bluuebunny/crossref_metadata_2025 huggingface dataset:
Phospholipase D-catalyzed hydrolysis of phosphatidylcholine provides the choline precursor for acetylcholine synthesis in a human neuronal cell line.
Authors: Lee H C , Fellenz-Maloney M P , Liscovitch M , Blusztajn J K
Published: May 2006

To identify the metabolic pathway that generates choline (Cho) for acetylcholine (AcCho) from its storage pool in membrane phosphatidylcholine (PtdCho), human neuronal cells (LA-N-2) were radioisotopically labeled with 1-O-hexadecyl-2-hydroxy-sn-glycero(3)phospho[14C]choline. The compound was efficiently taken up by the cells and metabolically labelled PtdCho, Cho, AcCho, and phosphocholine pools. In pulse-chase experiments, the specific radioactivities of the metabolites of 1-O-hexadecyl-2-hydroxy-sn-glycero(3)-phospho[14C]choline indicated that it was rapidly acylated to Ptd-Cho and then hydrolyzed first to free Cho and not to phosphocholine or glycerophosphocholine. This Cho was subsequently converted to AcCho and to phosphocholine. In the absence of exogenous Cho, at least 15% of the total cellular AcCho pool was synthesized by this pathway in 1 h. The data demonstrate that the liberation of the free Cho precursor for AcCho synthesis from PtdCho can be accomplished in a one-step process, indicating the involvement of a phospholipase D-type enzyme. In the presence of hemicholinium-3, which inhibits Cho transport, the amount of intracellular [14C]Cho metabolites that accumulated during the chase period was higher than in control cells, indicating that PtdCho hydrolysis liberated Cho directly into the cytoplasm. These data show that cholinergic cells are characterized by an intracellular pathway, catalyzed by a phospholipase D, that generates Cho for AcCho synthesis from PtdCho. Abnormalities in the regulation of this pathway may contribute to selective vulnerability of cholinergic neurons in certain neurodegenerative diseases, e.g., Alzheimer disease.
Lymphocytes and macrophages outnumber oligodendroglia in normal fish spinal cord.
Authors: Dowding A J , Scholes J
Published: May 2006

As shown by staining with a monoclonal antibody against fish CD45, leukocytes are present in very large numbers in the fish central nervous system. Their subtypes were distinguished by electron microscopy and found to include all major hematogenous forms except thrombocytes, the most numerous being tissue macrophages and lymphocytes. As a population, they differ fundamentally from ramified microglia, the restricted form of myeloid cells present in the central nervous system in mammals. They are rare in most grey matter regions but are concentrated in myelinated fiber tracts as well as in certain strata of the radial glial network. The macrophages engulf discarded myelin and outnumber the oligodendrocytes in normal spinal cord white matter, where the density of lymphocytes is > 5000-fold greater than reported in rat.
Essential role of hypothalamic muscarinic and alpha-adrenergic receptors in atrial natriuretic peptide release induced by blood volume expansion.
Authors: Antunes-Rodrigues J , Marubayashi U , Favaretto A L , Gutkowska J , McCann S M
Published: May 2006

Expansion of the blood volume induces natriuresis, which tends to return the blood volume to normal. This response is mediated at least in part by the release of atrial natriuretic peptide (ANP) into the circulation. Previous experiments have shown the participation of the anterior ventral third ventricular (AV3V) region of the hypothalamus in the ANP release that follows volume expansion. When injected into the AV3V region, the cholinergic drug carbachol induces natriuresis and the release of ANP. In the present experiments, microinjection of norepinephrine into the AV3V region induced natriuresis and an increase in plasma ANP. To determine whether cholinergic and alpha-adrenergic pathways are crucial to the volume expansion-induced release of ANP, certain receptor-blocking drugs were injected into the AV3V region of conscious rats. Thirty minutes later blood volume was expanded by intravenous injection of 2.0 ml/100 g of body weight of hypertonic saline (0.3 M NaCl). Microinjection of isotonic saline (2 microliters) into AV3V region of control animals 30 min prior to volume expansion had no effect on the 3-fold increase in plasma ANP concentrations measured 5 min after volume expansion. In contrast, although the receptor-blocking drugs did not alter the initial concentrations of plasma ANP 30 min later, just prior to volume expansion, blockade of muscarinic cholinergic receptors by intraventricular injection of 5 nmol (2 microliters) of atropine sulfate or methylatropine markedly reduced the response to volume expansion but did not obliterate it. Microinjection of the alpha receptor blocker phentolamine (5 nmol) into the AV3V 30 min prior to volume expansion also markedly suppressed the ANP response. Intraperitoneal (i.p.) injection of methylatropine (0.01 mmol/100 g of body weight), which does not cross the blood-brain barrier, also did not affect the basal levels of ANP 30 min after i.p. injection. But, in striking contrast with the blockade of the response to volume expansion induced by intraventricular injection of methylatropine, the response to volume expansion was markedly enhanced by i.p. injection of methylatropine. The results therefore indicate that hypothalamic muscarinic and alpha-adrenergic synapses are essential to release of ANP in response to volume expansion. These results are consistent with a hypothetical pathway for physiological control of ANP release which involves distension of baroreceptors within the right atria, carotid and aortic sinuses, and kidney which alters afferent input to brain stem noradrenergic neurons with axons projecting to the AV3V region. There they activate cholinergic interneurons by an alpha 1-adrenergic synapse. The cholinergic neurons in turn stimulate ANP neurons in this brain region via muscarinic receptors. The stimulation of these neurons activates efferent pathways which induce the release of ANP.
Arabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for indole-3-acetic acid.
Authors: Normanly J , Cohen J D , Fink G R
Published: May 2006

