bluuebunny/crossref_metadata_2025 huggingface dataset:
The LIM motif defines a specific zinc-binding protein domain.
Authors: Michelsen J W , Schmeichel K L , Beckerle M C , Winge D R
Published: May 2006

The cysteine-rich protein (CRP) contains two copies of the LIM sequence motif, CX2CX17HX2CX2CX2CX17-CX2C, that was first identified in the homeodomain proteins Lin-11, Is1-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are reminiscent of a metal-binding domain. We examined the metal-binding properties of CRP isolated from chicken smooth muscle (cCRP) and from a bacterial expression system and observed that cCRP is a specific Zn-binding metalloprotein. Four Zn(II) ions are maximally bound to cCRP, consistent with the idea that each LIM domain coordinates two metal ions. From spectroscopic studies of Co(II)- and 113Cd(II)-substituted cCRP, we determined that each metal ion is tetrahedrally coordinated with cysteinyl sulfurs dominating the ligand types. One metal site within each LIM motif has tetrathiolate (S4) coordination, the second site may either be S4 or S3N1. The LIM motif represents another example of a specific Zn-binding protein sequence.
Mature T cells of autoimmune lpr/lpr mice have a defect in antigen-stimulated suicide.
Authors: Russell J H , Rush B , Weaver C , Wang R
Published: May 2006

Antigen receptor-stimulated cell death of developing, immature T cells plays an important role in shaping the repertoire of antigens to which mature T cells will respond, but a role for receptor-stimulated death in controlling responses of mature T cells is controversial. Mutant lpr/lpr mice exhibit an autoimmune syndrome similar to systemic lupus erythematosus. Here we demonstrate that these mice have a defect in antigen-stimulated suicide of activated T cells in mature CD4+ and CD8+ T cell compartments. The defective suicide pathway is evident when the T cells are stimulated with antigen on antigen-presenting cells or with immobilized anti-CD3 in the absence of antigen-presenting cells. These studies, in concert with the work of others, suggest that antigen-stimulated death of mature cells may be important both in establishing peripheral tolerance and in limiting inflammation during normal immune responses.
The HOX11 gene encodes a DNA-binding nuclear transcription factor belonging to a distinct family of homeobox genes.
Authors: Dear T N , Sanchez-Garcia I , Rabbitts T H
Published: May 2006

A translocation involving human chromosome 10, band q24, in a subset of T-cell acute leukemias disrupts a region surrounding the putative oncogene HOX11, which encodes a protein with a homeodomain. The HOX11 protein binds to a specific DNA sequence, it localizes to the cell nucleus, and it transactivates transcription of a reporter gene linked to a cis-regulatory element, suggesting that HOX11 functions in vivo as a positive transcription activator. PCR analysis shows that the HOX11 homeodomain is a member of a distinct class of homeodomains, representatives of which occur in murine and Drosophila genomes. These all contain a threonine residue in place of the more common isoleucine or valine in helix 3 of the homeodomain. HOX11 therefore appears to belong to a family of DNA-binding transactivators of transcription.
Synthetic peptides corresponding to different mutated regions of the amyloid gene in familial Creutzfeldt-Jakob disease show enhanced in vitro formation of morphologically different amyloid fibrils.
Authors: Goldfarb L G , Brown P , Haltia M , Ghiso J , Frangione B , Gajdusek D C
Published: May 2006

We synthesized polypeptides corresponding to sequences encoded by normal and mutant alleles in the regions of codon 178 (Asp-->Asn) and codon 200 (Glu-->Lys) of the chromosome 20 amyloid gene that have been linked to familial Creutzfeldt-Jakob disease. Peptide suspensions from both regions spontaneously formed amyloid fibrils with different morphological characteristics and aggregation tendencies. Fibrillar arrays were denser and more profuse in mutant than in normal peptide suspensions and were even more marked when the homologous mutant and normal peptides were mixed together. Preparations from the region of codon 200 were in all cases more fibrillogenic than corresponding peptides from the region of codon 178. These in vitro observations support the hypothesis that amino acid changes from pathogenic single-allele point mutations in Creutzfeldt-Jakob disease may nucleate the in vivo folding behavior of the normal host protein to favor formation of insoluble amyloid fibrils.
The ras signaling pathway mimics insulin action on glucose transporter translocation.
Authors: Kozma L , Baltensperger K , Klarlund J , Porras A , Santos E , Czech M P
Published: May 2006

Recent observations suggest that insulin increases cellular levels of activated, GTP-bound Ras protein. We tested whether the acute actions of insulin on hexose uptake and glucose-transporter redistribution to the cell surface are mimicked by activated Ras. 3T3-L1 fibroblasts expressing an activated mutant (Lys-61) N-Ras protein exhibited a 3-fold increase in 2-deoxyglucose uptake rates compared with non-transfected cells. Insulin stimulated hexose uptake by approximately 2-fold in parental fibroblasts but did not stimulate hexose uptake in the N-Ras61K-expressing fibroblasts. Overexpression of N-Ras61K also mimicked the large effect of insulin on 2-deoxyglucose transport in 3T3-L1 adipocytes, and again the effects of the two agents were not additive. Total glucose transporter protein (GLUT) 1 was similar between parental and N-Ras61K-expressing 3T3-L1 fibroblasts or adipocytes, whereas total GLUT-4 protein was actually lower in the N-Ras61K-expressing compared with parental adipocytes. However, expression of N-Ras61K in 3T3-L1 adipocytes markedly elevated both GLUT-1 and GLUT-4 in plasma membranes relative to intracellular membranes, and insulin had no further effect. These modulations of glucose transporters by N-Ras61K expression are not due to upstream regulation of insulin receptors because receptor tyrosine phosphorylation and association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins were unaffected. These results show that activated Ras mimics the actions of insulin on membrane trafficking of glucose transporters, consistent with the concept that Ras proteins function as intermediates in this insulin signaling pathway.
Truncated erythropoietin receptor causes dominantly inherited benign human erythrocytosis.
Authors: de la Chapelle A , Träskelin A L , Juvonen E
Published: May 2006

