bluuebunny/crossref_metadata_2025 huggingface dataset:
Floral and ecological isolation between Aquilegia formosa and Aquilegia pubescens.
Authors: Hodges S A , Arnold M L
Published: May 2006

Habitat preference and pollination syndrome have been suggested as major factors in reproductive isolation among plant species. The columbine genus Aquilegia contains species that have been used as classic examples of reproductive isolation due to ecological and floral factors. In this analysis Aquilegia formosa, Aquilegia pubescens, and natural hybrid populations between these two species were assayed for genetic and morphological variation. Clinal variation was evident for three "random amplified polymorphic DNA" loci and five morphological characters along a transect extending from a lower altitude A. formosa population, through an intermediate hybrid population, to a higher altitude A. pubescens population. Similar clinal variation was also discovered for a transect that included A. formosa-like, hybrid, and A. pubescens-like populations at a single elevation. The change in the frequency of both sets of markers was closely associated with change in habitat. The molecular markers indicate the presence of bidirectional introgression between these two species. In contrast, there was apparently selection against introgression of four of the five alternate floral characters. Selection against the incorporation of floral characters from one species into the other species was suggested by the introgression of the DNA markers with little or no introgression of the four floral characters. These findings suggest the importance of adaptations associated with both pollination syndromes and habitat preference on species integrity.
Cell cycle regulation of V(D)J recombination-activating protein RAG-2.
Authors: Lin W C , Desiderio S
Published: May 2006

The antigen receptors of B and T lymphocytes are encoded in multiple germ-line DNA segments that are joined during lymphocyte development. The recombination-activating proteins RAG-1 and RAG-2 are both essential for this process, termed V(D)J rearrangement. Phosphorylation of the RAG-2 protein at Thr-490 by one or more cyclin-dependent kinases is associated with its rapid degradation. In an immature B-cell line and in normal thymocytes, RAG-2 protein accumulates preferentially in the G0/G1 phases of the cell cycle and declines by at least 20-fold before cells enter S phase. The amount of RAG-2 protein remains low throughout the S, G2, and M phases. The amount of RAG-1 protein shows considerably less fluctuation. The variation in RAG-2 protein is likely to be established, at least in part, by a posttranscriptional mechanism. These observations suggest that V(D)J rearrangement occurs entirely or preferentially within G0/G1.
Evidence for two independent domestications of cattle.
Authors: Loftus R T , MacHugh D E , Bradley D G , Sharp P M , Cunningham P
Published: May 2006

The origin and taxonomic status of domesticated cattle are controversial. Zebu and taurine breeds are differentiated primarily by the presence or absence of a hump and have been recognized as separate species (Bos indicus and Bos taurus). However, the most widely held view is that both types of cattle derive from a single domestication event 8000-10,000 years ago. We have examined mtDNA sequences from representatives of six European (taurine) breeds, three Indian (zebu) breeds, and four African (three zebu, one taurine) breeds. Similar levels of average sequence divergence were observed among animals within each of the major continental groups: 0.41% (European), 0.38% (African), and 0.42% (Indian). However, the sequences fell into two very distinct geographic lineages that do not correspond with the taurine-zebu dichotomy: all European and African breeds are in one lineage, and all Indian breeds are in the other. There was little indication of breed clustering within either lineage. Application of a molecular clock suggests that the two major mtDNA clades diverged at least 200,000, and possibly as much as 1 million, years ago. This relatively large divergence is interpreted most simply as evidence for two separate domestication events, presumably of different subspecies of the aurochs, Bos primigenius. The clustering of all African zebu mtDNA sequences within the taurine lineage is attributed to ancestral crossbreeding with the earlier B. taurus inhabitants of the continent.
Essential role for a heterogeneous nuclear ribonucleoprotein (hnRNP) in oogenesis: hrp40 is absent from the germ line in the dorsoventral mutant squid.
Authors: Matunis E L , Kelley R , Dreyfuss G
Published: May 2006

The Drosophila melanogaster hrp40 proteins are abundant nuclear pre-mRNA-binding proteins that are similar to the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins of vertebrates. Recently, hrp40 has been shown to be encoded by the squid gene, which is required for dorsoventral axis formation during oogenesis. Eggs and embryos from homozygous squid mothers are severely dorsalized, and complete deletion of the squid gene results in lethality. Here we have examined the expression and localization of hrp40 in wild-type and squid mutant ovaries. Using a monoclonal antibody specific for hrp40, the same isoforms of hrp40 are detected in both wild-type and squid ovaries, but the amount of hrp40 is reduced in squid ovaries. Furthermore, immunolocalization of hrp40 in wild-type egg chambers shows that hrp40 is present in the nurse cells, oocyte, and follicle cells. In contrast, in squid mutant egg chambers, hrp40 is absent from the germ-line-derived nurse cells and oocyte, but it is detected in the somatic follicle cells. The absence of hrp40 from the germ-line-derived cells of developing egg chambers is likely to lead to the striking dorsalized phenotype of squid eggs. In addition, dramatic stage-specific changes in the cellular localization of hrp40 are seen; the protein found in the nurse cell nuclei during early stages of oogenesis migrates to the cytoplasm at later stages. These findings reveal dynamic patterns of expression and localization of hnRNP proteins during development and provide evidence for an essential role for hnRNP proteins.
Modulation of cardiac Na+ channels expressed in a mammalian cell line and in ventricular myocytes by protein kinase C.
Authors: Qu Y , Rogers J , Tanada T , Scheuer T , Catterall W A
Published: May 2006