We used tryptophan auxotrophs of the dicot Arabidopsis thaliana (wall cress) to determine whether tryptophan has the capacity to serve as a precursor to the auxin, indole-3-acetic acid (IAA). Quantitative gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS) revealed that the trp2-1 mutant, which is defective in the conversion of indole to tryptophan, accumulated amide- and ester-linked IAA at levels 38-fold and 19-fold, respectively, above those of the wild type. Tryptophan and free IAA were isolated from the trp2-1 mutant grown in the presence of [15N]anthranilate and [2H5]tryptophan, and the relative 15N and 2H5 enrichments of tryptophan and IAA were determined via GC-SIM-MS. The 15N enrichment of tryptophan, 13% +/- 4%, was less than the 15N enrichment of the IAA pool, 39% +/- 4%; therefore, IAA biosynthesis occurs via a tryptophan-independent pathway. The amount of 2H5 incorporated by the plant into IAA from tryptophan (9% +/- 4%) was low and only slightly above the level of spontaneous, nonenzymatic conversion of [2H5]tryptophan to [2H5]IAA. These results show that the dicot Arabidopsis is similar to the monocot Zea mays in that the major route of IAA biosynthesis does not occur through tryptophan.
Shape and motion from image streams: a factorization method.
Authors: Tomasi C , Kanade T
Published: May 2006

Inferring scene geometry and camera motion from a stream of images is possible in principle, but it is an ill-conditioned problem when the objects are distant with respect to their size. We have developed a factorization method that can overcome this difficulty by recovering shape and motion without computing depth as an intermediate step. An image stream can be represented by the 2F x P measurement matrix of the image coordinates of P points tracked through F frames. Under orthographic projection this matrix is of rank 3. Using this observation, the factorization method uses the singular value decomposition technique to factor the measurement matrix into two matrices, which represent object shape and camera motion, respectively. The method can also handle and obtain a full solution from a partially filled-in measurement matrix, which occurs when features appear and disappear in the image sequence due to occlusions or tracking failures. The method gives accurate results and does not introduce smoothing in either shape or motion. We demonstrate this with a series of experiments on laboratory and outdoor image streams, with and without occlusions.
Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity.
Authors: Dawson T M , Steiner J P , Dawson V L , Dinerman J L , Uhl G R , Snyder S H
Published: May 2006

Immunosuppressants FK506 and cyclosporin A inhibit neurotoxicity of N-methyl-D-aspartate in primary cortical cultures, while having no effect on quisqualate- and kainate-mediated neurotoxicity. Rapamycin completely reverses the neuroprotective effect of FK506. Both FK506 and cyclosporin A inhibit NMDA-elicited/nitric oxide-mediated increases in cGMP levels in cortical cultures. FK506 has no effect on sodium nitroprusside-induced increases in cGMP. In a stably transfected human kidney 293 cell line overexpressing the gene encoding nitric oxide synthase [L-arginine, NADPH:oxygen oxidoreductase (nitric oxide-forming), EC 1.14.13.39], FK506 inhibits the calcium ionophore A23187, stimulated increases in nitrite (a breakdown product of nitric oxide), and potentiates phorbol ester-mediated inhibition of nitrite formation. FK506-mediated inhibition of nitric oxide formation is completely reversed by rapamycin. Calcineurin dephosphorylates protein kinase C-mediated phosphorylation of nitric oxide synthase. FK506 prevents the calcineurin-mediated dephosphorylation of nitric oxide synthase and thereby diminishes the enzyme's catalytic activity. These data establish nitric oxide synthase as a calcineurin substrate. Nitric oxide synthase catalytic activity is regulated by the phosphorylation state of the enzyme. Enhanced phosphorylation of nitric oxide synthase diminishes catalytic activity, and dephosphorylation (through activation of calcineurin) enhances catalytic activity. The neuroprotective effect of FK506 and cyclosporin A presumably involves the inhibition of calcineurin, preventing the dephosphorylation of nitric oxide synthase and its subsequent activation.
Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species.
Authors: Wink D A , Hanbauer I , Krishna M C , DeGraff W , Gamson J , Mitchell J B
Published: May 2006

