bluuebunny/crossref_metadata_2025 huggingface dataset:
Behavior of hematopoietic stem cells in a large animal.
Authors: Abkowitz J L , Persik M T , Shelton G H , Ott R L , Kiklevich J V , Catlin S N , Guttorp P
Published: May 2006

To study the behavior of hematopoietic stem cells in vivo, we transplanted glucose-6-phosphate dehydrogenase (G6PD) heterozygous (female Safari) cats with small amounts of autologous marrow. The G6PD phenotypes of erythroid burst-forming units and granulocyte/macrophage colony-forming units were repeatedly assayed for 3.5-6 years after transplantation to track contributions of stem cell clones to the progenitor cell compartment. Two phases of stem cell kinetics were observed, which were similar to the pattern reported in comparable murine studies. Initially there were significant fluctuations in contributions of stem cell clones. Later clonal contributions to hematopoiesis stabilized. The initial phase of clonal disequilibrium, however, extended for 1-4.5 years (and not 2-6 months as seen in murine experiments). After this subsided, all progenitor cells from some animals expressed a single parental G6PD phenotype, suggesting that blood cell production could be stably maintained by the progeny of one (or a few) cells. As the hematopoietic demand of a cat (i.e., number of blood cells produced per lifetime) is over 600 times that of a mouse, this provides evidence that an individual hematopoietic stem cell has a vast self-renewal and/or proliferative capacity. The long phase of clonal instability may reflect the time required for stem cells to replicate sufficiently to reconstitute a large stem cell reserve.
AUH, a gene encoding an AU-specific RNA binding protein with intrinsic enoyl-CoA hydratase activity.
Authors: Nakagawa J , Waldner H , Meyer-Monard S , Hofsteenge J , Jenö P , Moroni C
Published: May 2006

AU-rich elements within the 3' untranslated region of transcripts of lymphokines and some protooncogenes serve as signal for rapid mRNA degradation. By using an AUUUA matrix, we have affinity-purified a 32-kDa protein, microsequenced it, and cloned the corresponding cDNA. In vitro, the recombinant protein bound specifically to AU-rich transcripts, including those for interleukin 3, granulocyte/macrophage colony-stimulating factor, c-fos, and c-myc. Sequence analysis revealed an unexpected homology to enoyl-CoA hydratase (EC 4.2.1.17), and the recombinant protein showed a low degree of the enzymatic activity. Thus, this gene, designated AUH, encodes an RNA binding protein with intrinsic enzymatic activity. Protein immobilized on an AUUUA matrix was enzymatically active, suggesting that hydratase and AU-binding functions are located on distinct domains within a single polypeptide.
Loligomers: design of de novo peptide-based intracellular vehicles.
Authors: Sheldon K , Liu D , Ferguson J , Gariépy J
Published: May 2006

Defined branched peptides (loligomers) incorporating cytoplasmic translocation signals, nuclear localization sequences, and fluorescent probes were designed and synthesized to demonstrate the feasibility and simplicity of creating novel classes of intracellular vehicles. Loligomers containing all the above signals were rapidly internalized by Chinese hamster ovary (CHO) cells and accumulated in their nucleus. At 4 degrees C, the interaction of peptide constructs with CHO cells was limited to membrane association. Loligomers entered cells at higher temperatures by adsorptive endocytosis. Inhibitors of ATP synthesis affected cytoplasmic import only weakly but abolished nuclear uptake. The peptide signals guided both cytoplasmic and nuclear localization events. The properties exhibited by loligomers suggest a strategy for the facile design of "guided" classes of intracellular agents.
Epigenetic changes encompassing the IGF2/H19 locus associated with relaxation of IGF2 imprinting and silencing of H19 in Wilms tumor.
Authors: Taniguchi T , Sullivan M J , Ogawa O , Reeve A E
Published: May 2006

In most tissues IGF2 is expressed from the paternal allele while H19 is expressed from the maternal allele. We have previously shown that in some Wilms tumors the maternal IGF2 imprint is relaxed such that the gene is expressed biallelically. We have now investigated this subset of tumors further and found that biallelic expression of IGF2 was associated with undetectable or very low levels of H19 expression. The relaxation of IGF2 imprinting in Wilms tumors also involved a concomitant reversal in the patterns of DNA methylation of the maternally inherited IGF2 and H19 alleles. Furthermore, the only specific methylation changes that occurred in tumors with relaxation of IGF2 imprinting were solely restricted to the maternal IGF2 and H19 alleles. These data suggest that there has been an acquisition of a paternal epigenotype in these tumors as the result of a pathologic disruption in the normal imprinting of the IGF2 and H19 genes.
Synaptotagmin IV is an immediate early gene induced by depolarization in PC12 cells and in brain.
Authors: Vician L , Lim I K , Ferguson G , Tocco G , Baudry M , Herschman H R
Published: May 2006