Cardiac rH1 Na+ channel alpha subunits were expressed in cells of the Chinese hamster lung 1610 cell line by transfection, and a stable cell line expressing cardiac Na+ channels (SNa-rH1) was isolated. Mean Na+ currents of 2.2 +/- 1.0 nA were recorded, which corresponds to a cell surface density of approximately 1-2 channels active at the peak of the Na+ current per micron2. The expressed cardiac Na+ current was tetrodotoxin resistant (Kd = 1.8 microM) and had voltage-dependent properties similar to those of the Na+ current in neonatal ventricular myocytes. Activation of protein kinase C by 1-oleoyl-2-acetyl-sn-glycerol (OAG) (10 microM) decreased this current approximately 33% at a holding potential of -114 mV and 56% at -94 mV. This reduction in peak current was caused in part by an 8- to 14-mV shift of steady-state inactivation in the hyperpolarized direction. Na+ channel activation was unchanged. Effects of OAG in SNa-rH1 cells and in neonatal rat cardiac myocytes were similar, except that the time course of inactivation was slowed either transiently or persistently when protein kinase C was activated in myocytes bathed in low-Ca2+ (1 microM) or Ca(2+)-free solution but was unaffected in SNa-rH1 cells. The effects of OAG on cardiac Na+ current were blocked in cells that had been previously microinjected with a peptide inhibitor of protein kinase C but not with a peptide inhibitor of cAMP-dependent protein kinase, indicating that protein kinase C is responsible for the effect of OAG. Single-channel recordings from SNa-rH1 cells showed that the probability of channel opening was reduced by OAG, but the conductance was unaffected. OAG did not induce the late Na+ channel openings observed with PKC modulation of neuronal and skeletal muscle Na+ channels. Thus, the substantial reduction in Na+ current at normal diastolic depolarizations with 10 microM OAG is due to failure of channel opening in response to depolarization. Such Na+ current reductions may have profound effects on cardiac cell excitability.
Reconstitution of a yeast protein kinase cascade in vitro: activation of the yeast MEK homologue STE7 by STE11.
Authors: Neiman A M , Herskowitz I
Published: May 2006

The mating-factor response pathway of Saccharomyces cerevisiae employs a set of protein kinase similar to kinases that function in signal transduction pathways of metazoans. We have purified the yeast protein kinases encoded by STE11, STE7, and FUS3 as fusions to glutathione S-transferase (GST) and reconstituted a kinase cascade in which STE11 phosphorylates and activates STE7, which in turn phosphorylates the mitogen-activated protein kinase FUS3. GST-STE11 is active even when purified from cells that have not been treated with alpha-factor. This observation raises the possibility that STE11 activity is governed by an inhibitor which is regulated by pheromone. We also identify a STE11-dependent phosphorylation site in STE7 which is required for activity of STE7. Conservation of this site in the mammalian STE7 homologue MEK and other STE7 relatives suggests that this may be a regulatory phosphorylation site in all MAP kinase kinases.
Properties of angiotensin II receptors in glial cells from the adult corpus callosum.
Authors: Matute C , Pulakat L , Río C , Valcárcel C , Miledi R
Published: May 2006

The existence and the properties of angiotensin II receptors in the adult bovine and human corpus callosum (CC) were investigated by using Xenopus oocytes and primary glial cell cultures. In oocytes injected with CC mRNA, angiotensin II elicited oscillatory Cl- currents due to activation of the inositol phosphate/Ca(2+)-receptor-channel coupling system. The receptors expressed in oocytes and in CC cultures were pharmacologically similar to the AT1 receptor type as assayed by binding. Northern blot analysis and in situ hybridization studies in sections from CC and in glial cultures revealed that the receptors were molecularly related to the AT1 receptor and that they were present in astrocytes. In these cells, activation of the receptors with angiotensin II increased de novo DNA synthesis, promoted the release of aldosterone, and induced c-Fos expression. These findings indicate that CC astrocytes possess functional AT1 receptors that participate in various physiological processes.
Forces of tertiary structural organization in globular proteins.
Authors: Yue K , Dill K A
Published: May 2006