Nitric oxide, NO, which is generated by various components of the immune system, has been presumed to be cytotoxic. However, NO has been proposed to be protective against cellular damage resulting during ischemia reperfusion. Along with NO there is often concomitant formation of superoxide/hydrogen peroxide, and hence a synergistic relationship between the cytotoxic effects of nitric oxide and these active oxygen species is frequently assumed. To study more carefully the potential synergy between NO and active oxygen species in mammalian cell cytotoxicity, we utilized either hypoxanthine/xanthine cell cytotoxicity, we utilized either hypoxanthine/xanthine oxidase (a system that generates superoxide/hydrogen peroxide) or hydrogen peroxide itself. NO generation was accomplished by the use of a class of compounds known as "NONOates," which release NO at ambient temperatures without the requirement of enzyme activation or biotransformation. When Chinese hamster lung fibroblasts (V79 cells) were exposed to hypoxanthine/xanthine oxidase for various times or increasing amounts of hydrogen peroxide, there was a dose-dependent decrease in survival of V79 cells as measured by clonogenic assays. However, in the presence of NO released from (C2H5)2N[N(O)NO]-Na+ (DEA/NO), the cytotoxicity resulting from superoxide or hydrogen peroxide was markedly abrogated. Similarly, primary cultures of rat mesencephalic dopaminergic cells exposed either to hydrogen peroxide or to hypoxanthine/xanthine oxidase resulted in the degradation of the dopamine uptake and release mechanism. As was observed in the case of the V79 cells, the presence of NO essentially abrogated this peroxide-mediated cytotoxic effect on mesencephalic cells.
Mutants defective in the energy-conserving NADH dehydrogenase of Salmonella typhimurium identified by a decrease in energy-dependent proteolysis after carbon starvation.
Authors: Archer C D , Wang X , Elliott T
Published: May 2006

NADH dehydrogenase is the first component of the respiratory chain. It transfers electrons from NADH to ubiquinone and concomitantly establishes a proton motive force across the membrane. Salmonella typhimurium mutants defective in this enzyme were isolated in a screen for strains with increased expression of beta-galactosidase from a hemA-lacZ protein fusion. This unexpected phenotype results from stabilization of the hybrid protein during carbon starvation and is apparently due to an energy requirement for proteolytic attack. Sequence analysis of DNA fragments cloned from an insertion mutant indicates that S. typhimurium has a large cluster of genes encoding the energy-conserving NADH dehydrogenase, similar to one recently described in Paracoccus denitrificans. These findings establish the potential for genetic analysis of a complex enzyme whose function, especially in proton efflux, is poorly understood.
Antisense inhibition of the p65 subunit of NF-kappa B blocks tumorigenicity and causes tumor regression.
Authors: Higgins K A , Perez J R , Coleman T A , Dorshkind K , McComas W A , Sarmiento U M , Rosen C A , Narayanan R
Published: May 2006

The NF-kappa B transcription factor, composed of two proteins, p50 and p65, is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Various cell adhesion molecules have NF-kappa B binding sites and may play an important role in inflammatory response, tumorigenicity, and metastasis. In an earlier study, we demonstrated that adhesion of diverse transformed cells was blocked by antisense inhibition of the p65 subunit of NF-kappa B. Since cell-substratum interactions play an important role in tumorigenicity, we reasoned that antisense p65 could inhibit tumorigenicity. In diverse transformed cell lines, phosphorothioate antisense oligonucleotides to p65 inhibited in vitro growth, reduced soft-agar colony formation, and eliminated the ability of cells to adhere to an extracellular matrix. Stable transfectants of a fibrosarcoma cell line expressing dexamethasone-inducible antisense RNA to p65 showed inhibition of in vitro growth and in vivo tumor development. In response to inducible expression of antisense RNA, a pronounced tumor regression was seen in nude mice. The administration of antisense but not sense p65 oligonucleotides caused a pronounced inhibition of tumorigenicity in nude mice injected with diverse tumor-derived cell lines. Inhibitors of NF-kappa B function may thus be useful in the treatment of cancer.
Lipopolysaccharide-binding protein as a major plasma protein responsible for endotoxemic shock.
Authors: Gallay P , Heumann D , Le Roy D , Barras C , Glauser M P
Published: May 2006

