bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of inositol trisphosphate receptor subtype 3 to insulin and somatostatin secretory granules and regulation of expression in islets and insulinoma cells.
Authors: Blondel O , Moody M M , Depaoli A M , Sharp A H , Ross C A , Swift H , Bell G I
Published: May 2006

Calcium ions play a central role in stimulus-secretion coupling in pancreatic beta cells, and an elevation of cytosolic Ca2+ levels is necessary for insulin secretion. Inositol 1,4,5-trisphosphate mobilizes intracellular Ca2+ stores in the beta cell by binding to specific receptors that are ligand-activated Ca2+ channels. The inositol trisphosphate receptors comprise a family of structurally related proteins with distinct but overlapping tissue distributions. Previous studies indicated that the predominant inositol trisphosphate receptor subtype expressed in rat pancreatic islets was the protein designated IP3R-3. We have confirmed the expression of IP3R-3 in pancreatic islets by immunohistocytochemistry and localized this protein to the secretory granules of insulin-secreting beta cells and somatostatin-secreting delta cells by immunogold electron microscopy. Secretory granules contain high levels of Ca2+, and the presence of IP3R-3 in the granule provides a mechanism for mobilizing granule Ca2+ stores in response to glucose and/or hormones. The release of Ca2+ from granule stores would increase the Ca2+ concentration in the surrounding cytoplasm and promote rapid exocytosis of granules, especially those granules in close proximity to the plasma membrane. The levels of IP3R-3 were increased in pancreatic islets of diabetic rats and rats that had been refed after a period of fasting. They were also increased in rat insulinoma RINm5F cells cultured in 25 mM glucose compared with cells cultured in 5 mM glucose. The localization of IP3R-3 to secretory granules of insulin-secreting beta cells and somatostatin-secreting delta cells suggests that granule Ca2+ stores actively participate in the secretory process and that their release is regulated by inositol 1,4,5-trisphosphate. The regulation of IP3R-3 levels by glucose, diabetes, and refeeding may allow the beta cell to adjust the insulin secretory response to changing physiological conditions.
9-O-acetylated sialic acids have widespread but selective expression: analysis using a chimeric dual-function probe derived from influenza C hemagglutinin-esterase.
Authors: Klein A , Krishna M , Varki N M , Varki A
Published: May 2006

While 9-O-acetylation of sialic acids has been reported in some mammalian tissues, the distribution of this modification on specific cell types and molecules is largely unknown. The influenza C virus hemagglutinin-esterase is a membrane-bound glycoprotein that binds specifically to 9-O-acetylated sialic acids (hemagglutinin activity) and then hydrolyzes the O-acetyl group (receptor-destroying activity). A recombinant soluble form of influenza C virus hemagglutinin-esterase wherein the C-terminal transmembrane and cytoplasmic domains are replaced by the Fc portion of human IgG retains both its recognition and enzymatic functions. The latter activity can selectively remove 9-O-acetyl groups from bound or free sialic acids and, under specific conditions, 7-O-acetyl groups as well. Irreversible inactivation of the esterase unmasks stable recognition activity, giving a molecule that binds specifically to 9-O-acetylated sialic acids. These probes demonstrate widespread but selective expression of 9-O-acetylated sialic acids in certain cell types of rat tissues. Patterns of polarized or gradient expression further demonstrate the regulated nature of this modification. Direct probing of blots and thin-layer plates shows selective expression of 9-O-acetylation on certain glycoproteins and glycolipids in such tissues. Thus, 9-O-acetylation is more widespread than previously thought and occurs on specific molecules and cell types.
Contrasting the distribution of chloroplast DNA and allozyme polymorphism among local populations of Silene alba: implications for studies of gene flow in plants.
Authors: McCauley D E
Published: May 2006

The distribution of chloroplast DNA (cp-DNA) length variants was analyzed within and among 10 local populations of Silene alba, a dioecious angiosperm. The populations displayed considerable allele frequency variation, resulting in an estimate of Wright's Fst of 0.67 over a 25 x 25 km portion of the species' range. By contrast, a concurrent analysis of the genetic structure of these same populations based on seven polymorphic allozyme loci yielded an estimate of Fst of 0.13. The two Fst estimates are significantly different from one another when their respective confidence limits are estimated by jackknifing. The results of a breeding study were consistent with maternal inheritance of the cpDNA variants. With maternal inheritance the genetic structure of the cpDNA should reflect seed movement, whereas the genetic structure of the nuclear-encoded allozyme loci should reflect the movement of both seeds and pollen. Comparison of the two markedly different Fst estimates in the context of recent models of the population genetics of organelles suggests that the movement of both seeds and pollen contributes significantly to gene flow.
The relationship between genetic and physical distances in the cloned a1-sh2 interval of the Zea mays L. genome.
Authors: Civardi L , Xia Y , Edwards K J , Schnable P S , Nikolau B J
Published: May 2006