The tertiary structures of globular proteins have remarkable and complex symmetries. What forces cause them? We find that a very simple model reproduces some of those symmetries. Proteins are modeled as copolymers of specific sequences of hydrophobic (H) and polar (P) monomers (HP model) configured as self-avoiding flights on simple three-dimensional cubic lattices. The model has no parameters; we just seek the conformations that have the global maximum number of HH contacts for any given sequence. Finding global optima for chains in this model has not been computationally possible before for chains longer than 36-mers. We report here a procedure that can find all the globally optimal conformations, the number of which defines the degeneracy of a sequence, for chains up to 88 monomers long. It is about 37 orders of magnitude faster than previous exact methods. We find that degeneracy is an important aspect of sequence design. So far, we have found that four-helix bundles, alpha/beta-barrels, and parallel beta-helices are globally optimal conformations of polar/nonpolar sequences that have minimal degeneracy.
The chemistry of social regulation: multicomponent signals in ant societies.
Authors: Hölldobler B
Published: May 2006

Chemical signals mediating communication in ant societies are usually complex mixtures of substances with considerable variation in molecular composition and in relative proportions of components. Such multicomponent signals can be produced in single exocrine glands, but they can also be composed with secretions from several glands. This variation is often functional, identifying groups or specific actions on a variety of organizational levels. Chemical signals can be further combined with cues from other sensory modalities, such as vibrational or tactile stimuli. These kinds of accessory signals usually serve in modulatory communication, lowering the response threshold in the recipient for the actual releasing stimulus. Comparative studies suggest that modulatory signals evolved through ritualization from actions originally not related to the same behavioral context, and modulatory signals may further evolve to become independent releasing signals.
Analysis of chemical signals by nervous systems.
Authors: Hildebrand J G
Published: May 2006

Intraspecific and interspecific communication and recognition depend on olfaction in widely diverse species of animals. Olfaction, an ancient sensory modality, is based on principles of neural organization and function that appear to be remarkably similar throughout the zoosphere. Thus, the "primitives" of olfactory stimuli that determine the input information of olfaction, the kinds of "molecular images" formed at various levels in the olfactory pathway, and the cellular mechanisms that underlie olfactory information processing are comparable in invertebrates and vertebrates alike. A case in point is the male-specific olfactory subsystem in moths, which is specialized to detect and analyze the qualitative, quantitative, and temporal features of the con-specific females' sex-pheromonal chemical signal. This olfactory subsystem can be viewed, and is here presented, as a model in which common principles of organization and function of olfactory systems in general are exaggerated to serve the requirements of a chemical communication system that is crucial for reproductive success.
Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII.
Authors: Youil R , Kemper B W , Cotton R G
Published: May 2006

Each of four possible sets of mismatches (G.A/C.T, C.C/G.G, A.A/T.T, and C.A/G.T) containing the 8 possible single-base-pair mismatches derived from isolated mutations were examined to test the ability of T4 endonuclease VII to consistently detect mismatches in heteroduplexes. At least two examples of each set of mismatches were studied for cleavage in the complementary pairs of heteroduplexes formed between normal and mutant DNA. Four deletion mutations were also included in this study. The various PCR-derived products used in the formation of heteroduplexes ranged from 133 to 1502 bp. At least one example of each set showed cleavage of at least one strand containing a mismatch. Cleavage of at least one strand of the pairs of heteroduplexes occurred in 17 of the 18 known single-base-pair mutations tested, with an A.A/T.T set not being cleaved in any mismatched strand. We propose that this method may be effective in detecting and positioning almost all mutational changes when DNA is screened for mutations.
Effects of mechanical signaling on plant cell cytosolic calcium.
Authors: Haley A , Russell A J , Wood N , Allan A C , Knight M , Campbell A K , Trewavas A J
Published: May 2006

Mechanical signals are important influences on the development and morphology of higher plants. Using tobacco transformed with the Ca(2+)-sensitive luminescent protein aequorin, we recently reported the effects of mechanical signals of touch and wind on the luminescence and thus intracellular calcium of young seedlings. When mesophyll protoplasts are isolated from these transgenic tobacco plants and mechanically stimulated by swirling them in solution, cytoplasmic Ca2+ increases immediately and transiently up to 10 microM, and these transients are unaffected by an excess of EGTA in the medium. The size of the transient effect is related to the strength of swirling. Epidermal strips isolated from transgenic tobacco leaves and containing only viable guard cells and trichomes also respond to the strength of swirling in solution and can increase their cytoplasmic Ca2+ transiently up to 10 microM. Finally, the moss Physcomitrella patens containing recombinant aequorin exhibits transient increases in cytoplasmic Ca2+ up to 5 microM when swirled in solution. This effect is strongly inhibited by ruthenium red. Our data indicate that the effect of mechanical stimulation can be found in a number of different cell types and in a lower plant as well as tobacco and suggest that mechanoperception and the resulting increase in cytoplasmic Ca2+ may be widespread.
The ethylene hormone response in Arabidopsis: a eukaryotic two-component signaling system.
Authors: Chang C , Meyerowitz E M
Published: May 2006

