription is stimulated 2- to 5-fold and peaks within 1-3 hr of the initiation of DNA synthesis. We have delineated the proximal promoter element responsible for cell cycle-dependent transcription of a human histone H4 gene in vivo. Our results indicate that H4 cell cycle-dependent transcriptional regulation is mediated by an 11-base-pair element, the cell cycle element (5'-CTTTCG-GTTTT-3'), that resides in the in vivo protein-DNA interaction site, site II (nucleotides -64 to -24). The H4 cell cycle element functions as a master switch for expression of the FO108 human histone H4 gene in vivo; mutations within the H4 cell cycle element drastically reduce the level of expression as well as abrogate cell cycle-regulated transcription. Furthermore, these mutations result in a loss of binding in vitro of the cognate nuclear factor HiNF-M. In vivo competition analysis indicates that the cell cycle element mediates specific competition for a DNA-binding factor, presumably HiNF-M, that is a rate-limiting step in transcription of this H4 gene.
High-resolution tracking of microtubule motility driven by a single kinesin motor.
Authors: Malik F , Brillinger D , Vale R D
Published: May 2006

Kinesin is a microtubule-based motor protein that contains two identical force-generating subunits. The kinesin binding sites along the microtubule lie 8 nm apart (the dimension of the tubulin dimer), which implies that kinesin must translocate a minimum distance of 8 nm per hydrolysis cycle. Measurements of kinesin's microtubule-stimulated ATPase activity (approximately 20 ATP per sec) and velocity of transport (approximately 0.6 micron/sec), however, suggest that the net distance moved per ATP (approximately 30 nm) may be greater than one tubulin dimer under zero load conditions. To explore how kinesin translocates during its ATPase cycle, we constructed a microscope capable of tracking movement with 1-nm resolution at a bandwidth of 200 Hz and used this device to examine microtubule movement driven by a single kinesin motor. Regular stepwise movements were not observed in displacement traces of moving microtubules, although Brownian forces acting on elastic elements within the kinesin motor precluded detection of steps that were < 12 nm. Though individual steps of approximately 16 nm were occasionally observed, their infrequent occurrence suggests that kinesin rarely moves abruptly by distances of two or more tubulin subunits during its ATP hydrolysis cycle. Instead it is more likely that kinesin moves forward by the distance of only a single tubulin subunit under zero load conditions.
Transcription factor ISGF-3 formation requires phosphorylated Stat91 protein, but Stat113 protein is phosphorylated independently of Stat91 protein.
Authors: Improta T , Schindler C , Horvath C M , Kerr I M , Stark G R , Darnell J E
Published: May 2006

Transcription factor ISGF-3 is a multiprotein, interferon alpha-activated transcription complex consisting of a 48-kDa DNA-binding protein and two proteins termed Stats (for signal transducers and activators of transcription) that become phosphorylated on tyrosine in the cell cytoplasm, a 113-kDa and either a 91- or 84-kDa polypeptide, the latter two of which arise from differentially spliced mRNAs. Using cell lines lacking the Stat91 or Stat84 proteins, we show that mutations in several different sites in the 91-kDa protein block the interferon alpha-induced phosphorylation of the 91-kDa protein and subsequent ISGF-3 formation. Although correct tyrosine phosphorylation on residue 690 of the Stat113 protein occurs independent of the Stat91/84 protein, the Stat113 phosphoprotein by itself moves to the cell nucleus much less efficiently in the absence of phosphorylated Stat91/84 protein.
A G protein-coupled receptor with low density lipoprotein-binding motifs suggests a role for lipoproteins in G-linked signal transduction.
Authors: Tensen C P , Van Kesteren E R , Planta R J , Cox K J , Burke J F , van Heerikhuizen H , Vreugdenhil E
Published: May 2006