Erythropoietin regulates the proliferation and differentiation of erythroid precursor cells. Its effect is mediated by the erythropoietin receptor (EPOR), a member of a large family of cytokine receptors. The EPOR gene has recently been cloned, sequenced, and characterized. As shown experimentally, its intracellular C-terminal part contains a domain exerting negative control on erythropoiesis. Here we describe a G to A transition in nucleotide 6002 of the EPOR gene that converts a TGG codon for tryptophan into a TAG stop codon, predicting the truncation of the 70 C-terminal amino acids of the EPOR molecule. The mutation occurs in heterozygous form in the germ-line DNA of members of a large kindred in which primary erythrocytosis is segregating as a mild autosomal dominant trait. The mutation cosegregates with the disease phenotype in all 29 affected family members studied; it occurs in no unaffected family members or unrelated controls. This appears to be an example of a human condition caused by an EPOR mutation. Striking similarities exist between the human phenotype described here and phenotypes of cell lines expressing similarly truncated EPOR molecules produced experimentally. By analogy with these in vitro studies, one can hypothesize that the truncated EPOR molecules are activated by suppression of phosphorylation leading to loss of the down-modulation exerted by intact EPOR molecules. Experimental modifications of the EPOR gene may eventually have therapeutic applications.
Neurological disease induced in transgenic mice expressing the env gene of the Cas-Br-E murine retrovirus.
Authors: Kay D G , Gravel C , Pothier F , Laperrière A , Robitaille Y , Jolicoeur P
Published: May 2006

The Cas-Br-E murine leukemia virus induces a spongiform myeloencephalopathy in susceptible mice. We constructed transgenic mice harboring either the viral genome (in a replication-defective form) or only its env gene. Low levels of expression of either transgene resulted in mild neuropathology and/or signs of neurological disease in more than half of these mice. These results indicate that the disease can occur in the absence of virus replication and strongly suggest that the env gp70/p15E complex is sufficient to induce disease.
Storage of a sensory pattern by anti-Hebbian synaptic plasticity in an electric fish.
Authors: Bell C C , Caputi A , Grant K , Serrier J
Published: May 2006

Synaptic plasticity occurs in several regions of the vertebrate brain and is believed to mediate the storage of behaviorally significant information during learning. Synaptic plasticity is well demonstrated in most cases, but the behavioral meaning of the relevant neural signals and the behavioral role of the plasticity are uncertain. In this paper we describe a case of synaptic plasticity which involves identifiable sensory and motor signals and which appears to mediate the storage of an image of past sensory input. Corollary discharge signals associated with the motor command that drives the electric organ are prominent in the electrosensory lobe of mormyrid electric fish. Some of these corollary discharge signals elicit a negative image or representation of the electrosensory input pattern that has followed recent motor commands. When the temporal and spatial pattern of sensory input changes, the corollary discharge effect also changes in a corresponding manner. The cellular mechanisms by which the corollary discharge-evoked representation is stored were investigated by intracellular recording from cells of the electrosensory lobe and pairing intracellular current pulses with the corollary discharge signal. The results indicate that the representation of recent sensory input is stored by means of anti-Hebbian plasticity at the synapses between corollary discharge-conveying fibers and cells of the electrosensory lobe. The results also suggest that dendritic spikes and plasticity at inhibitory synapses are involved in the phenomenon.
Cosmological implications of light element abundances: theory.
Authors: Schramm D N
Published: May 2006

Primordial nucleosynthesis provides (with the microwave background radiation) one of the two quantitative experimental tests of the hot Big Bang cosmological model (versus alternative explanations for the observed Hubble expansion). The standard homogeneous-isotropic calculation fits the light element abundances ranging from 1H at 76% and 4He at 24% by mass through 2H and 3He at parts in 105 down to 7Li at parts in 1010. It is also noted how the recent Large Electron Positron Collider (and Stanford Linear Collider) results on the number of neutrinos (Nnu) are a positive laboratory test of this standard Big Bang scenario. The possible alternate scenario of quark-hadron-induced inhomogeneities is also discussed. It is shown that when this alternative scenario is made to fit the observed abundances accurately, the resulting conclusions on the baryonic density relative to the critical density (Omegab) remain approximately the same as in the standard homogeneous case, thus adding to the robustness of the standard model and the conclusion that Omegab approximately 0.06. This latter point is the driving force behind the need for nonbaryonic dark matter (assuming total density Omegatotal = 1) and the need for dark baryonic matter, since the density of visible matter Omegavisible < Omegab. The recent Population II B and Be observations are also discussed and shown to be a consequence of cosmic ray spallation processes rather than primordial nucleosynthesis. The light elements and Nnu successfully probe the cosmological model at times as early as 1 sec and a temperature (T) of approximately 10(10) K (approximately 1 MeV). Thus, they provided the first quantitative arguments that led to the connections of cosmology to nuclear and particle physics.
Causes and effects of the first quasars.
Authors: Rees M J
Published: May 2006