The simple gas ethylene affects numerous physiological processes in the growth and development of higher plants. With the use of molecular genetic approaches, we are beginning to learn how plants perceive ethylene and how this signal is transduced. Components of ethylene signal transduction are defined by ethylene response mutants in Arabidopsis thaliana. The genes corresponding to two of these mutants, etr1 and etr1, have been cloned. The ETR1 gene encodes a homolog of two-component regulators that are known almost exclusively in prokaryotes. The two-component regulators in prokaryotes are involved in the perception and transduction of a wide range of environmental signals leading to adaptive responses. The CTR1 gene encodes a homolog of the Raf family of serine/threonine protein kinases. Raf is part of a mitogen-activated protein kinase cascade known to regulate cell growth and development in mammals, worms, and flies. The ethylene response pathway may, therefore, exemplify a conserved protein kinase cascade regulated by a two-component system. The dominance of all known mutant alleles of ETR1 may be due to either constitutive activation of the ETR1 protein or dominant interference of wild-type activity. The discovery of Arabidopsis genes encoding proteins related to ETR1 suggests that the failure to recover recessive etr1 mutant alleles may be due to the presence of redundant genes.
Oxidative DNA damage and senescence of human diploid fibroblast cells.
Authors: Chen Q , Fischer A , Reagan J D , Yan L J , Ames B N
Published: May 2006

Human diploid fibroblast cells cease growth in culture after a finite number of population doublings. To address the cause of growth cessation in senescent IMR-90 human fibroblast cells, we determined the level of oxidative DNA damage by using 8-oxoguanine excised from DNA and 8-oxo-2'-deoxyguanosine in DNA as markers. Senescent cells excise from DNA four times more 8-oxoguanine per day than do early-passage young cells. The steady-state level of 8-oxo-2'-deoxyguanosine in DNA is approximately 35% higher in senescent cells than in young cells. Measurement of protein carbonyls shows that senescent cells did not appear to have elevated protein oxidation. To reduce the level of oxidative damage, we cultured cells under a more physiological O2 concentration (3%) and compared the replicative life span to the cells cultured at the O2 concentration of air (20%). We found that cells grown under 3% O2 achieved 50% more population doublings during their lifetime. Such an extension of life span resulted from the delayed onset of senescence and elevation of growth rate and saturation density of cells at all passages. The spin-trapping agent alpha-phenyl-t-butyl nitrone (PBN), which can act as an antioxidant, also effectively delayed senescence and rejuvenated near senescent cells. The effect is dose-dependent and is most pronounced for cells at the stage just before entry into senescence. Our data support the hypothesis that oxidative DNA damage contributes to replicative cessation in human diploid fibroblast cells.
Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes.
Authors: Jiang J , Gill B S , Wang G L , Ronald P C , Ward D C
Published: May 2006

Fluorescence in situ hybridization (FISH) is a powerful tool for physical mapping in human and other mammalian species. However, application of the FISH technique has been limited in plant species, especially for mapping single- or low-copy DNA sequences, due to inconsistent signal production in plant chromosome preparations. Here we demonstrate that bacterial artificial chromosome (BAC) clones can be mapped readily on rice (Oryza sativa L.) chromosomes by FISH. Repetitive DNA sequences in BAC clones can be suppressed efficiently by using rice genomic DNA as a competitor in the hybridization mixture. BAC clones as small as 40 kb were successfully mapped. To demonstrate the application of the FISH technique in physical mapping of plant genomes, both anonymous BAC clones and clones closely linked to a rice bacterial blight-resistance locus, Xa21, were chosen for analysis. The physical location of Xa21 and the relationships among the linked clones were established, thus demonstrating the utility of FISH in plant genome analysis.
Metaxin, a gene contiguous to both thrombospondin 3 and glucocerebrosidase, is required for embryonic development in the mouse: implications for Gaucher disease.
Authors: Bornstein P , McKinney C E , LaMarca M E , Winfield S , Shingu T , Devarayalu S , Vos H L , Ginns E I
Published: May 2006

We have identified a murine gene, metaxin, that spans the 6-kb interval separating the glucocerebrosidase gene (GC) from the thrombospondin 3 gene on chromosome 3E3-F1. Metaxin and GC are transcribed convergently; their major polyadenylylation sites are only 431 bp apart. On the other hand, metaxin and the thrombospondin 3 gene are transcribed divergently and share a common promoter sequence. The cDNA for metaxin encodes a 317-aa protein, without either a signal sequence or consensus for N-linked glycosylation. Metaxin protein is expressed ubiquitously in tissues of the young adult mouse, but no close homologues have been found in the DNA or protein data bases. A targeted mutation (A-->G in exon 9) was introduced into GC by homologous recombination in embryonic stem cells to establish a mouse model for a mild form of Gaucher disease. A phosphoglycerate kinase-neomycin gene cassette was also inserted into the 3'-flanking region of GC as a selectable marker, at a site later identified as the terminal exon of metaxin. Mice homozygous for the combined mutations die early in gestation. Since the same amino acid mutation in humans is associated with mild type 1 Gaucher disease, we suggest that metaxin protein is likely to be essential for embryonic development in mice. Clearly, the contiguous gene organization at this locus limits targeting strategies for the production of murine models of Gaucher disease.
Interactions between quantitative trait loci in soybean in which trait variation at one locus is conditional upon a specific allele at another.
Authors: Lark K G , Chase K , Adler F , Mansur L M , Orf J H
Published: May 2006