We have isolated and analyzed a cDNA from the central nervous system of the mollusc Lymnaea stagnalis encoding a putative receptor, which might be a natural hybrid between two different classes of receptor proteins. Preceded by a signal peptide, two types of repeated sequences are present in the N-terminal part of the protein. The first repeat displays a high sequence similarity to the extracellular binding domains of the low density lipoprotein receptor, which binds and internalizes cholesterol-containing apolipoproteins. The second repeat and the C-terminal part of the Lymnaea receptor are very similar to regions of a specific class of guanine nucleotide-binding protein-coupled receptors, the mammalian glycoprotein hormone receptors. The mRNA encoding the receptor is predominantly expressed in a small number of neurons within the central nervous system and to a lesser extent in the heart.
Spatial games and the maintenance of cooperation.
Authors: Nowak M A , Bonhoeffer S , May R M
Published: May 2006

The Prisoner's Dilemma (PD) is a widely employed metaphor for problems associated with the evolution of cooperative behavior. We have recently proposed an alternative approach to the PD, by exploring "spatial games" in which players--who are either pure cooperators, C, or pure defectors, D--interact with neighbors in some spatial array; in each generation, players add up the scores from all encounters, and in the next generation a given cell is retained by its previous owner or taken over by a neighbor, depending on who has the largest score. Over a wide range of the relevant parameters, we find that C and D persist together indefinitely (without any of the complex strategies that remember past encounters, and anticipate future ones, which characterize essentially all previous work on the iterated PD). Our earlier work, however, dealt with symmetric spatial lattices in two dimensions, deterministic winning and discrete time. We show here that the essential results remain valid in more realistic situations where the spatial distributions of cells are random in two or three dimensions, and where winning is partly probabilistic (rather than being determined by the largest local total). The essential results also remain valid (pace Huberman and Glance [Huberman, B. A. & Glance, N. S. (1993) Proc. Natl. Acad. Sci. USA 90, 7716-7718]) when interactions occur in continuous rather than discrete time.
Membrane-derived second messenger regulates x-ray-mediated tumor necrosis factor alpha gene induction.
Authors: Hallahan D E , Virudachalam S , Kuchibhotla J , Kufe D W , Weichselbaum R R
Published: May 2006

Cells adapt to adverse environmental conditions through a wide range of responses that are conserved throughout evolution. Physical agents such as ionizing radiation are known to initiate a stress response that is triggered by the recognition of DNA damage. We have identified a signaling pathway involving the activation of phospholipase A2 and protein kinase C in human cells that confers x-ray induction of the tumor necrosis factor alpha gene. Treatment of human cells with ionizing radiation or H2O2 was associated with the production of arachidonic acid. Inhibition of phospholipase A2 abolished radiation-mediated arachidonate production as well as the subsequent activation of protein kinase C and tumor necrosis factor alpha gene expression. These findings demonstrate that ionizing radiation-mediated gene expression in human cells is regulated in part by extranuclear signal transduction. One practical application of phospholipase A2 inhibitors is to ameliorate the adverse effects of radiotherapy associated with tumor necrosis factor alpha production.
The fragile X syndrome d(CGG)n nucleotide repeats form a stable tetrahelical structure.
Authors: Fry M , Loeb L A
Published: May 2006

The fragile X mental retardation syndrome is associated with the expansion of trinucleotide 5'-d(CGG)-3' repeats within the FMR1 gene and with hypermethylation of the cytosine residues of these repeats. The expansion and hypermethylation may account for the suppression of the transcription of the FMR1 gene and for the delay of its replication during the cell cycle. Here we show that d(CGG)n oligomers can form a stable Hoogsteen-bonded structure that exhibits properties consistent with those of tetraplex DNA. Oligomers, d(mCGG)n, (n = 4, 5, or 7), at pH 8.0 and in the presence of an alkali metal ion form stable species exhibiting a reduced electrophoretic mobility in nondenaturing polyacrylamide gels. These species are denatured by heating at 90 degrees C for 10 min. With a short d(mCGG)5 oligomer, the slowly migrating species is formed only when the cytosine residue is 5-methylated, whereas with the longer d(CGG)7 it is generated whether or not cytosine is 5-methylated. By contrast, complementary cytosine-rich oligomers do not form analogous complexes. The second-order association kinetics of the formation of the slowly migrating species of d(mCGG)5 suggests that it is an interstrand complex. Formation of intermediate-size complexes between d(mCGG)5 and d(mCGG)7 indicates that the stoichiometry of the slowly migrating structures is tetramolecular. Protection of the complex from methylation by dimethyl sulfate indicates the involvement of the N-7 positions of the guanine residues in Hoogsteen hydrogen bonding, a characteristic of quadruplex structures. If formed in vivo along the expanded and hypermethylated d(mCGG)n stretch, this tetraplex structure could suppress transcription and replication of the FMR1 gene in the fragile X syndrome cells.
Wortmannin binds specifically to 1-phosphatidylinositol 3-kinase while inhibiting guanine nucleotide-binding protein-coupled receptor signaling in neutrophil leukocytes.
Authors: Thelen M , Wymann M P , Langen H
Published: May 2006