A 470-kb segment from the long arm of chromosome 3 of Zea mays (inbred LH82), encompassing the a1-sh2 interval, was cloned as a yeast artificial chromosome. Comparison of the sizes of the restriction fragments generated from the cloned DNA fragment and from the DNA isolated from the maize inbred line LH82 established the colinearity of the a1-sh2 interval in these DNAs. By utilizing a chromosome fragmentation technique, a yeast artificial chromosome encompassing the a1-sh2 interval was separately fragmented at the a1 and sh2 loci. Comparison of the sizes of these fragmentation products established the physical distance between the a1 and sh2 loci to be 140 kb. Furthermore, these fragmentation experiments established the physical orientation of the a1 and sh2 genes relative to the maize centromere. The molecular cloning of the contiguous region between the a1 and sh2 loci made it possible to define the relationship between physical and genetic distances over a relatively large segment of the maize genome. In this interval, the relationship between physical and genetic distances is 1560 kb/centimorgan, which compares with 1460 kb/centimorgan for the entire maize genome, and 217 kb/centimorgan for a 1-kb segment within the a1 locus. Therefore, these findings are consistent with the hypothesis that genes per se are preferred sites for meiotic recombination rather than the hypothesis that genes reside in large recombinationally active segments of the genome.
Dopamine alters glutamate receptor desensitization in retinal horizontal cells of the perch (Perca fluviatilis).
Authors: Schmidt K F , Kruse M , Hatt H
Published: May 2006

The patch-clamp technique in combination with a fast liquid filament application system was used to study the effect of dopamine on the glutamate receptor desensitization in horizontal cells of the perch (Perca fluviatilis). Kinetics of ligand-gated ion channels in fish horizontal cells are modulated by dopamine. This modulation is presumably mediated by a cAMP-dependent protein phosphorylation. Before incubation with dopamine, the glutamate receptors of horizontal cells activate and desensitize with fast time constants. In the whole-cell recording mode, fast application of the agonists L-glutamate, quisqualate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid prior to the dopamine incubation gives rise to fast transient currents with peak values of about 200 pA that desensitize within 100 ms. Kainate as agonist produced higher steady-state currents but no transient currents. After incubation of the cells with dopamine for 3 min, the desensitization was significantly reduced and the agonists L-glutamate, quisqualate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid induced steady-state currents with amplitudes that were similar to the previously observed transient currents. Kainate-induced currents were only slightly affected. Fast desensitizing currents upon fast application of L-glutamate were also recorded from outside-out patches that were excised from horizontal cells before incubation with dopamine. The currents from excised patches desensitized to a steady-state level of about 0.2 of the peak amplitude with time constants of less than 2 ms. When the outside-out patches were excised from cells after dopamine incubation, steady-state currents were enhanced and no transient currents were observed. The results may indicate that the dopamine-dependent modulation of glutamate-induced currents, which is presumably mediated by a protein phosphorylation, is due to an alteration of the desensitization of the glutamate receptors.
Global arrest of translation during invertebrate quiescence.
Authors: Hofmann G E , Hand S C
Published: May 2006

Comparing the translational capacities of cell-free systems from aerobically developing embryos of the brine shrimp Artemia franciscana vs. quiescent embryos has revealed a global arrest of protein synthesis. Incorporation rates of [3H]leucine by lysates from 4-h anoxic embryos were 8% of those from aerobic (control) embryos, when assayed at the respective pH values measured for each treatment in vivo. Exposure of embryos to 4 h of aerobic acidosis (elevated CO2 in the presence of oxygen) suppressed protein synthesis to 3% of control values. These latter two experimental treatments promote developmental arrest of Artemia embryos and, concomitantly, cause acute declines in intracellular pH. When lysates from each treatment were assayed over a range of physiologically relevant pH values (pH 6.4-8.0), amino acid incorporation rates in lysates from quiescent embryos were consistently lower than values for the aerobic controls. Acute reversal of pH to alkaline values during the 6-min assays was not sufficient to return the incorporation rates of quiescent lysates to control values. Thus, a stable alteration in translational capacity of quiescent lysates is indicated. Addition of exogenous mRNA did not rescue the suppressed protein synthesis in quiescent lysates, which suggests that the acute blockage of amino acid incorporation is apparently not due to limitation in message. Thus, the results support a role for intracellular pH as an initial signaling event in translational control during quiescence yet, at the same time, indicate that a direct proton effect on the translational machinery is not the sole proximal agent for biosynthetic arrest in this primitive crustacean.
Regulation of scallop myosin by the regulatory light chain depends on a single glycine residue.
Authors: Jancso A , Szent-Györgyi A G
Published: May 2006

Specific Ca2+ binding and Ca2+ activation of ATPase activity in scallop myosin require a regulatory light chain (RLC) from regulated (molluscan or vertebrate smooth) myosin; hybrids containing vertebrate skeletal RLCs do not bind Ca2+ and their ATPase activity is inhibited. Chimeras between scallop and chicken skeletal RLCs restore Ca2+ sensitivity to RLC-free myosin provided that residues 81-117 are derived from scallop. Six mutants (R90M, A94K, D98P, N105K, M116Q, and G117C) were generated by replacing amino acids of the scallop RLC with the corresponding skeletal RLC residues in positions conserved in either regulated or nonregulated myosins. Ca2+ binding was abolished by a G117C and a G117A mutation; however, these mutants have a decreased affinity for the heavy chain. None of the other mutations affected RLC function. Replacement of the respective cysteine with glycine in the skeletal RLC has markedly changed the regulatory properties of the molecule. The single cysteine to glycine mutation conferred to this light chain the ability to restore Ca2+ binding and regulated ATPase activity, although Ca2+ activation of the actin-activated ATPase was lower than with scallop RLC. The presence of amino acids other than glycine at this position in vertebrate skeletal myosin RLCs may explain why these are not fully functional in the scallop system. The results are in agreement with x-ray crystallography data showing the central role of G117 in stabilizing the Ca(2+)-binding site of scallop myosin.
An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene.
Authors: Itzhaki H , Maxson J M , Woodson W R
Published: May 2006