The light we observe from the most distant known quasars set out when the Universe was about 200 times denser than it is now and less than one-tenth of its present age. The existence of these objects implies that galaxy formation had already, at that early epoch, proceeded to the stage when massive (>10(8)M[symbol, see text]) objects had accumulated in the centers of at least some young galaxies. A specific model is presented to show that the evolution and luminosity function of quasars are compatible with the cold dark matter cosmogony. Most big galaxies probably passed through a quasar phase; the remnant black holes in nearby galaxies may reveal themselves via the flares that occur whenever a star passes too close to them and gets tidally disrupted. The rich absorption spectra of quasars serve as a probe of the intervening medium. The gas responsible for the Lyman alpha absorption lines may be due to primordial gas gravitationally confined in minihalos of dark matter--shallow potential wells whose evolution and relation to dwarf galaxies are briefly discussed. The patchy heat input into the intergalactic medium from early quasars could modulate the environment in which galaxies form, leading to large-scale spatial correlations in the galaxy distribution. This review concludes with general comments on the prospects for a fully quantitative understanding of galaxy formation.
Galaxy clustering on large scales.
Authors: Efstathiou G
Published: May 2006

I describe some recent observations of large-scale structure in the galaxy distribution. The best constraints come from two-dimensional galaxy surveys and studies of angular correlation functions. Results from galaxy redshift surveys are much less precise but are consistent with the angular correlations, provided the distortions in mapping between real-space and redshift-space are relatively weak. The galaxy two-point correlation function, rich-cluster two-point correlation function, and galaxy-cluster cross-correlation function are all well described on large scales ( greater, similar 20h-1 Mpc, where the Hubble constant, H0 = 100h km.s-1.Mpc; 1 pc = 3.09 x 10(16) m) by the power spectrum of an initially scale-invariant, adiabatic, cold-dark-matter Universe with Gamma = Omegah approximately 0.2. I discuss how this fits in with the Cosmic Background Explorer (COBE) satellite detection of large-scale anisotropies in the microwave background radiation and other measures of large-scale structure in the Universe.
Identification and cloning of a protein kinase-encoding mouse gene, Plk, related to the polo gene of Drosophila.
Authors: Clay F J , McEwen S J , Bertoncello I , Wilks A F , Dunn A R
Published: May 2006

We have determined the nucleotide sequence of a cDNA encoding a protein kinase that is closely related to the enzyme encoded by the Drosophila melanogaster mutant polo and that we have designated Plk (polo-like kinase). Plk is also related to the products of the Saccharomyces cerevisiae cell cycle gene MSD2 (CDC5) and the recently described early growth response gene Snk. Together, Plk, polo, Snk, and MSD2 define a subfamily of serine/threonine protein kinases. Plk is expressed at high levels in a number of fetal and newborn mouse tissues but is not expressed in the corresponding adult organs. With the exception of adult hemopoietic tissues, the only adult tissues in which we could detect Plk expression were ovaries and testes. Taken together, the patterns of Plk expression suggest an association with proliferating cells. Since polo is required for mitosis in Drosophila it is possible that Plk is involved in some aspect of cell cycle regulation in mammalian cells.
Functional heterogeneity of calcium release by inositol trisphosphate in single Purkinje neurones, cultured cerebellar astrocytes, and peripheral tissues.
Authors: Khodakhah K , Ogden D
Published: May 2006

Purkinje neurones of the cerebellar cortex are rich in receptors for the Ca-mobilizing second messenger inositol trisphosphate (InsP3) in association with intracellular Ca stores. Cytosolic Ca ions are important in regulating neuronal excitability but it has proved difficult to demonstrate InsP3-evoked release of Ca in mammalian central neurones directly. Intracellular release of InsP3 by flash photolysis of caged InsP3, combined with whole-cell patch clamp and microspectrofluorimetry of Ca indicators, allows comparison of InsP3-evoked Ca release in single Purkinje cells in cerebellar slices with the same process in cultured astrocytes and peripheral tissues. In astrocytes, hepatocytes, exocrine cells, and vascular endothelium, minimal Ca release from stores requires photorelease of InsP3 at concentrations of 0.2-0.5 microM, and maximal efflux as judged by the rate of increase of Ca concentration is seen with 5-10 microM InsP3. In contrast in Purkinje cells, InsP3 concentrations of > or = 9 microM were required to produce minimal Ca release from stores under the same conditions, and Ca efflux increased with InsP3 concentrations up to 70-80 microM. Furthermore, the rate of increase and size of the Ca concentration in Purkinje cells are 10- to 30-fold greater than in astrocytes and peripheral tissues. The InsP3 sensitivity was not affected by changing exogenous cytosolic Ca buffering, suggesting that endogenous Ca binding cannot account for the difference. The results show a functional difference in InsP3-evoked Ca release between Purkinje cells and peripheral tissues.
"Caged" phenylephrine: development and application to probe the mechanism of alpha-receptor-mediated vasoconstriction.
Authors: Muralidharan S , Maher G M , Boyle W A , Nerbonne J M
Published: May 2006