Wortmannin (WT) and its derivative 17-hydroxywortmannin (HWT) inhibit at nanomolar concentrations superoxide formation and exocytosis in neutrophils stimulated with chemotactic agonists. Treatment of neutrophils with radiolabeled [3H]HWT resulted in specific and saturable binding that paralleled the inhibition of the respiratory burst. Both half-maximal binding and half-maximal inhibition were observed at 5 nM, and > 90% of maximal binding and inhibition was observed at 20 nM HWT. Fluorography of subcellular fractions that were separated on NaDodSO4/PAGE showed that [3H]HWT binds covalently to a 110-kDa cytosolic protein. The WT-binding protein was purified from human neutrophils and bovine brain homogenates by column chromatography. The pure protein was eluted from gel filtration columns with an apparent molecular mass of 200 kDa and showed a heterodimeric structure on Coomassie-stained NaDodSO4/PAGE. In addition to the 110 kDa wortmannin binding protein an equally intense band was seen migrating at 85 kDa. This band was identified on Western blots as p85 alpha, the regulatory subunit of phosphatidylinositol (PI) 3-kinase (ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137). The purified protein contained PI 3-kinase activity that was enriched > 20,000-fold from human neutrophil cytosol during preparation. The data impose a key role for PI 3-kinase-mediated signal transduction through guanine nucleotide-binding protein-coupled receptors and suggest that 3-phosphorylated inositol phospholipids are important second messengers for immediate responses in neutrophils. Furthermore, the results show that WT is a powerful and selective tool to study the function of PI 3-kinase.
Transcellular water transport in lung alveolar epithelium through mercury-sensitive water channels.
Authors: Folkesson H G , Matthay M A , Hasegawa H , Kheradmand F , Verkman A S
Published: May 2006

The movement of water between the air space and capillary compartments is important for the maintenance of air space hydration during respiration and for reabsorption of excess alveolar fluid. We have obtained immunocytochemical and functional evidence that plasma-membrane water channels are responsible for water transport in the intact lung. Northern and quantitative immunoblot analysis showed high expression of CHIP28 (channel-forming integral membrane protein of 28 kDa) water channels in rat lung; immunocytochemistry showed CHIP28 localization to epithelial cell plasma membranes. Stopped-flow light scattering measurements of osmotic water permeability (Pf) in freshly isolated rat alveolar type II epithelial cells indicated a high Pf of 0.015 +/- 0.002 cm/s (10 degrees C) that was weakly temperature-dependent (activation energy, 4 kcal/mol) and reversibly inhibited by 78 +/- 4% by 0.5 mM HgCl2. An in situ-perfused sheep lung model was used to determine the route for water movement in intact lung. Blood-to-air-space water transport was measured by sampling air space fluid after instillation into distal air spaces of hyperosmolar saline (900 mOsm) containing radioiodinated albumin and [14C]mannitol. In seven sets of experiments, air space osmolality and radioiodinated albumin equilibrated with a t1/2 of 0.85 +/- 0.1 min. In the contralateral lung perfused with 0.5 mM HgCl2, t1/2 increased to 2.7 +/- 0.4 min; the inhibitory effect of HgCl2 was fully reversed by 5 mM 2-mercaptoethanol. These results provide direct evidence for transcellular movement of water across the alveolar epithelium in intact lung through mercury-sensitive water channels.
Ntk: a Csk-related protein-tyrosine kinase expressed in brain and T lymphocytes.
Authors: Chow L M , Jarvis C , Hu Q , Nye S H , Gervais F G , Veillette A , Matis L A
Published: May 2006