Subtractive library construction and differential screening were used to identify a cDNA for a cell type-specific immediate early gene induced in rat PC12 pheochromocytoma cells. Sequencing identified the protein product of this gene as rat synaptotagmin IV (SytIV). Synaptotagmins are synaptic vesicle proteins thought to play a role in depolarization-induced, calcium-mediated exocytosis and neurotransmitter release. SytIV mRNA accumulation is transiently induced in PC12 cells by potassium depolarization, calcium ionophore, ATP, and forskolin. In contrast, growth factors and phorbol 12-myristate 13-acetate induce little or no SytIV mRNA accumulation. Kainic acid-induced seizures in rats are followed by accumulation of SytIV message in the hippocampus and piriform cortex. The SytIV gene may provide a direct link between depolarization-induced neuronal gene expression and subsequent modulation of synaptic structure and function.
Ultrasound backscatter microscope analysis of early mouse embryonic brain development.
Authors: Turnbull D H , Bloomfield T S , Baldwin H S , Foster F S , Joyner A L
Published: May 2006

The history of developmental and genetic analysis in the mouse has made it the model of choice for studying mammalian embryogenesis. Presently lacking is a simple technique for efficiently analyzing early mouse mutant phenotypes in utero. We demonstrate application of a real-time imaging method called ultrasound backscatter microscopy for visualizing mouse early embryonic neural tubes and hearts. This method was used to study live embryos in utero between 9.5 and 11.5 days of embryogenesis, with a spatial resolution close to 50 microns. Ultrasound backscatter microscope images of cultured embryos made it possible to visualize the heart chambers. This noninvasive imaging method was also used for analyzing a neural tube defect. The midhindbrain deletion associated with a null mutation of the Wnt-1 protooncogene was easily recognizable on ultrasound backscatter microscope images of 10.5- and 11.5-day embryos. Computer-generated volumetric renderings of the neural tube cavities were made from three-dimensional image data. This allowed a much clearer definition of the Wnt-1 mutant phenotype. These imaging techniques should be of considerable use in studying mouse development in utero.
Specific T-cell tolerance may reflect selective activation of lymphokine synthesis.
Authors: Vidard L , Colarusso L J , Benacerraf B
Published: May 2006

Selective T-cell unresponsiveness, as measured by interleukin 2 (IL-2) synthesis upon challenge with antigen, was induced in SJL mice by ovalbumin (OVA) in incomplete or complete Freund's adjuvant administered i.p. or s.c. Ten days later, the mice were given booster injections of 100 micrograms of OVA/complete Freund's adjuvant. On day 20, lymph node and spleen cells were challenged in vitro with serial dilutions of OVA. There was an antigen-specific dose-dependent down regulation of IL-2 production and T-cell proliferation in lymph node T cells. Concomitantly, 100 micrograms of OVA up regulated IL-4 and, to a lesser extent, interferon gamma (IFN-gamma) production, particularly by spleen T cells. Altogether, these data indicate that the drop of IL-2 production and T-cell proliferation, as well as the up regulation of IL-4 and IFN-gamma production, are complex manifestations of an evolving T-cell response. The maturation of the T-cell response leads to the production of different patterns of lymphokines, which may be significantly affected, as desired, by dosage, timing, and route of immunization, as well as by the choice of adjuvants.
CD8+ T cells suppress human immunodeficiency virus replication by inhibiting viral transcription.
Authors: Mackewicz C E , Blackbourn D J , Levy J A
Published: May 2006

CD8+ cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in cultured CD4+ cells by a noncytolytic mechanism that involves a secreted CD8(+)-cell antiviral factor (CAF). The results of this study suggest that CD8+ cells, as well as CAF, arrest HIV replication at the level of viral transcription. Culturing naturally infected CD4+ cells actively producing HIV with autologous CD8+ cells or a 50% dilution of culture fluids from these cells results in a > 80% reduction in the number of cells expressing HIV antigens and RNA. This effect was observed within 2 days after exposure to CD8+ cells but required 6 days in the presence of CAF-containing culture fluids to reach the same extent of HIV suppression. Northern blot analysis of CD4+ cell extracts revealed that all viral RNA species (unspliced and single and double spliced) were reduced in quantity to a similar extent. CAF-containing culture fluids also had a direct inhibitory effect on HIV long terminal repeat (LTR)-driven transcription in HIV-infected 1G5 cells carrying an LTR-luciferase construct. Suppression of basal levels of LTR-driven transcription was not detected. Thus, the results suggest that the noncytolytic CD8+ cell antiviral activity observed in HIV infection exerts its effects, at least in part, by specifically interrupting HIV transcription. These findings could help in developing therapies for HIV infection.
Covalent cross-linking of the Phytophthora megasperma oligopeptide elicitor to its receptor in parsley membranes.
Authors: Nürnberger T , Nennstiel D , Hahlbrock K , Scheel D
Published: May 2006