A large recombinant inbred population of soybean has been characterized for 220 restriction fragment-length polymorphism (RFLP) markers. Values for agronomic traits also have been measured. Quantitative trait loci (QTL) for height, yield, and maturity were located by their linkage to RFLP markers. QTL controlling large amounts of trait variation were analyzed for the dependence of trait variation on particular alleles at a second locus by comparing cumulative distributions of the trait for each genotype (four genotypes per pair of loci). Interesting pairs of loci were analyzed statistically with maximum likelihood and Monte Carlo comparison of additive and epistatic models. For each locus affecting height, variation was conditional upon the presence of a particular allele at a second unlinked locus that itself explained little or no trait variation. The results show that interactions between QTL are frequent and control large effects. Interactions distinguished between different QTL in a single linkage group and between QTL that affect different traits closely linked to one RFLP marker--i.e., distinguished between pleiotropy and closely linked genes. The implications for the evolution of inbreeding plants and for the construction of agronomic breeding strategies are discussed.
A secondary-structure model for the self-cleaving region of Neurospora VS RNA.
Authors: Beattie T L , Olive J E , Collins R A
Published: May 2006

Neurospora VS RNA performs an RNA-mediated self-cleavage reaction whose products contain 2',3'-cyclic phosphate and 5'-hydroxyl termini. This reaction is similar to those of hammerhead, hairpin, and hepatitis delta virus ribozymes; however, VS RNA is not similar in sequence to these other self-cleaving motifs. Here we propose a model for the secondary structure of the self-cleaving region of VS RNA, supported by site-directed mutagenesis and chemical modification structure probing data. The secondary structure of VS RNA is distinct from those of the other naturally occurring RNA self-cleaving domains. In addition to a unique secondary structure, several Mg-dependent interactions occur during the folding of VS RNA into its active tertiary conformation.
Bclx regulates the survival of double-positive thymocytes.
Authors: Ma A , Pena J C , Chang B , Margosian E , Davidson L , Alt F W , Thompson C B
Published: May 2006

The bclx gene has been shown to regulate programmed cell death in vitro. We now show that Bclx expression increases dramatically when T cells differentiate from CD4- CD8- (double negative) thymocytes to CD4+ CD8+ [double positive (DP)] thymocytes. In contrast single-positive (SP) thymocytes express negligible amounts of Bclx protein. This expression pattern contrasts with that of Bcl2, which is present in double-negative thymocytes, down-regulated in DP thymocytes, and reinduced upon maturation to SP thymocytes. Elimination of Bclx by gene targeting dramatically shortens the survival of DP thymocytes but not the survival of SP thymocytes or peripheral SP T cells. These data suggest that the induction of Bclx during thymic maturation plays a critical role in regulating the length of time DP thymocytes survive in the absence of selection.
Coenzyme Q reductase from liver plasma membrane: purification and role in trans-plasma-membrane electron transport.
Authors: Villalba J M , Navarro F , Córdoba F , Serrano A , Arroyo A , Crane F L , Navas P
Published: May 2006

A specific requirement for coenzyme Q in the maintenance of trans-plasma-membrane redox activity is demonstrated. Extraction of coenzyme Q from membranes resulted in inhibition of NADH-ascorbate free radical reductase (trans electron transport), and addition of coenzyme Q10 restored the activity. NADH-cytochrome c oxidoreductase (cis electron transport) did not respond to the coenzyme Q status. Quinone analogs inhibited trans-plasma-membrane redox activity, and the inhibition was reversed by coenzyme Q. A 34-kDa coenzyme Q reductase (p34) has been purified from pig-liver plasma membranes. The isolated enzyme was sensitive to quinone-site inhibitors. p34 catalyzed the NADH-dependent reduction of coenzyme Q10 after reconstitution in phospholipid liposomes. When plasma membranes were supplemented with extra p34, NADH-ascorbate free radical reductase was activated but NADH-cytochrome c oxidoreductase was not. These results support the involvement of p34 as a source of electrons for the trans-plasma-membrane redox system oxidizing NADH and support coenzyme Q as an intermediate electron carrier between NADH and the external acceptor ascorbate free radical.
Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces.
Authors: Cai W , Aburatani H , Stanton V P , Housman D E , Wang Y K , Schwartz D C
Published: May 2006

We have developed a surface mounting technology for the rapid construction of ordered restriction maps from individual DNA molecules. Optical restriction maps constructed from yeast artificial chromosome DNA molecules mounted on specially derivatized glass surfaces are accurate and reproducible, and the technology is amenable to automation. The mounting procedures described here should also be useful for fluorescence in situ hybridization studies. We believe these improvements to optical mapping will further stimulate the development of nonelectrophoretic approaches to genome analysis.
Position-dependent variegation of globin transgene expression in mice.
Authors: Robertson G , Garrick D , Wu W , Kearns M , Martin D , Whitelaw E
Published: May 2006