The activity of Src-related protein-tyrosine kinases is repressed by the phosphorylation of a conserved carboxyl-terminal tyrosine by another cytoplasmic protein-tyrosine kinase termed p50csk. In this study, we characterize Ntk, a protein-tyrosine kinase bearing striking similarities to p50csk. Like p50csk, Ntk possesses Src homology 3 and Src homology 2 domains and lacks the consensus tyrosine phosphorylation and myristoylation sites found in members of the Src family. Expression of ntk transcripts was maximal in brain, and was observed at significant levels in thymus and spleen. ntk RNA levels were dramatically reduced upon mitogenic stimulation of normal T lymphocytes and were minimal in transformed T-cell populations. Firm evidence that Ntk is a Csk-related enzyme was provided by the observation that it phosphorylated a Src-related polypeptide on the inhibitory carboxyl-terminal tyrosine. These findings indicate that Ntk is a Csk-related enzyme that may play an inhibitory role in the control of T-cell proliferation.
Light-generated oligonucleotide arrays for rapid DNA sequence analysis.
Authors: Pease A C , Solas D , Sullivan E J , Cronin M T , Holmes C P , Fodor S P
Published: May 2006

In many areas of molecular biology there is a need to rapidly extract and analyze genetic information; however, current technologies for DNA sequence analysis are slow and labor intensive. We report here how modern photolithographic techniques can be used to facilitate sequence analysis by generating miniaturized arrays of densely packed oligonucleotide probes. These probe arrays, or DNA chips, can then be applied to parallel DNA hybridization analysis, directly yielding sequence information. In a preliminary experiment, a 1.28 x 1.28 cm array of 256 different octanucleotides was produced in 16 chemical reaction cycles, requiring 4 hr to complete. The hybridization pattern of fluorescently labeled oligonucleotide targets was then detected by epifluorescence microscopy. The fluorescence signals from complementary probes were 5-35 times stronger than those with single or double base-pair hybridization mismatches, demonstrating specificity in the identification of complementary sequences. This method should prove to be a powerful tool for rapid investigations in human genetics and diagnostics, pathogen detection, and DNA molecular recognition.
Qualitative changes in the subunit composition of kappa B-binding complexes during murine B-cell differentiation.
Authors: Miyamoto S , Schmitt M J , Verma I M
Published: May 2006

We report here that the major kappa B-binding complex in murine mature B cells is composed of a p50-Rel heterodimer, whereas the major inducible form in pre-B cells is a p50-p65 heterodimer. Treatment of a pre-B-cell line with lipopolysaccharide changes the subunit composition of kappa B-binding complexes from p50-p65 to p50-Rel. This change is preceded by the enhanced Rel expression and correlates with the expression of the gene for the immunoglobin kappa light chain. The heterodimeric p50-Rel complex binds to the intronic enhancer-kappa B site in the immunoglobulin kappa light chain gene at least 20-fold more stably than does the p50-p65 dimer. These data support a model in which augmentation of Rel expression during the differentiation of pre-B cells to mature B cells leads to an exchange of kappa B-binding subunits resulting in the transcriptional activation of the gene for the immunoglobulin kappa light chain.
Rab5a is a common component of the apical and basolateral endocytic machinery in polarized epithelial cells.
Authors: Bucci C , Wandinger-Ness A , Lütcke A , Chiariello M , Bruni C B , Zerial M
Published: May 2006