An oligopeptide elicitor from Phytophthora megasperma f.sp. glycinea (Pep-13) that induces phytoalexin accumulation in cultured parsley cells was radioiodinated and chemically cross-linked to its binding site in microsomal and plasma membrane preparations with each of three homobifunctional reagents. Analysis by SDS/PAGE and autoradiography of solubilized membrane proteins demonstrated labeling of a 91-kDa protein, regardless of which reagent was used. Cross-linking of this protein was prevented by addition of excess unlabeled Pep-13. The competitor concentration found to half-maximally reduce the intensity of the cross-linked band was 6 nM, which is in good agreement with the IC50 value of 4.7 nM, obtained from ligand binding assays. No crosslinking of 125I-labeled Pep-13 was observed by using microsomal membranes from three other plant species, indicating species-specific occurrence of the binding site. Coupling of 125I-Pep-13 to the parsley 91-kDa protein required the same structural elements within the ligand as was recently reported for binding of 125I-Pep-13 to parsley microsomes, elicitor-induced stimulation of ion fluxes across the plasma membrane, the oxidative burst, the expression of defense-related genes, and phytoalexin production. These findings suggest that the 91-kDa protein identified in parsley membranes is the oligopeptide elicitor receptor mediating activation of a multicomponent defense response.
Limitations of Darwinian selection in a finite population.
Authors: Kimura M
Published: May 2006

In a finite population, the rate at which favorable mutations at several loci can evolve simultaneously is limited by the reproductive capacity of the species and the effective population number. The number of such loci is given by n < - (Ne/2) ln(1-LT), in which Ne is the effective population number and LT is the "substitution load" (roughly, the reproductive excess available for positive selection). If LT = 0.1, as seems reasonable for large mammals, n < 0.05Ne.
Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications.
Authors: Brown J R , Doolittle W F
Published: May 2006

Universal trees based on sequences of single gene homologs cannot be rooted. Iwabe et al. [Iwabe, N., Kuma, K.-I., Hasegawa, M., Osawa, S. & Miyata, T. (1989) Proc. Natl. Acad. Sci. USA 86, 9355-9359] circumvented this problem by using ancient gene duplications that predated the last common ancestor of all living things. Their separate, reciprocally rooted gene trees for elongation factors and ATPase subunits showed Bacteria (eubacteria) as branching first from the universal tree with Archaea (archaebacteria) and Eucarya (eukaryotes) as sister groups. Given its topical importance to evolutionary biology and concerns about the appropriateness of the ATPase data set, an evaluation of the universal tree root using other ancient gene duplications is essential. In this study, we derive a rooting for the universal tree using aminoacyl-tRNA synthetase genes, an extensive multigene family whose divergence likely preceded that of prokaryotes and eukaryotes. An approximately 1600-bp conserved region was sequenced from the isoleucyl-tRNA synthetases of several species representing deep evolutionary branches of eukaryotes (Nosema locustae), Bacteria (Aquifex pyrophilus and Thermotoga maritima) and Archaea (Pyrococcus furiosus and Sulfolobus acidocaldarius). In addition, a new valyl-tRNA synthetase was characterized from the protist Trichomonas vaginalis. Different phylogenetic methods were used to generate trees of isoleucyl-tRNA synthetases rooted by valyl- and leucyl-tRNA synthetases. All isoleucyl-tRNA synthetase trees showed Archaea and Eucarya as sister groups, providing strong confirmation for the universal tree rooting reported by Iwabe et al. As well, there was strong support for the monophyly (sensu Hennig) of Archaea. The valyl-tRNA synthetase gene from Tr. vaginalis clustered with other eukaryotic ValRS genes, which may have been transferred from the mitochondrial genome to the nuclear genome, suggesting that this amitochondrial trichomonad once harbored an endosymbiotic bacterium.
Dopamine- and cAMP-regulated phosphoprotein DARPP-32: phosphorylation of Ser-137 by casein kinase I inhibits dephosphorylation of Thr-34 by calcineurin.
Authors: Desdouits F , Siciliano J C , Greengard P , Girault J A
Published: May 2006

Although protein phosphatases appear to be highly controlled in intact cells, relatively little is known about the physiological regulation of their activity. DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of apparent M(r) 32,000, is phosphorylated in vitro by casein kinase I, casein kinase II, and cAMP-dependent protein kinase on sites phosphorylated in vivo. DARPP-32 phosphorylated on Thr-34 by cAMP-dependent protein kinase is a potent inhibitor of protein phosphatase 1 and an excellent substrate for calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. Here we provide evidence, using both purified proteins and brain slices, that phosphorylation of DARPP-32 on Ser-137 by casein kinase I inhibits the dephosphorylation of Thr-34 by calcineurin. This inhibition occurs only when phospho-Ser-137 and phospho-Thr-34 are located on the same DARPP-32 molecule and is not dependent on the mode of activation of calcineurin. The results demonstrate that the inhibition is due to a modification in the properties of the substrate which alters its dephosphorylation rate. Thus, casein kinase I may play a physiological role in striatonigral neurons as a modulator of the regulation of protein phosphatase 1 via DARPP-32.
Stimulation of T cells by antigen-presenting cells is kinetically controlled by antigenic peptide binding to major histocompatibility complex class II molecules.
Authors: McConnell H M , Wada H G , Arimilli S , Fok K S , Nag B
Published: May 2006