Expression of genes in eukaryotes has commonly been analyzed in a whole tissue, and levels of expression have been interpreted as the result of equivalent rates of transcription in every cell. We have produced transgenic mouse lines that express beta-galactosidase under the control of globin promoters linked to the major tissue-specific regulatory element of the alpha-globin locus, which permits the analysis of transgene expression in individual red blood cells. We find that expression of the transgene within all mouse lines is heterocellular. Individual cells either do not express the transgene at all or express it at a level characteristic of that line. The number of beta-galactosidase-expressing cells varies greatly between different lines of transgenic mice at any defined stage of development, but within a transgenic line, individual mice have strikingly similar numbers of expressing cells. This suggests that the degree of heterocellular expression is determined by the site of integration, as is seen in position-effect variegation.
Interferon gamma signals via a high-affinity multisubunit receptor complex that contains two types of polypeptide chain.
Authors: Marsters S A , Pennica D , Bach E , Schreiber R D , Ashkenazi A
Published: May 2006

Signaling by interferon gamma (IFN-gamma) requires two structurally related cell surface proteins: a ligand-binding polypeptide, known as the IFN-gamma receptor (IFN-gamma R), and an accessory factor. However, it is not known whether IFN-gamma forms a ternary complex with the IFN-gamma R and accessory factor to initiate signaling. Here we demonstrate complex formation between IFN-gamma and the two proteins, both in solution and at the cell surface. We observe complexes containing ligand, two molecules of IFN-gamma R (designated the IFN-gamma R alpha chain), and one or two molecules of accessory factor (designated the IFN-gamma R beta chain). Transfected cells expressing both IFN-gamma R chains bind IFN-gamma with higher affinity than do cells expressing alpha chain alone. Anti-beta-chain antibodies prevent the beta chain from participating in the ligand-receptor complex, reduce the affinity for IFN-gamma, and block signaling. Soluble alpha- or beta-chain extracellular domains also inhibit function. These results demonstrate that IFN-gamma signals via a high-affinity multisubunit complex that contains two types of receptor chain and suggest a potential approach to inhibiting specific actions of IFN-gamma by blocking the association of receptor subunits.
Library of libraries: approach to synthetic combinatorial library design and screening of "pharmacophore" motifs.
Authors: Sepetov N F , Krchnák V , Stanková M , Wade S , Lam K S , Lebl M
Published: May 2006

Construction of synthetic combinatorial libraries is described that allows for the generation of a library of motifs rather than a library of compounds. Peptide libraries based on this strategy were synthesized and screened with model targets streptavidin and anti-beta-endorphin antibody. The screens resulted in observation of expected motifs providing evidence of the effectiveness of the suggested approach.
Deficiency of retinoblastoma protein leads to inappropriate S-phase entry, activation of E2F-responsive genes, and apoptosis.
Authors: Almasan A , Yin Y , Kelly R E , Lee E Y , Bradley A , Li W , Bertino J R , Wahl G M
Published: May 2006

The retinoblastoma susceptibility gene (Rb) participates in controlling the G1/S-phase transition, presumably by binding and inactivating E2F transcription activator family members. Mouse embryonic fibroblasts (MEFs) with no, one, or two inactivated Rb genes were used to determine the specific contributions of Rb protein to cell cycle progression and gene expression. MEFs lacking both Rb alleles (Rb-/-) entered S phase in the presence of the dihydrofolate reductase inhibitor methotrexate. Two E2F target genes, dihydrofolate reductase and thymidylate synthase, displayed elevated mRNA and protein levels in Rb- MEFs. Since absence of functional Rb protein in MEFs is sufficient for S-phase entry under growth-limiting conditions, these data indicate that the E2F complexes containing Rb protein, and not the Rb-related proteins p107 and p130, may be rate limiting for the G1/S transition. Antineoplastic drugs caused accumulation of p53 in the nuclei of both Rb+/+ and Rb-/- MEFs. While p53 induction led to apoptosis in Rb-/- MEFs, Rb+/- and Rb+/+ MEFs underwent cell cycle arrest without apoptosis. These results reveal that diverse growth signals work through Rb to regulate entry into S phase, and they indicate that absence of Rb protein produces a constitutive DNA replication signal capable of activating a p53-associated apoptotic response.
Developmentally related responses of maize catalase genes to salicylic acid.
Authors: Guan L , Scandalios J G
Published: May 2006