A "caged" analogue of the alpha-adrenergic receptor agonist phenylephrine (PE) was prepared by exploiting the 2-nitrobenzyl protecting group and using a synthetic procedure developed to permit preferential derivatization at the amino group. On isolated adult rat mesenteric arterioles, caged-PE had no measurable effects at concentrations up to 100 microM; 0.5-ms light flashes in the presence of caged-PE, however, produced marked and dose-dependent vasoconstriction. Flash-induced vasoconstrictions were blocked by the alpha-receptor antagonist phentolamine and were unaffected by the beta-receptor antagonist propranolol, indicating that the light-induced responses reflect the selective activation of alpha-adrenergic receptors. After a single flash, a large transient decrease in vessel diameter was recorded, and in most vessels, this was followed by a smaller, sustained constriction. The sustained component of the contraction was selectively eliminated when Ca2+ was removed from the bath, which suggests that different mechanisms underlie the transient and the sustained responses to PE. The responses to single flashes of varying intensities occurred with a mean latency of 460 ms, which is consistent with the intermediacy of several steps between alpha-receptor activation and contraction. We anticipate that it will be possible to extend this approach to develop caged analogues of other neurotransmitters for mechanistic and kinetic studies.
Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein.
Authors: Sakaguchi K , Zambrano N , Baldwin E T , Shapiro B A , Erickson J W , Omichinski J G , Clore G M , Gronenborn A M , Appella E
Published: May 2006

The nucleocapsid (NC) protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is important for encapsidation of the virus genome, RNA dimerization, and primer tRNA annealing in vitro. Here we present evidence from gel mobility-shift experiments indicating that NCp7 binds specifically to an RNA sequence. Two complexes were identified in native gels. The more slowly migrating complex contained two RNA molecules and one peptide, while the more rapidly migrating one is composed of one RNA and one peptide. Further, mutational analysis of the RNA shows that the predicted stem and loop structure of stem-loop 1 plays a critical role. Our results show that NCp7 binds to a unique RNA structure within the psi region; in addition, this structure is necessary for RNA dimerization. We propose that NCp7 binds to the RNA via a direct interaction of one zinc-binding motif to stem-loop 1 followed by binding of the other zinc-binding motif to stem-loop 1, stem-loop 2, or the linker region of the second RNA molecule, forming a bridge between the two RNAs.
Structural changes in the actomyosin cross-bridges associated with force generation.
Authors: Brenner B , Yu L C
Published: May 2006

It is generally thought that to generate active force in muscle, myosin heads (cross-bridges) that are attached to actin undergo large-scale conformational changes. However, evidence for conformational changes of the attached cross-bridges associated with force generation has been ambiguous. In this study, we took advantage of the recent observation that cross-bridges that are weakly attached to actin in a relaxed muscle are apparently in attached preforce-generating states. The experimental conditions were chosen such that there were large fractions of cross-bridges attached under relaxing and activating conditions, and high-resolution equatorial x-ray diffraction patterns obtained under these conditions were compared. Changes brought about by activation in the two innermost intensities, I10 and I11, did not follow the familiar reciprocal changes. Instead, there was almost no change in I11, whereas I10 decreased by 34%. Together with the changes found in the higher-order reflections, the results suggest that the structure of the attached force-generating cross-bridges differs from that of the weakly bound, preforce-generating cross-bridges and possibly also differs from that of the cross-bridges in rigor. These observations support the concept that force generation involves a transition between distinct structural states of the actomyosin cross-bridges.
The growth factor-inducible immediate-early gene 3CH134 encodes a protein-tyrosine-phosphatase.
Authors: Charles C H , Sun H , Lau L F , Tonks N K
Published: May 2006

Stimulation of fibroblasts with serum growth factors results in the rapid activation of a set of immediate-early genes, among them 3CH134. We have purified a bacterially expressed form of the 3CH134-encoded polypeptide and demonstrated that it has intrinsic protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) activity in vitro. This activity is optimal at pH 7.5, is sensitive to vanadate and cysteinyl modifying agents, and is insensitive to a panel of serine/threonine phosphatase inhibitors. Purified 3CH134 protein displays a high degree of selectivity among the tyrosine-phosphorylated polypeptide substrates tested. Under our assay conditions, the rates of dephosphorylation are in the order EDNDYINASL peptide < myelin basic protein < reduced, carboxyamidomethylated, and maleylated lysozyme (RCML) < p42mapk. There is a 200-fold range in rates for these substrates, with p42mapk dephosphorylated 15-fold more rapidly than RCML. Although 3CH134 is most closely related to the tyrosine/serine dual-specificity phosphatase VH1, we failed to detect any 3CH134-directed activity on casein or RCML phosphorylated on serine/threonine residues by cAMP-dependent protein kinase. Since 3CH134 expression is controlled transcriptionally and posttranscriptionally, it may represent a class of PTPases whose activity is regulated at the level of protein synthesis and degradation.
Solubilized alpha beta Na,K-ATPase remains protomeric during turnover yet shows apparent negative cooperativity toward ATP.
Authors: Ward D G , Cavieres J D
Published: May 2006