In nonpolarized cells, the small GTPase Rab5a is localized to the plasma membrane, clathrin-coated vesicles, and early endosomes. Rab5a is required for early endosome fusion in vitro and regulates transport between the plasma membrane and early endosomes, in vivo. In polarized epithelial cells endocytosis occurs from separate apical and basolateral plasma membrane domains. Internalized molecules are initially delivered to distinct apical or basolateral early endosomes. In vitro, apical early endosomes can readily fuse with one another but not with the basolateral endosomes and vice versa, thereby indicating that the apical and basolateral early endocytic pathways are controlled by distinct machineries. Here, we have investigated the localization and function of Rab5a in polarized epithelial cells. Confocal immunofluorescence microscopy on mouse kidney sections revealed association of the protein with the apical and basolateral plasma membrane domains and underlying structures. In polarized Madin-Darby canine kidney I cells, endogenous and overexpressed Rab5a have the same distribution. Moreover, overexpression of the protein causes a 2-fold increase in fluid-phase uptake from both domains of the cell, thus showing that Rab5a functions in apical and basolateral endocytosis. Our data indicate that the apical and basolateral endocytic machineries of epithelial cells share common regulatory components and that Rab5a per se is not sufficient to target endocytic vesicles to apical or basolateral early endosomes.
Solution NMR structure of the major cold shock protein (CspA) from Escherichia coli: identification of a binding epitope for DNA.
Authors: Newkirk K , Feng W , Jiang W , Tejero R , Emerson S D , Inouye M , Montelione G T
Published: May 2006

Sequence-specific 1H and 15N resonance assignments have been determined for the major cold shock protein (CspA) from Escherichia coli with recently developed three-dimensional triple-resonance NMR experiments. By use of these assignments, five antiparallel beta-strands were identified from analysis of NMR data. Strands 1-4 have a classical 3-2-1-4 Greek key beta-sheet topology and there are two beta-bulges, at positions Lys10-Trp11 and Gly65-Asn66. Three-dimensional structures of CspA were generated from NMR data by using simulated annealing with molecular dynamics. The overall chain fold of CspA is a beta-barrel structure, with a tightly packed hydrophobic core. Two-dimensional isotope-edited pulsed-field gradient 15N-1H heteronuclear single-quantum coherence spectroscopy was used to characterize the 15N-1H fingerprint spectrum with and without a 24-base oligodeoxyribonucleotide, 5'-AACGGTTTGACGTACAGACCATTA-3'. Protein-DNA complex formation perturbs a subset of the amide resonances that are located mostly on one face of the CspA molecule. This portion of the CspA molecular surface includes two putative RNA-binding sequence motifs which contribute to an unusual cluster of eight surface aromatic side chains: Trp11, Phe12, Phe18, Phe20, Phe31, His33, Phe34, and Tyr42. These surface aromatic groups, and also residues Lys16, Ser44, and Lys60 located on this same face of CspA, are highly conserved in the family of CspA homologues. These isotope-edited pulsed-field gradient NMR data provide a low-resolution mapping of a DNA-binding epitope on CspA.
The cooperativity and allosteric inhibition of Escherichia coli phosphofructokinase depend on the interaction between threonine-125 and ATP.
Authors: Auzat I , Le Bras G , Garel J R
Published: May 2006

During the reaction catalyzed by the phosphofructokinase (EC 2.7.1.11) from Escherichia coli, the phosphoryl group transferred from ATP interacts with Thr-125 [Shirakihara, Y. & Evans, P. R. (1988) J. Mol. Biol. 204, 973-994]. The replacement of Thr-125 by serine changes the saturation by fructose 6-phosphate from cooperative to hyperbolic and abolishes the allosteric inhibition by phosphoenolpyruvate. The same changes, a saturation by fructose 6-phosphate that is no longer cooperative and an activity that is no longer inhibited by phosphoenolpyruvate, are observed with wild-type phosphofructokinase when adenosine 5'-[gamma-thio]triphosphate is used instead of ATP as the phosphoryl donor. These two perturbations of the ATP-Thr-125 interaction lead to the suppression of both the allosteric inhibition by phosphoenolpyruvate and the cooperativity of fructose-6-phosphate saturation, as if replacing the neutral oxygen of ATP by sulfur or removing the methyl group of Thr-125 had "locked" phosphofructokinase in its active conformation. The geometry of this ATP-Thr-125 interaction and/or the presence of the methyl group on the beta-carbon of Thr-125 are crucial for the regulatory properties of phosphofructokinase. This interaction could be a hydrogen bond between the neutral oxygen of the gamma-phosphate of ATP and the hydroxyl group of Thr-125.
Herpes simplex virus 1 gamma(1)34.5 gene function, which blocks the host response to infection, maps in the homologous domain of the genes expressed during growth arrest and DNA damage.
Authors: Chou J , Roizman B
Published: May 2006