Because lipopolysaccharide (LPS)-binding protein (LBP) sensitizes monocytes to LPS in vitro, it has been suggested that LBP initiates host defenses against Gram-negative bacteria. The role of LBP in vivo, and particularly in endotoxemic shock, is unknown, however. Therefore an IgG against murine LBP was prepared. It was found to neutralize binding of LPS and subsequent activation of murine macrophages in vitro. This anti-LBP protected mice against the lethal effect of LPS when given at the same time as LPS challenge, but it failed to protect mice when delayed 15 min after LPS challenge. The same preparation was also effective after challenge with lipid A but not after challenge with Staphylococcus aureus enterotoxin. The protection was correlated with a strong decrease of circulating tumor necrosis factor. These data demonstrate that in vivo LBP is a major mediator of the lethal effects of endotoxemia.
Three-dimensional model for the hormone binding domains of steroid receptors.
Authors: Goldstein R A , Katzenellenbogen J A , Luthey-Schulten Z A , Seielstad D A , Wolynes P G
Published: May 2006

We have used a motif-based structural search method to identify structural homologs of the hormone binding domains of the nuclear receptors from among a set of known protein structures and have found the closest similarity with members of the subtilisin-like serine proteases. These proteins consist of an open twisted sheet of parallel beta-strands flanked on both sides by alpha-helices. The alignment with the protease scaffold was refined by using multiple sequence prealignment of different sets of nuclear receptors, and alternative model structures were screened by considering their consistency with the results of biochemical experiments defining the ligand binding pocket. In the most favored model, nearly all of the residues thought to be involved in ligand binding map to a pocket of appropriate dimensions where the subtilisin-like proteases have their active site. The three-dimensional model that we propose for the hormone binding domains of the nuclear receptors provides a framework for the design of experiments to further investigate nuclear receptor structure and function.
Cloning and pharmacological characterization of a rat kappa opioid receptor.
Authors: Meng F , Xie G X , Thompson R C , Mansour A , Goldstein A , Watson S J , Akil H
Published: May 2006

A full-length cDNA was isolated from a rat striatal library by using low-stringency screening with two PCR fragments, one spanning transmembrane domains 3-6 of the mouse delta opioid receptor and the other unidentified but homologous to the mouse delta receptor from rat brain. The novel cDNA had a long open reading frame encoding a protein of 380 residues with 59% identity to the mouse delta receptor and topography consistent with a seven-helix guanine nucleotide-binding protein-coupled receptor. COS-1 cells transfected with the coding region of this clone showed high-affinity binding to kappa opioid receptor-selective ligands such as dynorphin A and U-50,488 and also nonselective opioid ligands such as bremazocine, ethylketocyclazocine, and naloxone. Not bound at all (or bound with low affinity) were dynorphin A-(2-13), enantiomers of naloxone and levophanol [i.e., (+)-naloxone and dextrorphan], and selective mu and delta opioid receptor ligands. Activation of the expressed receptor by kappa receptor agonists led to inhibition of cAMP. Finally, in situ hybridization revealed a mRNA distribution in rat brain that corresponded well to the distribution of binding sites labeled with kappa-selective ligands. These observations indicate that we have cloned a cDNA encoding a rat kappa receptor of the kappa 1 subtype.
Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.
Authors: Smit L S , Wilkinson D J , Mansoura M K , Collins F S , Dawson D C
Published: May 2006

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the traffic ATPase superfamily, possesses two putative nucleotide-binding folds (NBFs). The NBFs are sufficiently similar that sequence alignment of highly conserved regions can be used to identify analogous residues in the two domains. To determine whether this structural homology is paralleled in function, we compared the activation of chloride conductance by forskolin and 3-isobutyl-1-methylxanthine in Xenopus oocytes expressing CFTRs bearing mutations in NBF1 or NBF2. Mutation of a conserved glycine in the putative linker domain in either NBF produced virtually identical changes in the sensitivity of chloride conductance to activating conditions, and mutation of this site in both NBFs produced additive effects, suggesting that in the two NBFs this region plays a similar and critical role in the activation process. In contrast, amino acid substitutions in the Walker A and B motifs, thought to form an integral part of the nucleotide-binding pockets, produced strikingly different effects in NBF1 and NBF2. Substitutions for the conserved lysine (Walker A) or aspartate (Walker B) in NBF1 resulted in a marked decrease in sensitivity to activation, whereas the same changes in NBF2 produced an increase in sensitivity. These results are consistent with a model for the activation of CFTR in which both NBF1 and NBF2 are required for normal function but in which either the nature or the exact consequences of nucleotide binding differ for the two domains.
Cell-specific action and mutable structure of a transcription factor effector domain.
Authors: Monuki E S , Kuhn R , Lemke G
Published: May 2006