The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define sequences responsible for ethylene-responsive expression. Deletion analysis of the 5' flanking sequences of GST1 identified a single positive regulatory element of 197 bp between -667 and -470 necessary for ethylene-responsive expression. The sequences within this ethylene-responsive region were further localized to 126 bp between -596 and -470. The ethylene-responsive element (ERE) within this region conferred ethylene-regulated expression upon a minimal cauliflower mosaic virus-35S TATA-box promoter in an orientation-independent manner. Gel electrophoresis mobility-shift assays and DNase I footprinting were used to identify proteins that bind to sequences within the ERE. Nuclear proteins from carnation petals were shown to specifically interact with the 126-bp ERE and the presence and binding of these proteins were independent of ethylene or petal senescence. DNase I footprinting defined DNA sequences between -510 and -488 within the ERE specifically protected by bound protein. An 8-bp sequence (ATTTCAAA) within the protected region shares significant homology with promoter sequences required for ethylene responsiveness from the tomato fruit-ripening E4 gene.
Transgenic mice overexpressing phosphoenolpyruvate carboxykinase develop non-insulin-dependent diabetes mellitus.
Authors: Valera A , Pujol A , Pelegrin M , Bosch F
Published: May 2006

An increase in hepatic gluconeogenesis is believed to be an important factor responsible for the fasting hyperglycemia detected in patients with non-insulin-dependent diabetes mellitus (NIDDM). Phosphoenolpyruvate carboxykinase (GTP) (PEPCK; EC 4.1.1.32) is a regulatory enzyme of gluconeogenesis. To study the role of the expression of PEPCK gene in the development of NIDDM, we have produced lines of transgenic mice expressing a PEPCK minigene under control of its own promoter. Transgenic mice were hyperglycemic and had higher serum insulin concentrations. In addition, alterations in liver glycogen content and muscle glucose transporter GLUT-4 gene expression were detected. The overexpression of the PEPCK gene led to an increase in glucose production from pyruvate in hepatocytes in primary culture. When intraperitoneal glucose tolerance tests were performed, blood glucose levels were higher than those detected in normal mice. This animal model shows that primary alterations in the rate of liver glucose production may induce insulin resistance and NIDDM.
Role of antibody and T cells in the long-term inhibition of IgE synthesis.
Authors: Haba S , Nisonoff A
Published: May 2006

We have shown that the long-term inhibition of IgE synthesis associated with perinatal inoculation of syngeneic IgE is accompanied by the synthesis of autoantibodies to IgE. Synthesis of IgE can also be inhibited by passive transfer of syngeneic anti-IgE antibodies. In the present investigation we made use of adoptive transfer experiments to assess the relative roles of antibodies and T cells in the inhibitory process. It was found that spleen cells from IgE-suppressed mice (synthesizing anti-IgE antibodies) could adoptively transfer the state of inhibition to syngeneic adult mice. The inhibition occurred only under conditions in which the recipient mice synthesized anti-IgE antibodies. Separated B cells, CD4+ T cells, CD8+ T cells, or a mixture of B and CD8+ T cells were ineffective. However, strong inhibition of IgE synthesis (as indicated by serum levels and numbers of IgE-secreting cells in the spleen) was observed after transfer of a mixture of B cells and CD4+ (helper) T cells. The results indicate that in this experimental model anti-IgE antibodies are the suppressive agent and that T cells do not play a role other than that of providing help to B cells for anti-IgE synthesis.
Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.
Authors: Jasper F , Koncz C , Schell J , Steinbiss H H
Published: May 2006

Virulence proteins VirD1 and VirD2 are subunits of a relaxosome-like protein complex that mediates conjugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants. The VirD1-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conjugative DNA strand (the T-strand) at the third base of these repeats. Nuclear localization signals present in VirD2 target the T-strand to plant cell nuclei. In addition, VirD2 probably plays a role in the high-frequency integration of the T-DNA into the plant genome by illegitimate recombination. Whereas Agrobacterium transformation of dicots is very efficient, T-DNA integration in most monocots can barely be detected. To develop an artificial T-DNA delivery system for monocots, a technique for efficient in vitro production of T-strand DNAs was established by using VirD1 and VirD2 proteins purified from overexpressing Escherichia coli strains. The topoisomerase-like VirD2 enzyme was shown to mediate precise, sequence-specific cleavage of T-DNA border sequences carried by single-stranded DNA templates, even in the absence of VirD1 protein. During this reaction, VirD2 remains covalently bound to the 5' end of artificial T-strand DNAs. In contrast, VirD2, alone or in complex with VirD1, fails to nick linear double-stranded DNA templates in vitro.
Crucial role for DNA supercoiling in Mu transposition: a kinetic study.
Authors: Wang Z , Harshey R M
Published: May 2006