The response of the maize catalase genes (Cat1, Cat2, and Cat3) to salicylic acid (SA) was examined at two distinct developmental stages: embryogenesis and germination. A unique, germination-related differential response of each maize catalase gene to various doses of SA was observed. During late embryogenesis, total catalase activity in scutella increased dramatically with 1 mM SA treatment. The accumulation of Cat2 transcript and CAT-2 isozyme protein provided the major contribution to the observed increase in total catalase activity. This increase was paralleled by the enhanced growth of germinated embryos at that stage. In a CAT-2 null mutant line, a full compensation of total catalase activity by the CAT-1 isozyme was observed in the presence of SA. This suggests that catalase is important for maintenance of normal cellular processes under stress conditions. SA at 1 mM, which enhances growth of precociously germinated embryos, appeared to inhibit seed germination at 1 day after inhibition. Furthermore, Cat2 transcript accumulation was inhibited at this stage. SA is probably not a direct signal for the induction of the catalase genes. Other signals, possibly germination-related regulator(s), might be responsible for the induction of the catalase genes. The effect of SA on the activity of purified catalase protein was also examined.
The inositol 1,4,5-trisphosphate receptor is essential for T-cell receptor signaling.
Authors: Jayaraman T , Ondriasová E , Ondrias K , Harnick D J , Marks A R
Published: May 2006

Antigen-specific activation of T lymphocytes, via stimulation of the T-cell antigen receptor (TCR) complex, is marked by a rapid and sustained increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). It has been suggested that the second messenger inositol 1,4,5-trisphosphate (IP3) produced after TCR stimulation binds to the IP3 receptor (IP3R), an intracellular Ca(2+)-release channel, and triggers the increase in [Ca2+]i that activates transcription of the gene for T-cell growth factor interleukin 2 (IL-2). However, the role of the IP3R in T-cell signaling and possibly in plasma membrane Ca2+ influx in T cells remains unproven. Stable transfection of T cells (Jurkat) with antisense type 1 IP3R cDNA prevented type 1 IP3R expression, providing a tool for dissecting the role of IP3 signaling during T-cell activation. T cells lacking type 1 IP3R failed to increase [Ca2+]i or produce IL-2 after TCR stimulation. Moreover, depletion of intracellular Ca2+ stores without TCR activation stimulated Ca2+ influx in cells lacking the type 1 IP3R. These results establish that the type 1 IP3R is required for intracellular Ca2+ release that triggers antigen-specific T-cell proliferation but not for plasma membrane Ca2+ influx.
PXA1, a possible Saccharomyces cerevisiae ortholog of the human adrenoleukodystrophy gene.
Authors: Shani N , Watkins P A , Valle D
Published: May 2006

The adrenoleukodystrophy protein (ALDp) is an ATP-binding cassette (ABC) transporter in the human peroxisome membrane. It is defective in X chromosome-linked adrenoleukodystrophy (ALD), a neurodegenerative disorder with impaired peroxisomal oxidation of very long chain fatty acids. We report cloning and characterization of PXA1, a yeast gene encoding a protein (Pxa1p) exhibiting high similarity to ALDp. Disruption of PXA1 results in impaired growth on oleic acid and reduced ability to oxidize oleate. Pxa1p is peroxisome associated; however, in the PXA1 mutant yeast, as in ALD cells, peroxisomes are morphologically intact. Disruption of a second yeast gene, YKL741, which encodes a more distantly related ALDp homolog (Yk174p), in either wild-type or PXA1 mutant yeast, results in a growth phenotype identical to that of the PXA1 mutant. This result suggests that Yk1741p and Pxa1p may be subunits of the same transporter. Sequence analysis of Pxa1p, ALDp, and related ABC transporters reveals a possible fatty acid binding domain and a 14-amino acid EAA-like motif, previously described only in prokaryotes. Because of the similarities in sequence and function, we propose that Pxa1p is the Saccharomyces cerevisiae ortholog of ALDp.
Decreased human immunodeficiency virus type 1 plasma viremia during antiretroviral therapy reflects downregulation of viral replication in lymphoid tissue.
Authors: Cohen O J , Pantaleo G , Holodniy M , Schnittman S , Niu M , Graziosi C , Pavlakis G N , Lalezari J , Bartlett J A , Steigbigel R T
Published: May 2006

Although several immunologic and virologic markers measured in peripheral blood are useful for predicting accelerated progression of human immunodeficiency virus (HIV) disease, their validity for evaluating the response to antiretroviral therapy and their ability to accurately reflect changes in lymphoid organs remain unclear. In the present study, changes in certain virologic markers have been analyzed in peripheral blood and lymphoid tissue during antiretroviral therapy. Sixteen HIV-infected individuals who were receiving antiretroviral therapy with zidovudine for > or = 6 months were randomly assigned either to continue on zidovudine alone or to add didanosine for 8 weeks. Lymph node biopsies were performed at baseline and after 8 weeks. Viral burden (i.e., HIV DNA copies per 10(6) mononuclear cells) and virus replication in mononuclear cells isolated from peripheral blood and lymph node and plasma viremia were determined by semiquantitative polymerase chain reaction assays. Virologic and immunologic markers remained unchanged in peripheral blood and lymph node of patients who continued on zidovudine alone. In contrast, a decrease in virus replication in lymph nodes was observed in four of six patients who added didanosine to their regimen, and this was associated with a decrease in plasma viremia. These results indicate that decreases in plasma viremia detected during antiretroviral therapy reflect downregulation of virus replication in lymphoid tissue.
Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.
Authors: Hori R , Pyo S , Carey M
Published: May 2006

Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface of TFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.
Use of an oocyte expression assay to reconstitute inductive signaling.
Authors: Lustig K D , Kirschner M W
Published: May 2006

We have developed a paracrine signaling assay capable of mimicking inductive events in the early vertebrate embryo. RNA encoding one or more secreted proteins is microinjected into a Xenopus laevis oocyte. After a brief incubation to allow translation, a piece of embryonic tissue competent to respond to the signaling protein is grafted onto the oocyte. The secreted protein's effect on the grafted explant is then scored by assaying expression of tissue-specific markers. Explants of ectodermal tissue from blastula or gastrula stage embryos were grafted onto oocytes that had been injected with RNA encoding activin or noggin. We found that the paracrine assay faithfully reconstitutes mesoderm induction by activin and neural induction by noggin. Blastula-stage explants grafted onto activin-expressing oocytes expressed the mesodermal marker genes brachyury, goosecoid, and muscle actin. Gastrula-stage explants grafted onto noggin-expressing oocytes expressed neural cell adhesion molecule (NCAM) and formed cement gland. By injecting pools of RNA synthesized from a cDNA expression library into the oocyte, we also used the assay to screen for secreted neural-inducing proteins. We assayed 20,000 independent transformants of a library constructed from LiCl-dorsalized Xenopus laevis embryos, and we identified two cDNAs that induced neural tissue in ectodermal explants from gastrula-stage embryos. Both cDNAs encode noggin. These results suggest that the paracrine assay will be useful for the cloning of novel signaling proteins as well as for the analysis of known factors.
Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21.
Authors: Olopade O I , Pomykala H M , Hagos F , Sveen L W , Espinosa R , Dreyling M H , Gursky S , Stadler W M , Le Beau M M , Bohlander S K
Published: May 2006

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.
Increase of skeletal muscle relaxation speed by direct injection of parvalbumin cDNA.
Authors: Müntener M , Käser L , Weber J , Berchtold M W
Published: May 2006

Parvalbumin (PV) is a high affinity Ca(2+)-binding protein found at high concentration in fast-contracting/relaxing skeletal muscle fibers of vertebrates. It has been proposed that PV acts in the process of muscle relaxation by facilitating Ca2+ transport from the myofibrils to the sarcoplasmic reticulum. However, on the basis of metal-binding kinetics of PV in vitro, this hypothesis has been challenged. To investigate the function of PV in skeletal muscle fibers, direct gene transfer was applied in normal and regenerating rat soleus muscles which do not synthesize detectable amounts of PV. Two weeks after in vivo transfection with PV cDNA, considerable levels of PV mRNA and protein were detected in normal muscle, and even higher amounts were detected in regenerating muscle. Twitch half-relaxation time was significantly shortened in a dose-dependent way in transfected muscles, while contraction time remained unaltered. The observed shortening of half-relaxation time is due to PV and its ability to bind Ca2+, because a mutant protein lacking Ca(2+)-binding capacity did not promote any change in physiology. These results directly demonstrate the physiological function of PV as a relaxing factor in mammalian skeletal muscle.
Identification of human brain regions underlying responses to resistive inspiratory loading with functional magnetic resonance imaging.
Authors: Gozal D , Omidvar O , Kirlew K A , Hathout G M , Hamilton R , Lufkin R B , Harper R M
Published: May 2006

Compensatory ventilatory responses to increased inspiratory loading are essential for adequate breathing regulation in a number of pulmonary diseases; however, the human brain sites mediating such responses are unknown. Midsagittal and axial images were acquired in 11 healthy volunteers during unloaded and loaded (30 cmH2O; 1 cmH2O = 98 Pa) inspiratory breathing, by using functional magnetic resonance imaging (fMRI) strategies (1.5-tesla MR; repetition time, 72 msec; echo time, 45 msec; flip angle, 30 degrees; field of view, 26 cm; slice thickness, 5 mm; number of excitations, 1; matrix, 128 x 256). Digital image subtractions and region of interest analyses revealed significantly increased fMRI signal intensity in discrete areas of the ventral and dorsal pons, interpeduncular nucleus, basal forebrain, putamen, and cerebellar regions. Upon load withdrawal, certain regions displayed a rapid fMRI signal off-transient, while in others, a slower fMRI signal decay emerged. Sustained loading elicited slow decreases in fMRI signal across activated regions, while second application of an identical load resulted in smaller signal increases compared to initial signal responses (P < 0.001). A moderate inspiratory load is associated with consistent regional activation of discrete brain locations; certain of these regions have been implicated in mediation of loaded breathing in animal models. We speculate that temporal changes in fMRI signal may indicate respiratory after-discharge and/or habituation phenomena.
New physics of metals: fermi surfaces without Fermi liquids.
Authors: Anderson P W
Published: May 2006

I relate the historic successes, and present difficulties, of the renormalized quasiparticle theory of metals ("AGD" or Fermi liquid theory). I then describe the best-understood example of a non-Fermi liquid, the normal metallic state of the cuprate superconductors.