A prominent feature of the Na,K-ATPase reaction is an ATP dependence that suggests high- and low-affinity ATP requirements during the enzymic cycle. As only one ATP-binding domain has been identified in the alpha subunit and none has been identified in the beta subunit, it has seemed likely that the apparent negative cooperativity results from subunit interactions in an (alpha beta)2 diprotomer. To test this possibility, we have examined the behavior of solubilized alpha beta protomers of Na,K-ATPase down to 50 nM [gamma-32P]ATP. Active-enzyme analytical ultracentrifugation shows that the protomer is the active species and that no oligomerization occurs during turnover. However, we find that dual ATP effects can be clearly demonstrated and that nonhydrolyzable ATP analogs can stimulate the Na,K-ATPase activity of the soluble protomer. We conclude that the apparent negative cooperativity is inherent to the alpha beta protomer and that this should explain some of the complexities found with membrane-bound Na,K-ATPase and, perhaps, other P-type cation pumps.
Self-recognition in helicate self-assembly: spontaneous formation of helical metal complexes from mixtures of ligands and metal ions.
Authors: Kramer R , Lehn J M , Marquis-Rigault A
Published: May 2006

On treatment with copper(I) ions, mixtures of the [oligo(2,2')-bipyridine] strands 2-5 yield spontaneously the double helicates 2H(2)-2H(5) without significant crossover. Similarly, when a mixture of the two tris-bypyridine ligands 1 and 3a (containing different substitutions) is allowed to react with copper(I) and nickel(II) ions, only the double helicate 2H(3a) and the triple helicate 3H(1) are formed. Thus, these systems undergo self-assembly of helicates with self-recognition. The process represents a self/nonself-discrimination involving the preferential binding of like metal ions by like ligand strands in a mixture to selectively assemble into the corresponding helicates. In a broader perspective, these results point to a change in paradigm from pure compounds to instructed mixtures--that is from seeking chemical purity to designing programmed systems composed of mixtures of instructed components capable of spontaneously forming well--defined superstructures through self-processes.
A double-filter method for nitrocellulose-filter binding: application to protein-nucleic acid interactions.
Authors: Wong I , Lohman T M
Published: May 2006

Nitrocellulose-filter binding is a powerful technique commonly used to study protein-nucleic acid interactions; however, its utility in quantitative studies is often compromised by its lack of precision. To improve precision and accuracy, we have introduced two modifications to the traditional technique: the use of a 96-well dot-blot apparatus and the addition of a DEAE membrane beneath the nitrocellulose membrane. Using the dot-blot apparatus, an entire triplicate set of data spanning 20-24 titrant concentrations can be collected on a single 4.5 x 5 inch sheet of nitrocellulose, obviating the need to manipulate separate filters for each titration point. The entire titration can then be quantitated simultaneously with direct two-dimensional beta-emission imaging technology. The DEAE second membrane traps all DNA that does not bind to the nitrocellulose, enabling a direct determination of the total amount of DNA filtered. This measurement improves precision by allowing the amount of DNA retained by the nitrocellulose to be normalized against the total amount of DNA filtered. The DEAE membrane also permits a more accurate quantitation of filter-retention efficiency and nonspecific background retention based on free DNA rather than total DNA filtered. The general approach and methods of analysis to obtain equilibrium binding isotherms are discussed, using as examples our studies of the Escherichia coli Rep protein, a helicase, and its interactions with short oligodeoxynucleotides.
The FKB2 gene of Saccharomyces cerevisiae, encoding the immunosuppressant-binding protein FKBP-13, is regulated in response to accumulation of unfolded proteins in the endoplasmic reticulum.
Authors: Partaledis J A , Berlin V
Published: May 2006

The FKB2 gene of Saccharomyces cerevisiae encodes a homolog of mammalian FKBP-13, an FK506/rapamycin-binding protein that localizes to the lumen of the endoplasmic reticulum (ER). We have found that FKB2 mRNA levels increase in response to the accumulation of unfolded precursor proteins in the ER. FKB2 mRNA levels are elevated in cells blocked in N-glycosylation--i.e., in wild-type cells treated with tunicamycin and in the sec53-6 mutant grown at the nonpermissive temperature. Mutations that block other steps in secretion have no effect on FKB2 mRNA levels, indicating that increases in FKB2 mRNA are not the consequence of a general block in secretion. The increase in FKB2 mRNA in response to unfolded proteins in the ER is mediated through a 21-bp unfolded-protein response (UPR) element located in the 5' noncoding region of FKB2. UPR elements present in other ER chaperone genes, such as yeast KAR2 (BiP), mammalian GRP78 (BiP), and GRP94, function in an analogous manner to that in FKB2. As with KAR2, FKB2 mRNA levels are also elevated by heat shock. The similarities in the regulation of FKB2 and other ER chaperone genes suggest that FKBP-13 may play a role in protein trafficking in the ER.
EBNA-5, an Epstein-Barr virus-encoded nuclear antigen, binds to the retinoblastoma and p53 proteins.
Authors: Szekely L , Selivanova G , Magnusson K P , Klein G , Wiman K G
Published: May 2006