POU proteins are cell-specific transcription factors whose specificity of action has been attributed to protein-DNA and protein-protein interactions mediated by their DNA-binding (POU) domains. Here we report that transcriptional activation by SCIP, a POU protein expressed by developing Schwann cells, is dependent on an amino-terminal effector domain and that this domain mediates cell-specific transactivation in the complete absence of the POU domain. When fused to a heterologous DNA-binding domain, this SCIP domain is a potent transactivator in Schwann cells but is inactive in three heterologous cell types. The primary structure of the SCIP amino-terminal domain is novel but contains a polymorphic string of alanine residues similar to those found in several other transcription factors. Although previously hypothesized to be important for transcription factor activity, we find that the SCIP string is functionally irrelevant. We propose that homopolymers of alanine, and certain other amino acids, do not represent a motif required for transcription factor function but instead reflect regions of unstable DNA related to those associated with four recently characterized human genetic disorders.
Relationship between intrinsic connections and functional architecture revealed by optical imaging and in vivo targeted biocytin injections in primate striate cortex.
Authors: Malach R , Amir Y , Harel M , Grinvald A
Published: May 2006

In primate primary visual cortex, neurons sharing similar response properties are clustered together forming functional domains that appear as a mosaic of patches or bands, often traversing the entire cortical depth from the pia to the white matter. Similarly, each cortical site connects laterally through an extensive network of intrinsic projections that are organized in multiple clusters (patches) and reach distances of up to a few millimeters. The relationship between the functional domains and these laterally connected patches has remained a controversial issue despite intensive research efforts. To investigate this relationship, we obtained high-resolution functional maps of the cortical architecture by in vivo optical imaging. Subsequently, extracellular injections of the sensitive anterograde tracer biocytin were targeted into selected functional domains. Within the ocular dominance system, we found that long-range intrinsic connections tended to link the monocular regions of same-eye ocular dominance columns. Furthermore, we discovered that binocular domains formed a separate set of connections in area V1; binocular regions were selectively connected among themselves but were not connected to strictly monocular regions, suggesting that they constitute a distinct columnar system. In the other subsystem subserving orientation preference, patches of intrinsic connections tended to link domains sharing similar orientation preferences. Analyses of the precision of these connections indicated that in both functional subsystems, < 15% of the connections were between domains having orthogonal response properties. However, their selectivity was limited; approximately 30% +/- 10% of the interconnected patches contained neurons exhibiting orientation tuning that differed from those found at the injection sites by at least 45 degrees. At short range (up to 400 microns from the injection site), this casual trend seemed markedly accentuated; the local, synaptic-rich axonal and dendritic arbors crossed freely through columns of diverse functional properties. These complex sets of connections can endow cortical neurons with a rich diversity of response properties and broad tuning.
Neonatal imprinting predetermines the sexually dimorphic, estrogen-dependent expression of galanin in luteinizing hormone-releasing hormone neurons.
Authors: Merchenthaler I , Lennard D E , López F J , Negro-Vilar A
Published: May 2006

The incidence of colocalization of galanin (GAL) in luteinizing hormone-releasing hormone (LHRH) neurons is 4- to 5-fold higher in female than male rats. This fact and the finding that the degree of colocalization parallels estradiol levels during the estrous cycle suggest that GAL is an estrogen-inducible product in a subset of LHRH neurons. To analyze further this paradigm we evaluated the effects of gonadectomy and steroid replacement therapy in male and female rats. Ovariectomy resulted in a significant decrease in the number of cells colocalizing LHRH and GAL, whereas estradiol replacement to such animals restored the incidence of colocalization to that observed in controls. In males, however, estradiol treatment failed to enhance the incidence of colocalization of GAL and LHRH, indicating, therefore, that the colocalization of these peptides is gender-determined. This possibility--i.e., gender-specific determination of LHRH neurons coexpressing GAL--was evaluated by neonatal manipulation of hypothalamic steroid imprinting. As mentioned above, male rats did not respond to estrogen or testosterone by increasing GAL/LHRH colocalization as females did. Neonatally orchidectomized rats, whose hypothalami have not been exposed to testosterone during the critical period, when treated with estrogen in adulthood showed an increase in colocalization of GAL and LHRH similar to that seen in female animals. These observations indicate that the colocalization of LHRH/GAL is neonatally determined by an epigenetic mechanism that involves the testis. In summary, this sex difference in the incidence of colocalization of GAL and LHRH represents a unique aspect of sexual differentiation in that only certain phenotypic characteristics of a certain cellular lineage are dimorphic. The subpopulation of LHRH neurons that also produces GAL represents a portion of the LHRH neuronal system that is sexually differentiated and programed to integrate, under steroidal control, a network of LHRH neurons that could synchronize their activity to control the estrous cycle in rats.
Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies.
Authors: Prusiner S B , Groth D , Serban A , Koehler R , Foster D , Torchia M , Burton D , Yang S L , DeArmond S J
Published: May 2006