Activation of CD4+ T cells by antigenic peptide involves the interaction of major histocompatibility complex (MHC) class II-peptide complexes on the surface of antigen-presenting cells (APCs) with T-cell receptors. This report describes the kinetics of T-cell triggering by exogenous antigenic peptides in the presence of APCs. A rapid specific increase in extracellular acidification rate is observed within minutes upon exposure of A.E7 T cells (restricted for IEk and moth cytochrome c peptide containing residues 88-103) and 4R3.9 T cells (restricted for IAk and myelin basic protein peptide containing residues 1-14 [AcMBP-(1-14)]) to their cognate peptides in the presence of CH27 cells bearing both IAk and IEk MHC class II molecules. Pretreatment of cloned T cells, but not APCs, with herbimycin A resulted in complete inhibition of triggering events, indicating that the acidification response is mediated by T-cell second messenger pathways. This rapid assay for 4R3.9 T-cell stimulation showed increased T-cell triggering activity for AcMBP-(1-14)-A4 and MBP-(1-14)-M4 peptides compared to the native AcMBP-(1-14)-K4. By using the previously determined kinetic constants for MBP-(1-14)-A4 reactions with IAk, it is possible to show that at the lowest peptide concentrations the kinetics of T-cell triggering are limited by the kinetics of the peptide binding to MHC class II molecules.
In vivo cytokine gene transfer by gene gun reduces tumor growth in mice.
Authors: Sun W H , Burkholder J K , Sun J , Culp J , Turner J , Lu X G , Pugh T D , Ershler W B , Yang N S
Published: May 2006

Implantation of tumor cells modified by in vitro cytokine gene transfer has been shown by many investigators to result in potent in vivo antitumor activities in mice. Here we describe an approach to tumor immunotherapy utilizing direct transfection of cytokine genes into tumor-bearing animals by particle-mediated gene transfer. In vivo transfection of the human interleukin 6 gene into the tumor site reduced methylcholanthrene-induced fibrosarcoma growth, and a combination of murine tumor necrosis factor alpha and interferon gamma genes inhibited growth of a renal carcinoma tumor model (Renca). In addition, treatment with murine interleukin 2 and interferon gamma genes prolonged the survival of Renca tumor-bearing mice and resulted in tumor eradication in 25% of the test animals. Transgene expression was demonstrated in treated tissues by ELISA and immunohistochemical analysis. Significant serum levels of interleukin 6 and interferon gamma were detected, demonstrating effective secretion of transgenic proteins from treated skin into the bloodstream. This in vivo cytokine gene therapy approach provides a system for evaluating the antitumor properties of various cytokines in different tumor models and has potential utility for human cancer gene therapy.
Ras membrane targeting is essential for glucose signaling but not for viability in yeast.
Authors: Bhattacharya S , Chen L , Broach J R , Powers S
Published: May 2006

Ras proteins are small GTP binding proteins that serve as critical relays in a variety of signal transduction pathways in eukaryotic cells. Like most metazoan Ras proteins, yeast Ras is post-translationally modified by addition of a farnesyl and a palmitoyl moiety, and these modifications are required for targeting the protein to the cytoplasmic face of the plasma membrane and for biological activity of the protein. We have constructed mutants of the yeast (Saccharomyces cerevisiae) Ras that are farnesylated in vivo but are not palmitoylated. These mutant proteins are not localized to the plasma membrane but function in the cell as well as the wild-type protein. Such mutants are viable but fail to induce a transient increase in intracellular cAMP concentration in response to glucose addition, although this deficiency does not yield a marked growth phenotype. These results are consistent with the hypothesis that the essential role of the farnesyl moiety on yeast Ras is to enhance productive interaction between Ras and its essential downstream target, adenylyl cyclase, rather than to localize Ras to the plasma membrane.
Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity.
Authors: Seidel H M , Milocco L H , Lamb P , Darnell J E , Stein R B , Rosen J
Published: May 2006

Signal transducers and activators of transcription (STAT proteins) bind to palindromic sequence elements related to interferon gamma (IFN-gamma) activation sites, which were first identified in the promoters of IFN-gamma-inducible genes. Although the sequences of the natural palindromic STAT-binding elements vary considerably, they conform to the general structure TT(N)5AA. We have systematically examined the effects of the spacing between the TT and AA core half sites on the binding of the STAT complexes activated by IFN-gamma, interleukin (IL) 6, granulocyte-macrophage colony-stimulating factor, and IL-4. We show that (i) as suggested earlier, a core palindromic TT--AA motif with a 5-bp spacing displays general STAT binding, (ii) a palindromic motif with a spacing of 4 bp selectively binds to complexes containing Stat3, and (iii) a motif with a 6-bp spacing selectively binds the STAT complexes activated by IL-4. We have examined natural elements in the promoters of cytokine-responsive genes that differ in half-site spacing and found that they display binding properties predicted from the synthetic binding sites. Furthermore, the observed differential selective binding characteristics for the most part correlate with the ability to mediate transcriptional activation of transfected test genes in response to the cytokines tested. Our results thus demonstrate that the specificity of STAT-directed transcription in response to particular cytokines or cytokine families depends in part on the spacing of half sites within the conserved response element sequence.
A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene.
Authors: Schneider S S , Schick C , Fish K E , Miller E , Pena J C , Treter S D , Hui S M , Silverman G A
Published: May 2006