The gamma(1)34.5 gene of herpes simplex virus is dispensable in some cell lines (e.g., Vero). In others (e.g., human neuroblastoma cell line SK-N-SH), the gamma(1)34.5- deletion mutant triggers a premature total shutoff of all protein synthesis, thereby rendering the cell nonviable and reducing drastically viral yields. The inability to prevent the cellular stress response that causes the infected cell to die may be responsible for the inability of the deletion mutant to multiply and cause pathology in the central nervous system of mice. The gamma(1)34.5 gene consists of an amino-terminal domain, a variable linker sequence consisting of 3 amino acids repeated 5-10 times, and a carboxyl-terminal domain homologous to the corresponding domain of MyD116, a gene expressed in myeloid leukemia cells induced to differentiate by interleukin 6, and growth arrest and DNA damage gene 34 (GADD34), a gene induced by growth arrest and DNA damage. We have constructed several viral mutants from which various domains of the gamma(1)34.5 gene had been deleted or rendered mute by the insertion of a stop codon. Studies on those mutants show that the domain of the gamma(1)34.5 gene necessary to preclude the total shutoff of protein synthesis corresponds to the carboxyl-terminal domain of the gamma(1)34.5 gene homologous to the corresponding coding domain of the MyD116 and GADD34 genes.
Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator.
Authors: Poulsen J H , Fischer H , Illek B , Machen T E
Published: May 2006

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel regulated by protein kinase A. The most common mutation in cystic fibrosis (CF), deletion of Phe-508 (delta F508-CFTR), reduces Cl- secretion, but the fatal consequences of CF have been difficult to rationalize solely in terms of this defect. The aim of this study was to determine the role of CFTR in HCO3- transport across cell membranes. HCO3- permeability was assessed from measurements of intracellular pH [pHi; from spectrofluorimetry of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5-(and -6)carboxyfluorescein] and of channel activity (patch clamp; cell attached and isolated, inside-out patches) on NIH 3T3 fibroblasts and C127 mammary epithelial cells transfected with wild-type CFTR (WT-CFTR) or delta F508-CFTR, and also on mock-transfected cells. When WT-CFTR-transfected cells were acidified (pulsed with NH4Cl) and incubated in Na(+)-free (N-methyl-D-glucamine substitution) solutions (to block Na(+)-dependent pHi regulatory mechanisms), pHi remained acidic (pH approximately 6.5) until the cells were treated with 20 microM forskolin (increases cellular [cAMP]); pHi then increased toward (but not completely to) control level (pHi 7.2) at a rate of 0.055 pH unit/min. Forskolin had no effect on rate of pHi recovery in delta F508 and mock-transfected cells. This Na(+)-independent, forskolin-dependent pHi recovery was not observed in HCO3-/CO2-free medium. Forskolin-treated WT-CFTR-transfected (but not delta F508-CFTR or mock-transfected) cells in Cl(-)-containing, HCO3(-)-free solutions showed Cl- channels with a linear I/V relationship and a conductance of 10.4 +/- 0.5 pS in symmetrical 150 mM Cl-. When channels were incubated with different [Cl-] and [HCO3-] on the inside and outside, the Cl-/HCO3- permeability ratio (determined from reversal potentials of I/V curves) was 3.8 +/- 1.0 (mean +/- SEM; n = 9); the ratio of conductances was 3.9 +/- 0.5 (at 150 mM Cl- and 127 mM HCO3-. We conclude that in acidified cells the WT-CFTR functions as a base loader by allowing a cAMP-dependent influx of HCO3- through channels that conduct HCO3- about one-quarter as efficiently as it conducts Cl-. Under physiological conditions, the electrochemical gradients for both Cl- and HCO3- are directed outward, so CFTR likely contributes to the epithelial secretion of both ions. HCO3- secretion may be important for controlling pH of the luminal, but probably not the cytoplasmic, fluid in CFTR-containing epithelia. In CF, a decreased secretion of HCO3- may lead to decreased pH of the luminal fluid.
A conserved heptamer motif for ribosomal RNA transcription termination in animal mitochondria.
Authors: Valverde J R , Marco R , Garesse R
Published: May 2006