Mice, homozygous for prion protein (PrP) gene ablation (Prn-p0/0), develop normally and remain well > 500 days after inoculation with murine scrapie prions. In contrast, wild-type mice developed scrapie < 165 days after inoculation and most Prn-p0/+ mice, heterozygous for disruption of the PrP gene, exhibited signs of central nervous system dysfunction between 400 and 465 days after inoculation. In situ immunoblots showed widespread deposition of scrapie PrP (PrPSc) in the brains of both wild-type Prn-p+/+ and Prn-p0/+ mice, while neither cellular PrP (PrPC) nor PrPSc was detected in the brains of Prn-p0/0 mice. In contrast to Prn-p+/+ and Prn-p0/+ mice, Prn-p0/0 mice failed to propagate prion infectivity as measured by bioassays. Syrian hamster (SHa) PrP transgenes rendered Prn-p0/0 mice susceptible to prions containing SHaPrPSc. Immunization of Prn-p0/0 mice with purified, infectious mouse or SHa prions dispersed in Freund's adjuvant produced antisera that bound mouse, SHa, and human PrP on Western blots. Presumably, the lack of PrPC expression in Prn-p0/0 mice prevents them from becoming tolerant to the immunogen. The resistance of Prn-p0/0 mice to developing scrapie after inoculation with murine prions supports the hypothesis that PrPSc is essential for both transmission and pathogenesis of the prion diseases.
Expression and biochemical properties of a protein serine/threonine phosphatase encoded by bacteriophage lambda.
Authors: Barik S
Published: May 2006

The predicted amino acid sequence encoded by the open reading frame 221 (orf221) of bacteriophage lambda exhibited a high degree of similarity to the catalytic subunits of a variety of protein serine/threonine phosphatases belonging to PP1, PP2A, and PP2B groups. Cloning and expression of the orf221 gene in Escherichia coli provided direct evidence that the gene codes for a protein serine/threonine phosphatase. The single-subunit recombinant enzyme was purified in soluble form and shown to possess a unique repertoire of biochemical properties--e.g., an absolute requirement for Mn2+, resistance to okadaic acid, inhibitors 1 and 2, and ability to dephosphorylate casein, adenovirus E1A proteins, and the alpha subunit of phosphorylase kinase. No phosphotyrosine phosphatase activity was observed. Mutational and biochemical analyses identified the conserved residues 73-77 and Cys138 to be important for activity. The name PP-lambda is proposed for this unusual prokaryotic enzyme.
The classical human phosphoglucomutase (PGM1) isozyme polymorphism is generated by intragenic recombination.
Authors: March R E , Putt W , Hollyoake M , Ives J H , Lovegrove J U , Hopkinson D A , Edwards Y H , Whitehouse D B
Published: May 2006

The molecular basis of the classical human phosphoglucomutase 1 (PGM1) isozyme polymorphism has been established. In 1964, when this genetic polymorphism was first described, two common allelozymes PGM1 and PGM1 2 were identified by starch gel electrophoresis. The PGM1 2 isozyme showed a greater anodal electrophoretic mobility than PGM1 1. Subsequently, it was found that each of these allelozymes could be split, by isoelectric focusing, into two subtypes; the acidic isozymes were given the suffix + and the basic isozymes were given the suffix -. Hence, four genetically distinct isozymes 1+, 1-, 2+, and 2- were identified. We have now analyzed the whole of the coding region of the human PGM1 gene by DNA sequencing in individuals of known PGM1 protein phenotype. Only two mutations have been found, both C to T transitions, at nt 723 and 1320. The mutation at position 723, which changes the amino acid sequence from Arg to Cys at residue 220, showed complete association with the PGM1 2/1 protein polymorphism: DNA from individuals showing the PGM1 1 isozyme carried the Arg codon CGT, whereas individuals showing the PGM1 2 isozyme carried the Cys codon TGT. Similarly, the mutation at position 1320, which leads to a Tyr to His substitution at residue 419, showed complete association with the PGM1+/- protein polymorphism: individuals with the + isozyme carried the Tyr codon TAT, whereas individuals with the - isozyme carried the His codon CAT. The charge changes predicted by these amino acid substitutions are entirely consistent with the charge intervals calculated from the isoelectric profiles of these four PGM1 isozymes. We therefore conclude that the mutations are solely responsible for the classical PGM1 protein polymorphism. Thus, our findings strongly support the view that only two point mutations are involved in the generation of the four common alleles and that one allele must have arisen by homologous intragenic recombination between these mutation sites.
Thymocyte development in major histocompatibility complex-deficient mice: evidence for stochastic commitment to the CD4 and CD8 lineages.
Authors: Crump A L , Grusby M J , Glimcher L H , Cantor H
Published: May 2006