The squamous cell carcinoma antigen (SCCA) is a member of the ovalbumin family of serine proteinase inhibitors (serpins). A neutral form of the protein is found in normal and some malignant squamous cells, whereas an acidic form is detected exclusively in tumor cells and in the circulation of patients with squamous cell tumors. In this report, we describe the cloning of the SCCA gene from normal genomic DNA. Surprisingly, two genes were found. They were tandemly arrayed and flanked by two other closely related serpins, plasminogen activator inhibitor type 2 (PAI2) and maspin at 18q21.3. The genomic structure of the two genes, SCCA1 and SCCA2, was highly conserved. The predicted amino acid sequences were 92% identical and suggested that the neutral form of the protein was encoded by SCCA1 and the acidic form was encoded by SCCA2. Further characterization of the region should determine whether the differential expression of the SCCA genes plays a causal role in development of more aggressive squamous cell carcinomas.
Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine.
Authors: Overly C C , Lee K D , Berthiaume E , Hollenbeck P J
Published: May 2006

Organelle acidification is an essential element of the endosomal-lysosomal pathway, but our understanding of the mechanisms underlying progression through this pathway has been hindered by the absence of adequate methods for quantifying intraorganelle pH. To address this problem in neurons, we developed a direct quantitative method for accurately determining the pH of endocytic organelles in live cells. In this report, we demonstrate that the ratiometric fluorescent pH indicator 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) is the most advantageous available probe for such pH measurements. To measure intraorganelle pH, cells were labeled by endocytic uptake of HPTS, the ratio of fluorescence emission intensities at excitation wavelengths of 450 nm and 405 nm (F450/405) was calculated for each organelle, and ratios were converted to pH values by using standard curves for F450/405 vs. pH. Proper calibration is critical for accurate measurement of pH values: standard curves generated in vitro yielded artifactually low organelle pH values. Calibration was unaffected by the use of culture medium buffered with various buffers or different cell types. By using this technique, we show that both acidic and neutral endocytically derived organelles exist in the axons of sympathetic neurons in different steady-state proportions than in the cell body. Furthermore, we demonstrate that these axonal organelles have a bimodal pH distribution, indicating a rapid acidification step in their maturation that reduces the average pH of a fraction of the organelles by 2 pH units while leaving few organelles of intermediate pH at steady state. Finally, we demonstrate a spatial gradient or organelle pH along axons, with the relative frequency of acidic organelles increasing with proximity to the cell body.
Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals.
Authors: Ruat M , Molliver M E , Snowman A M , Snyder S H
Published: May 2006

We have molecularly cloned a calcium sensing receptor (CaSR) from a rat striatal cDNA library. Rat CaSR displays 92% overall homology to its bovine counterpart with seven putative transmembrane domains characteristic of the superfamily of guanine nucleotide-binding proteins and significant homology with the metabotropic glutamate receptors. Northern blot analysis reveals two transcripts in thyroid, kidney, lung, ileum, and pituitary. In brain highest regional expression of the RNA occurs in the hypothalamus and the corpus striatum. Immunohistochemistry reveals discrete punctate localizations throughout the brain that appear to be associated with nerve terminals. No staining is evident in cell bodies of neurons or glia. Cerebral arteries display an intense network of CaSR immunoreactive fibers associated with vessel innervation. CaSR on nerve terminal membranes may regulate neurotransmitter disposition in response to Ca2+ levels in the synaptic space.
Mutation spectrum of the gene encoding the beta subunit of rod phosphodiesterase among patients with autosomal recessive retinitis pigmentosa.
Authors: McLaughlin M E , Ehrhart T L , Berson E L , Dryja T P
Published: May 2006

Mutations in the gene encoding the beta subunit of rod cGMP phosphodiesterase are known causes of photoreceptor degeneration in two animal models of retinitis pigmentosa, the rd (retinal degeneration) mouse and the Irish setter dog with rod/cone dysplasia. Here we report a screen of 92 unrelated patients with autosomal recessive retinitis pigmentosa for defects in the human homologue of this gene. We identified seven different mutations that cosegregate with the disease. They were found among four patients with each patient heterozygously carrying two mutations. All of these mutations are predicted to affect the putative catalytic domain, probably leading to a decrease in phosphodiesterase activity and an increase in cGMP levels within rod photoreceptors. Mutations in the gene encoding the beta subunit of rod phosphodiesterase are the most common identified cause of autosomal recessive retinitis pigmentosa, accounting for approximately 4% of cases in North America.
Two routes of chlorophyllide synthesis that are differentially regulated by light in barley (Hordeum vulgare L.).
Authors: Holtorf H , Reinbothe S , Reinbothe C , Bereza B , Apel K
Published: May 2006