DNA supercoiling plays an indispensable role in an early step of bacteriophage Mu transposition. This step involves formation of a nucleoprotein complex in which the Mu ends synapse and undergo two concerted single-strand cleavages. We describe a kinetic analysis of the role of supercoiling in the Mu-end synapsis reaction as measured by the cleavage assay. We observe a dependence of the reaction rate on superhelical density as well as on the length of Mu donor plasmid DNA. The reaction has a high activation enthalpy (approximately 67 kcal/mol). These results imply that the free energy of supercoiling is used directly to lower the activation barrier of the rate-limiting step of the reaction. Only the free energy of supercoiling associated with DNA outside the Mu ends appears to be utilized, implying that the Mu ends come together before the supercoiling energy is used. Our results suggest an essential function for the bacterial sequences attached to the ends of Mu virion DNA.
Tethering human immunodeficiency virus 1 integrase to a DNA site directs integration to nearby sequences.
Authors: Bushman F D
Published: May 2006

Certain retrovirus and retrotransposons display strong biases in the selection of host DNA sites for integration. To probe the possibility that simple tethering of the retroelement integrase protein to a target DNA site is sufficient to direct integration, the activities of a hybrid composed of human immunodeficiency virus 1 integrase and lambda repressor were analyzed. In in vitro reactions containing several target DNAs, the lambda repressor-integrase hybrid was found to direct integration selectively to targets containing lambda operators. Addition of lambda repressor blocked selective integration, indicating that binding to the operators was required. The lambda repressor-integrase hybrid protein directed integration primarily to sites near the operators on the same face of the B-DNA helix, indicating that target DNA was probably captured by looping out the intervening sequences. Such hybrid integrase proteins may be useful for directing retroviral integration to specific sequences in vivo.
Early changes in protein synthesis induced by basic fibroblast growth factor, nerve growth factor, and epidermal growth factor in PC12 pheochromocytoma cells.
Authors: Hondermarck H , McLaughlin C S , Patterson S D , Bradshaw R A
Published: May 2006

Nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) stimulate neuronal differentiation, whereas epidermal growth factor (EGF) promotes only mitogenic responses in PC12 pheochromocytoma cells. The early changes in protein synthesis induced by bFGF, NGF, and EGF in these cells have been determined by two-dimensional PAGE of [35S]methionine-labeled proteins and computerized image analysis. The rate of synthesis of only 29 proteins (out of approximately 1500 identified) was found to be modulated during the first several hours of growth factor stimulation. Individually, 12 were affected by EGF, 23 were affected by bFGF, and 20 were affected by NGF. Eight of these were regulated by all three growth factors, while 10 proteins were commonly induced by bFGF and NGF, in accordance with the essentially identical morphological responses induced by these two factors. In addition, the effects of bFGF and NGF were about equally divided between increases and decreases in the rate of synthesis of individual proteins, whereas EGF caused significantly more positive (increased) responses. All proteins modulated by NGF or FGF alone were negative in their response and those induced by only EGF were positive. Of particular interest, the rate of synthesis of two proteins of 55 kDa and pI 5.45 and 5.50 was dramatically and transiently induced during the first 2 hr of bFGF and NGF treatment and was not affected by EGF. This study indicates that all three factors elicit early increases and decreases in the synthesis of a quite limited number of proteins and provides molecular evidence for the specificity of a differentiative vs. a proliferative growth factor-induced signaling pathway in these cells.
Role of the cdc25C phosphatase in G2 arrest induced by nitrogen mustard.
Authors: O'Connor P M , Ferris D K , Hoffmann I , Jackman J , Draetta G , Kohn K W
Published: May 2006

G2 arrest induced by nitrogen mustard in human lymphoma CA46 cells is associated with a failure to activate hyperphosphorylated cdc2/cyclin B1 complexes. We investigated the possibility that this might be due to a suppression of cdc25C phosphatase activity. cdc25C from interphase cells migrated as a 54- to 57-kDa doublet in SDS gels and exhibited basal phosphatase activity. cdc25C from mitotic cells migrated as a 66-kDa hyperphosphorylated species and exhibited elevated phosphatase activity. cdc25C hyperphosphorylation and activation were mediated by cdc2, supporting the view of a cdc2-cdc25C autocatalytic feedback loop. Immunofluorescence and cell fractionation studies suggested cdc2-cdc25C interaction occurred within the cytoplasm. Cells arrested in G2 phase following nitrogen mustard treatment or cells arrested in S phase with aphidicolin failed to dephosphorylate and activate cdc2, and this correlated with failure to convert cdc25C into the most active hyperphosphorylated species. Our findings suggest that checkpoints guarding against mitotic entry in the presence of unreplicated or damaged DNA suppress formation of the cdc2-cdc25C autocatalytic feedback loop that normally brings about rapid activation of cdc2.
Signaling among neighboring plants and the development of size inequalities in plant populations.
Authors: Ballaré C L , Scopel A L , Jordan E T , Vierstra R D
Published: May 2006