The mechanism resulting in commitment of precursor cells in the thymus to either the CD4 or CD8 lineage remains poorly understood. In principle, this may reflect a stochastic process or may reflect instructional signals from host major histocompatibility complex (MHC) molecules. We have examined the role of MHC products in subset commitment by using mice deficient in class I or class II MHC products. Normal numbers of committed CD4 intermediates (CD4+ CD8lo) develop in the thymus in the absence of class II molecules. Similarly, CD8 transitional cells (CD4loCD8+) are present in the thymus of mice lacking class I products. These findings suggest that commitment of CD4+8+ precursor cells to either lineage is a stochastic process that does not depend on instructive signals from MHC molecules (i.e., expression of alternative differentiative options by uncommitted precursor cells is independent of this environmental signal). These studies also suggest that an interaction between the T-cell antigen receptor (TCR) and MHC molecules that is independent of CD4/CD8 coreceptor engagement enhances stochastic coreceptor downregulation substantially and leads to upregulation of TCR expression as a prelude to selective events that require joint coreceptor/TCR engagement. We suggest that this initial interaction molds the TCR repertoire of stochastically generated T-cell subsets toward recognition of self-MHC products.
Direct transfer of transforming growth factor beta 1 gene into arteries stimulates fibrocellular hyperplasia.
Authors: Nabel E G , Shum L , Pompili V J , Yang Z Y , San H , Shu H B , Liptay S , Gold L , Gordon D , Derynck R
Published: May 2006

The arterial wall responds to thrombosis or mechanical injury through the induction of specific gene products that increase cellular proliferation and connective tissue formation. These changes result in intimal hyperplasia that is observed in restenosis and the early phases of atherosclerosis. Transforming growth factor beta 1 (TGF-beta 1) is a secreted multi-functional protein that plays an important role in embryonal development and in repair following tissue injury. However, the function of TGF-beta 1 in vascular cell growth in vivo has not been defined. In this report, we have evaluated the role of TGF-beta 1 in the pathophysiology of intimal and medial hyperplasia by gene transfer of an expression plasmid encoding active TGF-beta 1 into porcine arteries. Expression of TGF-beta 1 in normal arteries resulted in substantial extracellular matrix production accompanied by intimal and medial hyperplasia. Increased procollagen, collagen, and proteoglycan synthesis in the neointima was demonstrated by immunohistochemistry relative to control transfected arteries. Expression of TGF-beta 1 induced a distinctly different program of gene expression and biologic response from the platelet-derived growth factor B (PDGF B) gene: procollagen synthesis induced by TGF-beta 1 was greater, and cellular proliferation was less prominent. These findings show that TGF-beta 1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo and could play a reparative role in the response to arterial injury.
Nitric oxide synthases reveal a role for calmodulin in controlling electron transfer.
Authors: Abu-Soud H M , Stuehr D J
Published: May 2006

Nitric oxide (NO) is synthesized within the immune, vascular, and nervous systems, where it acts as a wide-ranging mediator of mammalian physiology. The NO synthases (EC 1.14.13.39) isolated from neurons or endothelium are calmodulin dependent. Calmodulin binds reversibly to neuronal NO synthase in response to elevated Ca2+, triggering its NO production by an unknown mechanism. Here we show that calmodulin binding allows NADPH-derived electrons to pass onto the heme group of neuronal NO synthase. Calmodulin-triggered electron transfer to heme was independent of substrate binding, caused rapid enzymatic oxidation of NADPH in the presence of O2, and was required for NO synthesis. An NO synthase isolated from cytokine-induced macrophages that contains tightly bound calmodulin catalyzed spontaneous electron transfer to its heme, consistent with bound calmodulin also enabling electron transfer within this isoform. Together, these results provide a basis for how calmodulin may regulate NO synthesis. The ability of calmodulin to trigger electron transfer within an enzyme is unexpected and represents an additional function for calcium-binding proteins in biology.
Cloning of glycoprotein D cDNA, which encodes the major subunit of the Duffy blood group system and the receptor for the Plasmodium vivax malaria parasite.
Authors: Chaudhuri A , Polyakova J , Zbrzezna V , Williams K , Gulati S , Pogo A O
Published: May 2006