Epstein-Barr virus (EBV) immortalized human lymphoblastoid cell lines express six virally encoded nuclear proteins, designated EBV nuclear antigens 1-6 (EBNA-1-6). We show that the EBNA-5 protein (alternatively designated EBNA-LP) that is required for B-cell transformation can form a molecular complex with the retinoblastoma (RB) and p53 tumor suppressor proteins. Using EBNA-5 deletion mutants, we have found that a 66-amino acid-long peptide, encoded by the W repeat of the EBV genome, is sufficient for binding. Point mutations of RB and p53 that inhibit their complexing with other DNA viral oncoproteins do not affect their binding to EBNA-5. p53 competes with RB for EBNA-5 binding. Our data suggest that the mechanisms involved in EBV transformation may include impairment of RB and p53 function.
Okadaic acid regulation of the retinoblastoma gene product is correlated with the inhibition of growth factor-induced cell proliferation in mouse fibroblasts.
Authors: Kim T A , Velasquez B R , Wenner C E
Published: May 2006

Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, was used to study the mechanism of action of transforming growth factor beta (TGF-beta) on cell cycle progression in C3H/10T1/2 mouse embryonic fibroblasts, where TGF-beta exerts a growth-stimulatory effect. Concentrations of okadaic acid as low as 5 nM inhibited TGF-beta (5 ng/ml)- or 10% serum-induced [3H]thymidine incorporation into postconfluent, quiescent cells. Further, these inhibitory effects were observed when okadaic acid was added as late as 10 hr after TGF-beta or serum stimulation. Since C3H/10T1/2 fibroblasts undergo the G1/S transition at 10-14 hr after TGF-beta and 8-12 hr after serum stimulation, these observations indicate that a phosphatase activity may be required for S-phase entry. In a parallel experiment, okadaic acid partially inhibited TGF-beta-induced [14C]leucine incorporation by 20-65%, depending upon the okadaic acid concentration. In conjunction with the effect of okadaic acid on DNA and protein synthesis, Western blot analysis indicated that okadaic acid inhibited phosphorylation of the retinoblastoma gene product and decreased its protein level, even when added 10 hr after TGF-beta or 8 hr after serum stimulation. These findings strongly suggest that protein phosphatases play a pivotal role for S-phase entry in mouse fibroblasts. Moreover, protein phosphatases may be required in the intermediate steps of TGF-beta or serum growth factor signal-transduction pathways for the stimulation of phosphorylation of the retinoblastoma protein, especially in late G1.
Cloning of the STE5 gene of Saccharomyces cerevisiae as a suppressor of the mating defect of cdc25 temperature-sensitive mutants.
Authors: Perlman R , Yablonski D , Simchen G , Levitzki A
Published: May 2006

The STE5 gene of Saccharomyces cerevisiae was cloned using a screening procedure designed to isolate genes of the S. cerevisiae pheromone response pathway. We screened a yeast genomic high-copy-number plasmid library for genes that allow mating of cdc25ts mutants at the restrictive temperature without affecting the cell-cycle-arrest phenotype. One of the genes cloned was identified by genetic analysis as STE5. STE5 encodes a predicted open reading frame of 916 amino acids and exhibits significant homology to Far1 protein. RNA blot analysis reveals that STE5 gene transcription is regulated by the mating type of the cell and depends on an intact pheromone-response pathway.
Activation of the tyrosine aminotransferase gene is dependent on synergy between liver-specific and hormone-responsive elements.
Authors: Nitsch D , Boshart M , Schütz G
Published: May 2006

Tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) gene activity is stimulated by glucocorticoids and glucagon and is repressed by insulin. Expression and responsiveness to the different signal transduction pathways are restricted to the liver, in which the gene is activated shortly after birth. Here we provide a model for the basis of this tissue specificity of the hormonal control. In the two enhancers mediating hormone induction of TAT gene activity we find the hormone response elements in combination with binding sites for constitutive liver-enriched transcription factors: proteins of the hepatocyte nuclear factor 3 family bind in the vicinity of the glucocorticoid response element located 2.5 kb upstream of the transcription start site, while hepatocyte nuclear factor 4 interacts with an essential element in the cAMP-responsive enhancer at -3.6 kb. By juxtaposing the liver-specific element and the target sequence of the signal transduction pathway the regulatory properties of either enhancer can be reconstituted. Thus, the interdependence of the respective enhancer motifs restricts the hormonal activation of the TAT gene to the liver. The coincidence of the onset of TAT gene expression around birth with the perinatal changes in the concentrations of glucocorticoids, glucagon, and insulin suggests cooperation of signal transduction pathways and cell type-specific transcription factors in the developmental activation of the TAT gene.
Interactions between three common subunits of yeast RNA polymerases I and III.
Authors: Lalo D , Carles C , Sentenac A , Thuriaux P
Published: May 2006

The AC40 and AC19 subunits (encoded by RPC40 and RPC19) are shared by yeast RNA polymerases I and III and have a local sequence similarity to prokaryotic alpha subunits. Mutational analysis of the corresponding "alpha motif" indicated that its integrity is essential on AC40 subunit but is not essential on AC19 subunit. By applying the two-hybrid method, these two polypeptides were shown to associate in vivo. Extragenic suppression of rpc19 and rpc40 mutations confirmed that AC19 and AC40 subunits interact with each other in vivo and revealed an interaction with ABC10 beta subunit [encoded by RPB10; Woychick, N. A. & Young, R.A. (1990) J. Biol. Chem. 265, 17816-17819], one of the five polypeptides common to all three nuclear RNA polymerases. A correction of the RPB10 sequence showed that ABC10 beta subunit is a 70-amino acid polypeptide, as confirmed by peptide microsequencing. These results suggest that the assembly of RNA polymerase I and III requires the association of ABC10 beta subunit with an AC19/AC40 heterodimer.
Functional domains of the AraC protein.
Authors: Bustos S A , Schleif R F
Published: May 2006