Transgenic tobacco plants that express an oat phytochrome gene (phyA) under control of the cauliflower mosaic virus (CaMV) 35S promoter and display altered photophysiology were used to test the role of light as a vehicle of information in dominance relationships between neighboring plants. Compared with the isogenic wild type, phyA-overexpressing plants showed dramatically reduced morphological responsivity to changes in the red/far red ratio of the incident light and to the proximity of neighboring plants in spacing experiments. In transgenic canopies an increase in stand density caused the small plants of the population to be rapidly suppressed by their neighbors. In wild-type canopies, plants responded to increased density with large morphological changes, and there appeared to be an inverse relationship between the magnitude of this morphological response and the ranking of the individual plant in the population size hierarchy. In these wild-type populations, size inequality increased only moderately with density within the time frame of the experiments. Our results suggest that, in crowded stands, the ability of individual plants to acquire information about their light environment via phytochrome plays a central role in driving architectural changes that, at the population level, delay the development of size differences between neighbors.
Comparative aspects of Na+/K+ pump-mediated uncoupled Na+ efflux in red blood cells and kidney proteoliposomes.
Authors: Martin W H , Richards D E , Marín R , Jack-Hays M , Hoffman J F
Published: May 2006

Ouabain-sensitive uncoupled Na+ efflux has been studied in human, pig, and rat red cells and in vesicles containing reconstituted kidney Na+/K+ pumps obtained from these same species. The red cells from the different species gave qualitatively similar results; the uncoupled Na+ efflux was 15-30% of the Na+/K+ exchange rate, and this flux was inhibited at 5 mM extracellular Na+ (Na+o). At higher levels of Na+o there was a monotonic increase in the Na+ efflux. As has previously been observed in human red cells, the uncoupled efflux from pig red cells consists of Na+ and anion cotransport, suggesting that anion cotransport may be a general characteristic of uncoupled Na+ efflux in red cells. The uncoupled Na+ efflux carried out by pig and rat kidney Na+/K+ pumps differs from the red cell activity in that it represents no more than 2-4% of the Na+/K+ exchange rate and that 5 mM Na+o does not inhibit this efflux. Furthermore, the efflux does not appear to be dependent on anion cotransport. Vesicles containing human kidney Na+/K+ pumps differ from vesicles derived from pig or rat kidneys in that the Na+ efflux is not inhibited or stimulated by Na+ present on the opposite side; it thus appears that the Na+,K(+)-ATPase in these vesicles may be incapable of Na+/Na+ exchange. These results indicate that the ligand and kinetic properties of the uncoupled Na+ efflux mode of red cells are markedly different from kidney-derived Na+/K+ pumps reconstituted into proteoliposomes. The basis for these differences may be inherent in the Na+/K+ pumps themselves or represent differences between the two types of preparations studied.
Differential gene expression in an actinorhizal symbiosis: evidence for a nodule-specific cysteine proteinase.
Authors: Goetting-Minesky M P , Mullin B C
Published: May 2006

Nodules formed on the roots of actinorhizal plants as a consequence of nitrogen-fixing symbioses with the actinomycete Frankia appear to result from modification of the developmental pathway that leads to lateral root formation. Presently no information exists about factors that control this developmental switch or, until now, about genes that are differentially expressed as a result of an altered developmental pathway. Differential screening of an Alnus glutinosa nodule cDNA library revealed altered levels of gene expression in nodules as compared with roots and allowed isolation of host plant nodule-specific cDNA sequences. The deduced amino acid sequence of one full-length cDNA, AgNOD-CP1, represents a nodule-specific cysteine proteinase similar to cysteine proteinases of the papain superfamily. Residues critical to catalysis, active site, and disulfide bridges are conserved. Suggested roles for this enzyme are as a defense response to Frankia invasion, as a component of tissue remodeling in root and nodule tissues, as a cell cycle component, or as an element of protein turnover. Complexity of hybridization patterns revealed by Southern blot analysis suggests that the gene for AgNOD-CP1 is a member of a multigene family. Northern hybridization results indicate that this gene may have been recruited for a role specific to this symbiosis, a phenomenon observed in the Rhizobium-legume symbioses, perhaps common to many microbe-plant interactions.
Cognitive processes and cerebral cortical fundi: findings from positron-emission tomography studies.
Authors: Markowitsch H J , Tulving E
Published: May 2006

Positron-emission tomography (PET) studies of regional cerebral blood flow have provided evidence relevant to localization of cognitive functions. The critical loci identified in these studies are typically described in terms of macroanatomically labeled cortical and subcortical regions. We report the results of a meta-analysis of localization of changes in blood flow, based on nearly 1000 cerebral cortical peaks of activity obtained from groups of subjects in 30 PET studies. The results showed that, on average, 47% of these peaks were localized within the fundus regions of cortical sulci. This is an unexpectedly high proportion because fundal regions compose < 8% of the cortical mantle. Further analysis suggested a coarse correlation between the extent of fundal activation observed in different studies and the estimated cognitive complexity of the tasks used in the studies. These findings are potentially interesting because (i) the preponderance of fundal activation has implications for the interpretation of the PET data, (ii) they suggest that cortical sulcal and fundal regions may play a distinctive role in higher cognitive processing, or (iii) both of the above.
The G alpha subunit G alpha 4 couples to pterin receptors and identifies a signaling pathway that is essential for multicellular development in Dictyostelium.
Authors: Hadwiger J A , Lee S , Firtel R A
Published: May 2006