NADPH-protochlorophyllide oxidoreductase (POR; EC 1.6.99.1) catalyzes the only known light-dependent step in chlorophyll synthesis of higher plants, the reduction of protochlorophyllide (Pchlide) to chlorophyllide. In barley, two distinct immunoreactive POR proteins were identified. In contrast to the light-sensitive POR enzyme studied thus far (POR-A), levels of the second POR protein remained constant in seedlings during the transition from dark growth to the light and in green plants. The existence of a second POR-related protein was verified by isolating and sequencing cDNAs that encode a second POR polypeptide (POR-B) with an amino acid sequence identity of 75% to the POR-A. In the presence of NADPH and Pchlide, the in vitro-synthesized POR-A and POR-B proteins could be reconstituted to ternary enzymatically active complexes that reduced Pchlide to chlorophyllide only after illumination. Even though the in vitro activities of the two enzymes were similar, the expression of their genes during the light-induced transformation of etiolated to green seedlings was distinct. While the POR-A mRNA rapidly declined during illumination of dark-grown seedlings and soon disappeared, POR-B mRNA remained at an approximately constant level in dark-grown and green seedlings. Thus these results suggest that chlorophyll synthesis is controlled by two light-dependent POR enzymes, one that is active only transiently in etiolated seedlings at the beginning of illumination and the other that also operates in green plants.
Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP).
Authors: Rolfe M , Beer-Romero P , Glass S , Eckstein J , Berdo I , Theodoras A , Pagano M , Draetta G
Published: May 2006

The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.
Refinement of odor molecule tuning by dendrodendritic synaptic inhibition in the olfactory bulb.
Authors: Yokoi M , Mori K , Nakanishi S
Published: May 2006

Mitral/tufted cells (M/T cells) and granule cells form reciprocal dendrodendritic synapses in the main olfactory bulb; the granule cell is excited by glutamate from the M/T cell and in turn inhibits M/T cells by gamma-aminobutyrate. The trans-synaptically excited granule cell is thought to induce lateral inhibition in neighboring M/T cells and to refine olfactory information. It remains, however, elusive how significantly and specifically this synaptic regulation contributes to the discrimination of different olfactory stimuli. This investigation concerns the mechanism of olfactory discrimination by single unit recordings of responses to a series of normal aliphatic aldehydes from individual rabbit M/T cells. This analysis revealed that inhibitory responses are evoked in a M/T cell by a defined subset of odor molecules with structures closely related to the excitatory odor molecules. Furthermore, blockade of the reciprocal synaptic transmission by the glutamate receptor antagonist or the gamma-aminobutyrate receptor antagonist markedly suppressed the odor-evoked inhibition, indicating that the inhibitory responses are evoked by lateral inhibition via the reciprocal synaptic transmission. The synaptic regulation in the olfactory bulb thus greatly enhances the tuning specificity of odor responses and would contribute to discrimination of olfactory information.
Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide.
Authors: Facci L , Dal Toso R , Romanello S , Buriani A , Skaper S D , Leon A
Published: May 2006

Mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play important roles in tissue inflammation and in neuroimmune interactions. Very little is known about endogenous molecules and mechanisms capable of modulating mast cell activation. Palmitoylethanolamide, found in peripheral tissues, has been proposed to behave as a local autacoid capable of downregulating mast cell activation and inflammation. A cognate N-acylamide, anandamide, the ethanolamide of arachidonic acid, occurs in brain and is a candidate endogenous agonist for the central cannabinoid receptor (CB1). As a second cannabinoid receptor (CB2) has been found in peripheral tissues, the possible presence of CB2 receptors on mast cells and their interaction with N-acylamides was investigated. Here we report that mast cells express both the gene and a functional CB2 receptor protein with negative regulatory effects on mast cell activation. Although both palmitoylethanolamide and anandamide bind to the CB2 receptor, only the former downmodulates mast cell activation in vitro. Further, the functional effect of palmitoylethanolamide, as well as that of the active cannabinoids, was efficiently antagonized by anandamide. The results suggest that (i) peripheral cannabinoid CB2 receptors control, upon agonist binding, mast cell activation and therefore inflammation; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for the CB2 receptor on mast cells; (iii) modulatory activities on mast cells exerted by the naturally occurring molecule strengthen a proposed autacoid local inflammation antagonism (ALIA) mechanism; and (iv) palmitoylethanolamide and its derivatives may provide antiinflammatory therapeutic strategies specifically targeted to mast cells ("ALIAmides").
The alpha and gamma 1 isoforms of protein phosphatase 1 are highly and specifically concentrated in dendritic spines.
Authors: Ouimet C C , da Cruz e Silva E F , Greengard P
Published: May 2006

Protein phosphatase 1 (PP1) is a highly conserved enzyme that has been implicated in diverse biological processes in the brain as well as in nonneuronal tissues. The present study used light and electron microscopic immunocytochemistry to characterize the distribution of two PP1 isoforms, PP1 alpha and PP1 gamma 1, in the rat neostriatum. Both isoforms are heterogeneously distributed in brain with the highest immunoreactivity being found in the neostriatum and hippocampal formation. Further, both isoforms are highly and specifically concentrated in dendritic spines. Weak immunoreactivity is present in dendrites, axons, and some axon terminals. Immunoreactivity for PP1 alpha is also present in the perikaryal cytoplasm and nuclei of most medium- and large-sized neostriatal neurons. The specific localization of PP1 in dendritic spines is consistent with a central role for this enzyme in signal transduction. The data support the concept that, in the course of evolution, spines developed as specialized signal transduction organelles enabling neurons to integrate diverse inputs from multiple afferent nerve terminals.
Prostate cancer in a transgenic mouse.
Authors: Greenberg N M , DeMayo F , Finegold M J , Medina D , Tilley W D , Aspinall J O , Cunha G R , Donjacour A A , Matusik R J , Rosen J M
Published: May 2006

Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop an animal model for prostate cancer, several lines of transgenic mice were generated by using the prostate-specific rat probasin promoter to derive expression of the simian virus 40 large tumor antigen-coding region. Mice expressing high levels of the transgene display progressive forms of prostatic disease that histologically resemble human prostate cancer, ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. Prostate tumors have been detected specifically in the prostate as early as 10 weeks of age. Immunohistochemical analysis of tumor tissue has demonstrated that dorsolateral prostate-specific secretory proteins were confined to well-differentiated ductal epithelial cells adjacent to, or within, the poorly differentiated tumor mass. Prostate tumors in the mice also display elevated levels of nuclear p53 and a decreased heterogeneous pattern of androgen-receptor expression, as observed in advanced human prostate cancer. The establishment of breeding lines of transgenic mice that reproducibly develop prostate cancer provides an animal model system to study the molecular basis of transformation of normal prostatic cells and the factors influencing the progression to metastatic prostate cancer.
Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells.
Authors: Bergson C , Mrzljak L , Lidow M S , Goldman-Rakic P S , Levenson R
Published: May 2006

To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases.
Lipocortin 1 mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat.
Authors: Wu C C , Croxtall J D , Perretti M , Bryant C E , Thiemermann C , Flower R J , Vane J R
Published: May 2006

Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall (approximately 30 mmHg) in mean arterial blood pressure, (ii) a reduction (> 75%) in the pressor responses to norepinephrine (1 microgram/kg i.v.), and (iii) an induction of nitric oxide synthase (iNOS) as measured in the lung. Dexamethasone (1 mg/kg i.p. at 2 h prior to LPS) attenuated the hypotension and the vascular hyporeactivity to norepinephrine and reduced (by approximately 77%) the expression of iNOS in the lung. These effects of dexamethasone were prevented by pretreatment of LPS-treated rats with a neutralizing antiserum to lipocortin 1 (anti-LC1; 60 mg/kg s.c. at 24 h prior to LPS) but not by a control nonimmune sheep serum. Stimulation of J774.2 macrophages with LPS (1 microgram/ml for 24 h) caused the expression of iNOS and cyclooxygenase 2 (COX-2) protein and significantly increased nitrite generation; this was prevented by dexamethasone (0.1 microM at 1 h prior to LPS), which also increased cell surface lipocortin 1. Pretreatment of J774.2 cells with anti-LC1 (1:60 dilution at 4 h prior to LPS) also abolished the inhibitory effect of dexamethasone on iNOS expression and nitrite accumulation but not that on COX-2 expression. A lipocortin 1 fragment (residues 1-188 of human lipocortin 1; 20 micrograms/ml at 1 h prior to LPS) also blocked iNOS in J774.2 macrophages activated by LPS (approximately 78% inhibition), and this too was prevented by anti-LC1. We conclude that the extracellular release of endogenous lipocortin 1 (i) mediates the inhibition by dexamethasone of the expression of iNOS, but not of COX-2, and (ii) contributes substantially to the beneficial actions of dexamethasone in rats with endotoxic shock.
DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions.
Authors: Kowalczykowski S C , Krupp R A
Published: May 2006

DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange. A gratuitous allosteric effector consisting of the noncovalent complex of ADP and aluminum fluoride, ADP.AIF4-, can both induce the high-affinity DNA-binding state of RecA protein and support the homologous pairing and exchange of up to 800-900 bp of DNA. These results demonstrate that induction of the functionally active, high-affinity DNA-binding state of RecA protein is needed for RecA protein-promoted DNA-strand exchange and that there is no requirement for a high-energy nucleotide cofactor for the exchange of DNA strands. Consequently, the free energy needed to activate the DNA substrates for DNA-strand exchange is not derived from ATP hydrolysis. Instead, the needed free energy is derived from ligand binding and is transduced to the DNA via the associated ligand-induced structural transitions of the RecA protein-DNA complex; ATP hydrolysis simply destroys the effector ligand. This concept has general applicability to the mechanism of energy transduction by proteins.
Proprotein convertases in amphioxus: predicted structure and expression of proteases SPC2 and SPC3.
Authors: Oliva A A , Steiner D F , Chan S J
Published: May 2006