The AraC protein, which regulates the L-arabinose operons in Escherichia coli, was dissected into two domains that function in chimeric proteins. One provides a dimerization capability and binds the ligand arabinose, and the other provides a site-specific DNA-binding capability and activates transcription. In vivo and in vitro experiments showed that a fusion protein consisting of the N-terminal half of the AraC protein and the DNA-binding domain of the LexA repressor dimerizes, binds well to a LexA operator, and represses expression of a LexA operator-beta-galactosidase fusion gene in an arabinose-responsive manner. In vivo and in vitro experiments also showed that a fusion protein consisting of the C-terminal half of the AraC protein and the leucine zipper dimerization domain from the C/EBP transcriptional activator binds to araI and activates transcription from a PBAD promoter-beta-galactosidase fusion gene. Dimerization was necessary for occupancy and activation of the wild-type AraC binding site.
Identification of a human achaete-scute homolog highly expressed in neuroendocrine tumors.
Authors: Ball D W , Azzoli C G , Baylin S B , Chi D , Dou S , Donis-Keller H , Cumaraswamy A , Borges M , Nelkin B D
Published: May 2006

Basic helix-loop-helix transcription factors of the achaete-scute family are instrumental in Drosophila neurosensory development and are candidate regulators of development in the mammalian central nervous system and neural crest. We report the isolation and initial characterization of a human achaete-scute homolog that is highly expressed in two neuroendocrine cancers, medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). The human gene, which we have termed human achaete-scute homology 1 (hASH1), was cloned from a human MTC cDNA library. It encodes a predicted protein of 238 aa that is 95% homologous to mammalian achaete-scute homolog (MASH) 1, a rodent basic helix-loop-helix factor. The 57-residue basic helix-loop-helix domain is identical to that in the rodent gene, and the basic and helical regions, excluding the loop, are 72-80% identical to Drosophila achaete-scute family members. The proximal coding region of the hASH1 cDNA contains a striking 14-copy repeat of the triplet CAG that exhibits polymorphism in human genomic DNA. Thus, hASH1 is a candidate locus for disease-causing mutations via triplet repeat amplification. Analysis of rodent-human somatic cell hybrids permitted assignment of hASH1 to human chromosome 12. Northern blots revealed hASH1 transcripts in RNA from a human MTC cell line, two fresh MTC tumors, fetal brain, and three lines of human SCLC. In contrast, cultured lines of non-SCLC lung cancers and a panel of normal adult human tissues showed no detectable hASH1 transcripts. Expression of hASH1 may provide a useful marker for cancers with neuroendocrine features and may contribute to the differentiation and growth regulation of these cells.
Circadian rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria.
Authors: Kondo T , Strayer C A , Kulkarni R D , Taylor W , Ishiura M , Golden S S , Johnson C H
Published: May 2006

We have used a luciferase reporter gene and continuous automated monitoring of bioluminescence to demonstrate unequivocally that cyanobacteria exhibit circadian behaviors that are fundamentally the same as circadian rhythms of eukaryotes. We also show that these rhythms can be studied by molecular methods in Synechococcus sp. PCC7942, a strain for which genetic transformation is well established. A promoterless segment of the Vibrio harveyi luciferase structural genes (luxAB) was introduced downstream of the promoter for the Synechococcus psbAI gene, which encodes a photosystem II protein. This reporter construction was recombined into the Synechococcus chromosome, and bioluminescence was monitored under conditions of constant illumination following entrainment to light and dark cycles. The reporter strain, AMC149, expressed a rhythm of bioluminescence which satisfies the criteria of circadian rhythms: persistence in constant conditions, phase resetting by light/dark signals, and temperature compensation of the period. Rhythmic changes in levels of the native psbAI message following light/dark entrainment supported the reporter data. The behavior of this prokaryote disproves the dogma that circadian mechanisms must be based on eukaryotic cellular organization. Moreover, the cyanobacterial strain described here provides an efficient experimental system for molecular analysis of the circadian clock.
Pathogenetic role of Arg-Gly-Asp-recognizing integrins in acute renal failure. off.
Authors: Goligorsky M S , DiBona G F
Published: May 2006

Reorientation of the alpha 3 subunit of integrins from predominantly basal to the apical cell surface of cultured renal tubular epithelial cells subjected to oxidant stress has previously been demonstrated. The present study was designed to assess functional competence of ectopically expressed apical integrins. Cell-cell adhesion assay revealed enhanced cytoatractant properties of stressed cells. Stressed epithelial cells exhibited specific recognition and binding of laminin-coated latex beads. These processes were inhibited with the peptide Gly-Arg-Gly-Asp-Asn-Pro (GRGDNP) suggesting a role of RGD-recognizing integrins in augmented adhesion to stressed cells. Given that such enhanced adhesion in in vivo acute renal failure may govern tubular obstruction by desquamated epithelium, a physiological marker of patency of tubular lumen, proximal tubular pressure, was monitored in rats subjected to 60 min of renal ischemia followed by reperfusion. Proximal tubular pressure increased 2-fold after 2 hr of reperfusion in animals that had undergone 60 min of ischemia. Infusion of GRGDNP into the renal artery during reperfusion period virtually abolished an increase in proximal tubular pressure observed in ischemic acute renal failure. These in vitro and in vivo findings are consistent with the hypothesis that RGD-recognizing integrins play an important role in the pathogenesis of tubular obstruction in ischemic acute renal failure.
Relaxation kinetics of the Na+/glucose cotransporter.
Authors: Loo D D , Hazama A , Supplisson S , Turk E , Wright E M
Published: May 2006