In this paper, we show that the G alpha subunit G alpha 4 couples to pterin receptors and identifies a signalling pathway that is essential for multicellular development in Dictyostelium. G alpha 4 is developmentally regulated, is essential for proper morphogenesis and spore production, and functions cell nonautonomously. We show that G alpha 4 is coupled to receptors (alpha FAR) that activate chemotaxis and adenylyl and guanylyl cyclases in response to folate during the early stages of development and to a late class of folate receptors (beta FAR) that have different specificities for pterins. G alpha 4 is preferentially expressed in cells randomly distributed within the aggregate that are a component of the anterior-like cell population, and it is not detectably expressed in prespore cells. Our results suggest that an endogenous factor, possibly a pterin, produced during multicellular development is a requisite signal for multicellular development, acting through G alpha 4. We propose that the G alpha 4-expressing cells function as a regulatory cell type controlling prespore cell fate, possibly in response to an endogenous pterin. Our results indicate that G alpha 4 and G alpha 2 have parallel functions in mediating cellular responses to folate (pterins) and cAMP, respectively.
Molecular cloning of a putative vesicular transporter for acetylcholine.
Authors: Roghani A , Feldman J , Kohan S A , Shirzadi A , Gundersen C B , Brecha N , Edwards R H
Published: May 2006

Classical neurotransmitters such as acetylcholine (ACh) require transport into synaptic vesicles for regulated exocytotic release. The Caenorhabditis elegans gene unc-17 encodes a protein with homology to mammalian transporters that concentrate monoamine neurotransmitters into synaptic vesicles. Mutations in unc-17 protect against organophosphorus toxicity, indicating a role in cholinergic neurotransmission. Using the relationship of unc-17 to the vesicular amine transporters, we first isolated a related sequence from the electric ray Torpedo californica [Torpedo vesicular ACh transporter (TorVAChT)] that is expressed by the electric lobe but not by peripheral tissues. Using the relationship of the Torpedo sequence to unc-17, we then isolated the cDNA for a rat homologue (rVAChT). Northern blot analysis shows expression of these sequences in the basal forebrain, basal ganglia, and spinal cord but not cerebellum or peripheral tissues. In situ hybridization shows expression of rVAChT mRNA in all cholinergic cell groups, including those in the basal forebrain, brainstem, and spinal cord that previously have been shown to express choline acetyltransferase mRNA. The human VAChT gene also localizes to chromosome 10 near the gene for choline acetyltransferase. Taken together, these observations support a role for rVAChT in vesicular ACh transport and indicate its potential as a novel marker for cholinergic neurons.
DNA length, bending, and twisting constraints on IS50 transposition.
Authors: Goryshin IYu  , Kil Y V , Reznikoff W S
Published: May 2006

Transposition is a multistep process in which a transposable element DNA sequence moves from its original genetic location to a new site. Early steps in this process include the formation of a transposition complex in which the end sequences of the transposable element are brought together in a structurally precise fashion through the action of the element-encoded transposase protein and the cleavage of the element free from the adjoining DNA. If transposition complex formation must precede DNA cleavage (or nicking), then changing the length of the donor DNA between closely spaced ends should have dramatic effects on the frequency of the transposition. This question has been examined by studying the effects of altering donor DNA length on IS50 transposition. Donor DNA < or = 64 bp severely impaired transposition. Donor DNA > or = 200 bp demonstrated high transposition frequencies with only modest length dependencies. Constructs with donor DNA lengths between 66 and 174 bp demonstrated a dramatic periodic effect on transposition (periodicity approximately 10.5 bp).
Rabies ribonucleocapsid as an oral immunogen and immunological enhancer.
Authors: Hooper D C , Pierard I , Modelska A , Otvos L , Fu Z F , Koprowski H , Dietzschold B
Published: May 2006

The administration of rabies ribonucleocapsid (RNP) by oral as well as parenteral routes was found to prime specific T cells and elicit N-protein-specific antibodies. per os and intramuscular immunization led to the production of antibodies of the IgA and IgG isotypes, respectively. Mice primed orally with RNP produced significantly enhanced amounts of virus-neutralizing antibody, compared with non-immune controls, upon subsequent parenteral booster immunization with inactivated rabies virus. Thus oral immunization with rabies RNP primed cells capable of mediating a secondary systemic response to rabies virus. The results of experiments in which peptide and protein antigens were administered either physically coupled to or mixed with RNP indicate that RNP has an inherent capacity to enhance immune responses.
Isoform-specific interactions of apolipoprotein E with microtubule-associated protein tau: implications for Alzheimer disease.
Authors: Strittmatter W J , Saunders A M , Goedert M , Weisgraber K H , Dong L M , Jakes R , Huang D Y , Pericak-Vance M , Schmechel D , Roses A D
Published: May 2006