A search of sequence data bases for a tridecamer transcription termination signal, previously described in human mtDNA as being responsible for the accumulation of mitochondrial ribosomal RNAs (rRNAs) in excess over the rest of mitochondrial genes, has revealed that this termination signal occurs in equivalent positions in a wide variety of organisms from protozoa to mammals. Due to the compact organization of the mtDNA, the tridecamer motif usually appears as part of the 3' adjacent gene sequence. Because in phylogenetically widely separated organisms the mitochondrial genome has experienced many rearrangements, it is interesting that its occurrence near the 3' end of the large rRNA is independent of the adjacent gene. The tridecamer sequence has diverged in phylogenetically widely separated organisms. Nevertheless, a well-conserved heptamer--TGGCAGA, the mitochondrial rRNA termination box--can be defined. Although extending the experimental evidence of its role as a transcription termination signal in humans will be of great interest, its evolutionary conservation strongly suggests that mitochondrial rRNA transcription termination could be a widely conserved mechanism in animals. Furthermore, the conservation of a homologous tridecamer motif in one of the last 3' secondary loops of nonmitochondrial 23S-like rRNAs suggests that the role of the sequence has changed during mitochondrial evolution.
Induction of cytochrome CYPIA1 and formation of toxic metabolites of benzo[a]pyrene by rat aorta: a possible role in atherogenesis.
Authors: Thirman M J , Albrecht J H , Krueger M A , Erickson R R , Cherwitz D L , Park S S , Gelboin H V , Holtzman J L
Published: May 2006

Cigarette smoking is a leading risk factor for atherosclerosis. Endothelial injury may be the initial event in this process. The carcinogenic metabolites of the polycyclic aromatic hydrocarbons found in cigarette smoke tars could cause this injury. We tested this model by examining the effect of 3-methylcholanthrene administration on aortic polycyclic aromatic hydrocarbon metabolism. Immunoblotting with a monoclonal antibody (mAb 1-7-1) specific for cytochromes CYPIA1 and CYPIA2 showed that aortic microsomes from treated, but not from control, animals contained CYPIA1; the CYPIA1 was primarily in the endothelium. Aortic microsomes from induced animals metabolized benzo[a]pyrene (BaP) to the 7R,8S,9,10-tetrahydrotetrol-, 7,8-dihydrodiol-, 1,6 quinone-, 3,6 quinone-, 6,12 quinone-, 3-hydroxy-, and 9-hydroxy-BaP. mAb 1-7-1 inhibited the formation of the tetrahydrotetrol, the dihydrodiol-BaP, and the 3-hydroxy-BaP but did not inhibit the quinones or the 9-hydroxy-BaP. Arachidonic acid did not affect metabolism. These data suggest that the aortas of induced animals metabolize the BaP in cigarette smoke to carcinogenic and toxic products and that this metabolism may initiate vessel injury and lead to the accelerated atherosclerosis seen in cigarette smokers.
MYCN is retained in single copy at chromosome 2 band p23-24 during amplification in human neuroblastoma cells.
Authors: Corvi R , Amler L C , Savelyeva L , Gehring M , Schwab M
Published: May 2006