An important class of integral membrane proteins, cotransporters, couple solute transport to electrochemical potential gradients; e.g., the Na+/glucose cotransporter uses the Na+ electrochemical potential gradient to accumulate sugar in cells. So far, kinetic analysis of cotransporters has mostly been limited to steady-state parameters. In this study, we have examined pre-steady-state kinetics of Na+/glucose cotransport. The cloned human transporter (hSGLT1) was expressed in Xenopus oocytes, and voltage-clamp techniques were used to monitor current transients after step changes in membrane potential. Transients exhibited a voltage-dependent time constant (tau) ranging between 2 and 10 ms. The charge movement Q was fitted to a Boltzmann relation with maximal charge Qmax of approximately 20 nC, apparent valence z of 1, and potential V0.5 of -39 mV for 50% Qmax. Lowering external Na+ from 100 to 10 mM reduced Qmax 40%, shifted V0.5 from -39 to -70 mV, had no effect on z, and reduced the voltage dependence of tau. Qmax was independent of, but tau was dependent on, temperature (a 10 degrees C increase increased tau by a factor of approximately 2.5 at -50 mV). Addition of sugar or phlorizin reduced Qmax. Analyses of hSGLT1 pre-steady-state kinetics indicate that transfer upon a step of membrane potential in the absence of sugar is due to two steps in the reaction cycle: Na+ binding/dissociation (30%) and reorientation of the protein in the membrane field (70%). The rate-limiting step appears to be Na+ binding/dissociation. Qmax provides a measure of transporter density (approximately 10(4)/microns 2). Charge transfer measurements give insight into the partial reactions of the Na+/glucose cotransporter, and, combined with genetic engineering of the protein, provide a powerful tool for studying transport mechanisms.
Tumor-suppressor genes: news about the interferon connection.
Authors: Lengyel P
Published: May 2006

The interferons are a family of secreted, multifunctional proteins which are components of the defenses of vertebrates against viral, bacterial, and parasitic infections and certain tumors. They exert their various activities by inducing the synthesis of a large variety of proteins. There are direct and indirect indications that several of these proteins may have tumor-suppressor activities. The interferon-inducible proteins implicated include: (i) a double-stranded RNA-activatable protein kinase that can phosphorylate and thereby inactivate the eukaryotic peptide chain initiation factor eIF-2; (ii) the interferon regulatory factors IRF-1 and IRF-2, which can modulate the expression of the interferons and of some interferon-inducible proteins; and (iii) RNase L, a latent endoribonuclease which can be activated by (2'-5')oligoadenylates, the products of a family of enzymes which are also interferon-inducible. It is note-worthy that some of the proteins encoded by tumor virus oncogenes (e.g., E1A from adenovirus, EBNA-2 from Epstein-Barr virus, and terminal protein from hepatitis B virus) impair the induction of at least some proteins by interferons.
Expression cloning in yeast of a cDNA encoding a broad specificity amino acid permease from Arabidopsis thaliana.
Authors: Frommer W B , Hummel S , Riesmeier J W
Published: May 2006

To study amino acid transport in plants at the molecular level, we have isolated an amino acid permease cDNA from Arabidopsis thaliana by complementation of a yeast mutant defective in proline uptake with a cDNA. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows no significant homologies to other known transporters. Expression of the cDNA enables the yeast mutant to take up L-[14C]proline. Competition studies argue for a broad but stereospecific substrate recognition by the permease, which resembles neutral or general amino acid transport systems from Chlorella and higher plants. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism.
Drosophila transforming growth factor beta superfamily proteins induce endochondral bone formation in mammals.
Authors: Sampath T K , Rashka K E , Doctor J S , Tucker R F , Hoffmann F M
Published: May 2006

Both decapentaplegic (dpp) protein and 60A protein have been implicated in pattern formation during Drosophila melanogaster embryogenesis. Within the C-terminal domain, dpp and 60A are similar to human bone morphogenetic protein 2 (75% identity) and human osteogenic protein 1 (70% identity), respectively. Both recombinant human bone morphogenetic protein 2 and recombinant human osteogenic protein 1 have been shown to induce bone formation in vivo and to restore large diaphyseal segmental defects in various animal models. We examined whether the Drosophila proteins, dpp and 60A, have the capacity to induce bone formation in mammals by using the rat subcutaneous bone induction model. Highly purified recombinant dpp and 60A induced the formation of cartilage, bone, and bone marrow in mammals, as determined by histological observations and by measurements of the specific activity of alkaline phosphatase and calcium content of the implants, thereby demonstrating that related proteins from phylogenetically distant species are capable of inducing bone formation in mammals when placed in sites where progenitor cells are available.