The apolipoprotein E (apoE) type 4 allele (APOE4) is a susceptibility gene for late-onset familial and sporadic Alzheimer disease. ApoE is found in some neurofibrillary tangle-bearing neurons, one of the major pathologic hallmarks of the disease. Neurofibrillary tangles contain paired helical filaments formed from hyperphosphorylated microtubule-associated protein tau. In vitro, tau binds avidly to apoE3, but not to apoE4, forming a bimolecular complex. Tau phosphorylated with a brain extract does not bind either isoform. ApoE3 binds to the microtubule-binding repeat region of tau, which is also the region that is thought to cause self-assembly into the paired helical filament. Binding studies with fragments of ApoE demonstrate that the tau-binding region of apoE3 corresponds to its receptor-binding domain and is distinct from the region that binds lipoprotein particles or beta/A4 peptide. Isoform-specific interactions of apoE with tau may regulate intraneuronal tau metabolism in Alzheimer disease and alter the rate of formation of paired helical filaments and neurofibrillary tangles.
Enteric immunization of mice against influenza with recombinant vaccinia.
Authors: Meitin C A , Bender B S , Small P A
Published: May 2006

Intrajejunal administration to mice of a recombinant vaccinia virus containing the influenza virus hemagglutinin gene induced IgA antibody in nasal, gut, and vaginal secretions. It also induced IgG antibody in serum and cell-mediated immunity. The immunization provided significant protection against an influenza virus challenge. This work suggests that enteric-coated recombinant vaccinia could be an orally administered, inexpensive, multivalent, temperature-stable, safe, and effective vaccine for children that could be particularly useful in developing nations, where multiple injections are not easily administered. Oral administration of vaccines should also reduce children's fear of shots at the doctor's office.
The solubilization of model Alzheimer tangles: reversing the beta-sheet conformation induced by aluminum with silicates.
Authors: Fasman G D , Moore C D
Published: May 2006

Neurofibrillary tangles are one of two lesions found in the brain of Alzheimer disease victims. With synthetic peptide fragments of human neurofilament NF-M17 (Glu-Glu-Lys-Gly-Lys-Ser-Pro- Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly, phosphorylated and unphosphorylated), CD studies were done to examine the effect of sodium orthosilicate on the conformational state produced by Al3+ on fragments of neuronal proteins. Previous studies had shown a conformational transition from alpha-helix and random to beta-pleated sheet upon addition of Al3+ to both phosphorylated and unphosphorylated peptides. If sufficient quantities of Al3+ are added, the peptide precipitates from solution. The ability to reverse or slow the progression of aggregation was examined. Al3+ binding was reversed with 1-2 molar equivalents of sodium orthosilicate (with respect to Al3+), altering the conformation from beta-sheet to random coil and resulting in a CD spectrum similar to that of the initial peptide. The tight binding of the SiO4(4-) with the Al3+ provides the mechanism for this transition. These results provide additional information toward understanding the role of aluminum in the Alzheimer diseased brain and suggest the investigation of the possible use of silicates as a therapeutic agent.
NMR analysis of tRNA acceptor stem microhelices: discriminator base change affects tRNA conformation at the 3' end.
Authors: Puglisi E V , Puglisi J D , Williamson J R , RajBhandary U L
Published: May 2006

An important step in initiation of protein synthesis in Escherichia coli is the specific formylation of the initiator methionyl-tRNA (Met-tRNA) by Met-tRNA transformylase. The determinants for formylation are clustered mostly in the acceptor stem of the initiator tRNA. Here we use NMR spectroscopy to characterize the conformation of two RNA microhelices, which correspond to the acceptor stem of mutants of E. coli initiator tRNA and which differ only at the position corresponding to the "discriminator base" in tRNAs. One of the mutant tRNAs is an extremely poor substrate for Met-tRNA transformylase, whereas the other one is a much better substrate. We show that one microhelix forms a structure in which its 3'-ACCA sequence extends the stacking of the acceptor stem. The other microhelix forms a structure in which its 3'-UCCA sequence folds back such that the 3'-terminal A22 is in close proximity to G1. These results highlight the importance of the discriminator base in determining tRNA conformation at the 3' end. They also suggest a correlation between tRNA structure at the 3' end and its recognition by Met-tRNA transformylase.
A low-affinity estrogen-binding site in pregnant rat uteri: analysis and partial purification.
Authors: Gray W G , Biswas E E , Bashirelahi N , Biswas S B
Published: May 2006

We have identified a low-affinity (type II) estrogen-binding site (EBS) that is expressed at high levels during pregnancy in rat uteri. Although this activity was detectable in nonpregnant rat uteri, it was present in amounts (0.094 pmol/g of uteri) that were severalfold lower than the high-affinity type I estrogen receptor (0.57 pmol/g of uteri). During pregnancy, at 19-20 days of gestation, the low-affinity type II EBS became the major (> or = 88%) estrogen-binding site in rat uteri. The increase in the level of low-affinity EBS (7.9 pmol/g) in uteri was approximately 85-fold with an approximately 20-fold increase in the specific activity (0.39 pmol/mg) of this form, whereas the high-affinity form remained relatively unchanged. We report here a method of purification of type II EBS from pregnant rat uteri and present an analysis of its DNA and steroid-binding properties. Estradiol-binding studies and Scatchard analysis showed that the type II EBS had an apparent estradiol-binding affinity of > or = 24 nM. Gel filtration and SDS/PAGE analysis indicated that the type II EBS was a monomeric 73-kDa protein. The estradiol binding remained apparently uninhibited in the presence of a large excess of tamoxifen, nafoxidine, or dihydrotestosterone. Estradiol, diethylstilbestrol, and quercitin (a type II EBS-specific inhibitor) competed efficiently. The purified low-affinity EBS did not have sequence-specific DNA-binding activity with the estrogen-responsive element, which indicated that it differs in function from the type I estrogen receptor.
Low levels of exogenous histone H1 in yeast cause cell death.
Authors: Miloshev G , Venkov P , van Holde K , Zlatanova J
Published: May 2006