Amplification of the human N-myc protooncogene, MYCN, is frequently seen either in extrachromosomal double minutes or in homogeneously staining regions of aggressively growing neuroblastomas. MYCN maps to chromosome 2 band p23-24, but homogeneously staining regions have never been observed at this band, suggesting transposition of MYCN during amplification. We have employed fluorescence in situ hybridization to determine the status of MYCN at 2p23-24 in five human neuroblastoma cell lines. All five lines carried, in addition to amplified MYCN in homogeneously staining regions or double minutes, single-copy MYCN at the normal position. In one line there was coamplification of MYCN together with DNA of the host chromosome 12, to which MYCN had been transposed. Our results suggest a model of amplification where MYCN is retained at its original location. They further sustain the view that either the initial events of MYCN amplification or the further evolution of amplified MYCN copies follow mechanisms different from those leading to amplification of drug-resistance genes.
Spatial distribution of two maternal messengers in Paracentrotus lividus during oogenesis and embryogenesis.
Authors: Di Carlo M , Romancino D P , Montana G , Ghersi G
Published: May 2006

We demonstrated that two mRNAs that are synthesized during the vitellogenic period of oogenesis and that code for cell surface proteins are asymmetrically distributed in the unfertilized egg of Paracentrotus lividus. At fertilization, these RNAs rapidly localize in the cortical zone at the animal pole of the egg. They are then detected in the mesomeres and the macromeres, but not in the micromeres, and thereafter are found in the ectoderm but not in the vegetal plate, mesenchyme cells, or early intestine. They disappear in late gastrula. The proteins synthesized by these mRNAs show the same territorial location during the period examined here, which included the unfertilized egg and the 16-blastomere stage. These conclusions were reached on the basis of in situ hybridization and immunostaining experiments, as well as Northern and Western blot analyses of isolated blastomeres. The possible significance of this asymmetric distribution of these two mRNAs and proteins in the establishment of the animal/vegetal axis is discussed.
Sorting single molecules: application to diagnostics and evolutionary biotechnology.
Authors: Eigen M , Rigler R
Published: May 2006

A method is described that provides for detection and identification of single molecules in solution. The method is based on fluorescence correlation spectroscopy, which records spatio-temporal correlations among fluctuating light signals, coupled with devices for trapping single molecules in an electric field. This technique is applied to studies of molecular evolution, where it allows fast screening of large mutant spectra in which targets are labeled by specific fluorescent ligands. The method expands the horizon in molecular diagnostics by making it possible to monitor concentrations down to (less than) 10(-15) M without any need for amplification.
DNA methylation prevents the amplification of TROP1, a tumor-associated cell surface antigen gene.
Authors: Alberti S , Nutini M , Herzenberg L A
Published: May 2006

We tested the hypothesis that different genes can have different abilities to be amplified after transfection under comparable selection conditions. DNA from human lymphoid or choriocarcinoma cell lines was transfected into L cells. Transfectants for CD5, CD8A, TROP1, and TROP2, genes expressed on lymphocytes or trophoblast and carcinomas, were selected by fluorescence-activated cell sorting. To select for amplification of the transfected gene we cloned twice by fluorescence-activated cell sorting the transfectants with the highest expression. We analyzed a total of 38 families (1768 clones) derived from the original transfectants. We then analyzed by Southern blotting the clones with the highest increase in surface expression and determined the copy number of each transfected gene. CD5, CD8A, and TROP2 were amplified with high frequency and progressively, whereas TROP1 essentially was not amplified at all. We examined the hypothesis that DNA methylation prevents the amplification of the TROP1 gene by treating JAR choriocarcinoma cells with 5-azacytidine to decrease DNA methylation. DNA extracted at different times after the treatment was used for transfection. When DNA that showed demethylation of the TROP1 gene was used, 16 Trop-1 transfectants were obtained and 6 of them were found to contain up to 40 copies of the TROP1 gene per haploid genome. Thus, we showed that transfectants obtained from a demethylated TROP1 gene were amplified efficiently and progressively. We propose that DNA methylation affects DNA amplification either by altering the recognition of methylated DNA sequences or by changing the conformation of the chromatin of methylated segments. We speculate that DNA methylation is a determinant of gene amplification in vivo, for example in tumor cells.