SPC2 and SPC3 are two members of a family of subtilisin-related proteases which play essential roles in the processing of prohormones into their mature forms in the pancreatic B cell and many other neuroendocrine cells. To investigate the phylogenetic origins and evolutionary functions of SPC2 and SPC3 we have identified and cloned cDNAs encoding these enzymes from amphioxus (Branchiostoma californiensis), a primitive chordate. The amino acid sequence of preproSPC2 contains 689 aa and is 71% identical to human SPC2. In contrast, amphioxus prproSPC3 consists of 774 aa and exhibits 55% identity to human SPC3. These results suggest that the primary structure of SPC2 has been more highly conserved during evolution than that of SPC3. To further investigate the function(s) of SPC2 and SPC3 in amphioxus, we have determined the regional expression of these genes by using a reverse transcriptase-linked polymerase chain reaction (RT-PCR) assay. Whole amphioxus was dissected longitudinally into four equal-length segments and RNA was extracted. Using RT-PCR to simultaneously amplify SPC2 and SPC3 DNA fragments, we found that the cranial region (section 1) expressed equal amounts of SPC2 and SPC3 mRNAs, whereas in the caudal region (section 4) the SPC2-to-SPC3 ratio was 5:1. In the mid-body sections 2 and 3 the SPC2-to-SPC3 ratio was 1:5. By RT-PCR we also determined that amphioxus ILP, a homologue of mammalian insulin/insulin-like growth factor, was expressed predominately in section 3. These results suggest that the relative levels of SPC2 and SPC3 mRNAs are specifically regulated in various amphioxus tissues. Furthermore, the ubiquitous expression of these mRNAs in the organism indicates that they are involved in the processing of other precursor proteins in addition to proILP.
Swapping structural determinants of ribonucleases: an energetic analysis of the hinge peptide 16-22.
Authors: Mazzarella L , Vitagliano L , Zagari A
Published: May 2006

Bovine seminal ribonuclease (BS-RNase) is a homodimeric enzyme strictly homologous to the pancreatic ribonuclease (RNase A). Native BS-RNase is an equilibrium mixture of two distinct dimers differing in the interchange of the N-terminal segments and in their biological properties. The loop 16-22 plays a fundamental role on the relative stability of the two isomers. Both the primary and tertiary structures of the RNase A differ substantially from those of the seminal ribonuclease in the loop region 16-22. To analyze the possible stable conformations of this loop in both enzymes, structure predictions have been attempted, according to a procedure described by Palmer and Scheraga [Palmer, K. A. & Scheraga, H. A. (1992) J. Comput. Chem. 13, 329-350]. Results compare well with experimental x-ray structures and clarify the structural determinants that are responsible for the swapping of the N-terminal domains and for the peculiar properties of BS-RNase. Minimal modifications of RNase A sequence needed to form a stable swapped dimer are also predicted.
Transposable elements are stable structural components of Drosophila melanogaster heterochromatin.
Authors: Pimpinelli S , Berloco M , Fanti L , Dimitri P , Bonaccorsi S , Marchetti E , Caizzi R , Caggese C , Gatti M
Published: May 2006

We determined the distribution of 11 different transposable elements on Drosophila melanogaster mitotic chromosomes by using high-resolution fluorescent in situ hybridization (FISH) coupled with charge-coupled device camera analysis. Nine of these transposable elements (copia, gypsy, mdg-1, blood, Doc, I, F, G, and Bari-1) are preferentially clustered into one or more discrete heterochromatic regions in chromosomes of the Oregon-R laboratory stock. Moreover, FISH analysis of geographically distant strains revealed that the locations of these heterochromatic transposable element clusters are highly conserved. The P and hobo elements, which are likely to have invaded the D. melanogaster genome at the beginning of this century, are absent from Oregon-R heterochromatin but clearly exhibit heterochromatic clusters in certain natural populations. Together these data indicate that transposable elements are major structural components of Drosophila heterochromatin, and they change the current views on the role of transposable elements in host genome evolution.
The nucleotide sequence of chromosome I from Saccharomyces cerevisiae.
Authors: Bussey H , Kaback D B , Zhong W , Vo D T , Clark M W , Fortin N , Hall J , Ouellette B F , Keng T , Barton A B
Published: May 2006

Chromosome I from the yeast Saccharomyces cerevisiae contains a DNA molecule of approximately 231 kbp and is the smallest naturally occurring functional eukaryotic nuclear chromosome so far characterized. The nucleotide sequence of this chromosome has been determined as part of an international collaboration to sequence the entire yeast genome. The chromosome contains 89 open reading frames and 4 tRNA genes. The central 165 kbp of the chromosome resembles other large sequenced regions of the yeast genome in both its high density and distribution of genes. In contrast, the remaining sequences flanking this DNA that comprise the two ends of the chromosome and make up more than 25% of the DNA molecule have a much lower gene density, are largely not transcribed, contain no genes essential for vegetative growth, and contain several apparent pseudogenes and a 15-kbp redundant sequence. These terminally repetitive regions consist of a telomeric repeat called W', flanked by DNA closely related to the yeast FLO1 gene. The low gene density, presence of pseudogenes, and lack of expression are consistent with the idea that these terminal regions represent the yeast equivalent of heterochromatin. The occurrence of such a high proportion of DNA with so little information suggests that its presence gives this chromosome the critical length required for proper function.