To elucidate the function of lysine-rich histone, yeast cells, which are believed to lack this histone, were transformed with an expression vector carrying the sea urchin histone H1 gene under control of an inducible promoter. Expression of full-length protein was tested by immunoblotting and the intracellular distribution was monitored by immunoelectron microscopy. Even low amounts of exogenous H1 led to dramatic changes in intracellular morphology and cell death. The cells that survived had lost either the plasmid or the ability to express the exogenous protein. Thus, even low amounts of canonical histone H1 are lethal to yeast cells.
The 47-kDa major lipoprotein immunogen of Treponema pallidum is a penicillin-binding protein with carboxypeptidase activity.
Authors: Weigel L M , Radolf J D , Norgard M V
Published: May 2006

The recent model of Treponema pallidum molecular architecture proposes that the vast majority of the bacterium's integral membrane proteins are lipoprotein immunogens anchored in the cytoplasmic membrane while the outer membrane contains only a limited number of surface-exposed transmembrane proteins. This unique model explains, in part, the organism's remarkable ability to evade host immune defenses and establish persistent infection. Our strategy for refining this model involves demonstrating that the physiological functions of treponemal membrane proteins are consistent with their proposed cellular locations. In this study, we used an ampicillin-digoxigenin conjugate to demonstrate by chemiluminescence that the 47-kDa lipoprotein immunogen of T. pallidum (Tpp47) is a penicillin-binding protein. Reexamination of the Tpp47 primary sequence revealed the three amino acid motifs characteristic of penicillin-binding proteins. A recombinant, nonlipidated, soluble form of Tpp47 was used to demonstrate that Tpp47 is a zinc-dependent carboxypeptidase. Escherichia coli expressing Tpp47 was characterized by cell wall abnormalities consistent with altered peptidoglycan biosynthesis. Though the inability to cultivate T. pallidum in vitro and the lack of genetic exchange systems continue to impede treponemal research, this study advances strategies for utilizing E. coli molecular genetics as a means of elucidating the complex relationships between syphilis pathogenesis and T. pallidum membrane biology.
Growth inhibition and induction of differentiation of t(8;21) acute myeloid leukemia cells by the DNA-binding domain of PEBP2 and the AML1/MTG8(ETO)-specific antisense oligonucleotide.
Authors: Sakakura C , Yamaguchi-Iwai Y , Satake M , Bae S C , Takahashi A , Ogawa E , Hagiwara A , Takahashi T , Murakami A , Makino K
Published: May 2006

The translocation from chromosome 8 to chromosome 21, t(8;21), associated with acute myeloid leukemia results in production of an AML1/MTG8(ETO) fusion transcript. The product of the AML1 gene contains an evolutionarily conserved 128-amino acid region referred to as the "Runt domain," which is necessary for binding to DNA at the PEBP2 site. A fragment of the AML1 protein containing mainly the Runt domain and the antisense oligonucleotide complementary to the fusion transcript strongly inhibited the growth and induced differentiation of cell lines derived from acute myeloid leukemia containing t(8;21). These results indicate that the transcriptional regulation through the PEBP2 site is critically important for growth and differentiation of t(8;21) leukemic cells and that the product of the chimeric gene is responsible for the maintenance of the leukemic phenotype.
Isolation of unknown genes from human bone marrow by differential screening and single-pass cDNA sequence determination.
Authors: Orr S L , Hughes T P , Sawyers C L , Kato R M , Quan S G , Williams S P , Witte O N , Hood L
Published: May 2006

A cDNA sequencing project was initiated to characterize gene expression in human bone marrow and develop strategies to isolate novel genes. Forty-eight random DNAs from total human bone marrow were subjected to single-pass DNA sequence analysis to determine a limited complexity of mRNAs expressed in the bone marrow. Overall, 8 cDNAs (17%) showed no similarity to known sequences. Information from DNA sequence analysis was used to develop a differential prescreen to subtract unwanted cDNAs and to enrich for unknown cDNAs. Forty-eight cDNAs that were negative with a complex probe were subject to single-pass DNA sequence determination. Of these prescreened cDNAs, the number of unknown sequences increased to 23 (48%). Unknown cDNAs were also characterized by RNA expression analysis using 25 different human leukemic cell lines. Of 13 unknown cDNAs tested, 10 were expressed in all cell types tested and 3 revealed a hematopoietic lineage-restricted expression pattern. Interestingly, while a total of only 96 bone marrow cDNAs were sequenced, 31 of these cDNAs represent sequences from unknown genes and 12 showed significant similarities to sequences in the data bases. One cDNA revealed a significant similarity to a serine/threonine-protein kinase at the amino acid level (56% identity for 123 amino acids) and may represent a previously unknown kinase. Differential screening techniques coupled with single-pass cDNA sequence analysis may prove to be a powerful and simple technique to examine developmental gene expression.