cDNA clones encoding the major subunit of the Duffy blood group were isolated from a human bone marrow cDNA library using a PCR-amplified DNA fragment encoding an internal peptide sequence of glycoprotein D (gpD) protein. The open reading frame of the 1267-bp cDNA clone indicated that gpD protein was composed of 338 amino acids, predicting a M(r) of 35,733, which was the same as a deglycosylated gpD protein. Portions of the predicted amino acid sequence, matched with six CNBr/pepsin peptides obtained from affinity-purified gpD protein. In ELISA analysis, an anti-Duffy murine monoclonal antibody reacted with a synthetic peptide deduced from the cDNA clone. Hydropathy analysis suggested the presence of 9 membrane-spanning alpha-helices. In bone marrow RNA blot analysis, the gpD cDNA detected a 1.27-kb mRNA in Duffy-positive but not in Duffy-negative individuals. It also identified the same size mRNA in adult kidney, adult spleen, and fetal liver; in brain, it detected a prominent 8.5-kb and a minor 2.2-kb mRNA. In Southern blot analysis, gpD cDNA identified a single gene in Duffy-positive and -negative individuals. Duffy-negative individuals, therefore, have the gpD gene, but it is not expressed in bone marrow. The same or a similar gene is active in adult kidney, adult spleen, and fetal liver of Duffy-positive individuals. Whether this is true in Duffy-negative individuals remains to be demonstrated. A GenBank sequence search yielded a significant protein sequence homology to human and rabbit interleukin-8 receptors.
Myelin protein zero gene mutated in Charcot-Marie-tooth type 1B patients.
Authors: Su Y , Brooks D G , Li L , Lepercq J , Trofatter J A , Ravetch J V , Lebo R V
Published: May 2006

Autosomal dominant of Charcot-Marie-Tooth disease (CMT), whose gene is type 1B (CMT1B), has slow nerve conduction with demyelinated Schwann cells. In this study the abundant peripheral myelin protein zero (MPZ) gene, MPZ, was mapped 130 kb centromeric to the Fc receptor immunoglobulin gene cluster in band 1q22, and a major MPZ point mutation was found to cosegregate with CMT1B in one large CMT1B family. The MPZ point mutation in 18 of 18 related CMT1B pedigree 1 patients converts a positively charged lysine in codon 96 to a negatively charged glutamate. The same MPZ locus cosegregates with the CMT1B disease gene in a second CMT1B family [total multipoint logarithm of odds (lod) = 11.4 at theta = 0.00] with a splice junction mutation. Both mutations occur in MPZ protein regions otherwise conserved identically in human, rat, and cow since these species diverged 100 million years ago. MPZ protein, expressed exclusively in myelinated peripheral nerve Schwann cells, constitutes > 50% of myelin protein. These mutations are anticipated to disrupt homophilic MPZ binding and result in CMT1B peripheral nerve demyelination.
Transcriptional organization of a 450-kb region of the human X chromosome in Xq28.
Authors: Bione S , Tamanini F , Maestrini E , Tribioli C , Poustka A , Torri G , Rivella S , Toniolo D
Published: May 2006

In this paper, we report the transcriptional organization of a 450-kb gene cluster in Xq28, flanked by the glucose-6-phosphate dehydrogenase and the color vision genes. CpG islands previously identified and mapped to distal Xq28 have helped in construction of a continuous contig of cosmids and in identification of cDNAs corresponding to eight transcripts. Thirteen to 16 small genes with CpG islands are clustered in a region of 250-300 kb. Many are highly expressed in muscle or brain and may be the genes responsible for muscle or neurological disorders mapped to distal Xq28. Our analysis indicates that, in this region of the genome, genes not related in sequence are organized in transcriptional domains of 100 kb and that this organization may be important for establishing and regulating gene expression in relation to tissue distribution and X chromosome inactivation.
A difference Fourier-transform infrared study of two redox-active tyrosine residues in photosystem II.
Authors: MacDonald G M , Bixby K A , Barry B A
Published: May 2006

Photosystem II, the photosynthetic water-oxidizing complex, contains two redox-active tyrosine residues. Although current models suggest that these tyrosines are located in symmetric positions in the reaction center, there are functional differences between them. To elucidate those structural factors that give rise to this functional asymmetry, we have used difference Fourier-transform infrared spectroscopy to obtain the vibrational difference spectrum associated with the oxidation of each of these redox-active residues. Isotopic labeling was employed to definitively assign vibrational lines to the redox-active tyrosines. This work has shown that the vibrational spectra of the two redox-active species are significantly different from each other. This result suggests that the structure of the redox-active residue helps to determine its role in electron transfer in the reaction center.
Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice.
Authors: Wikenheiser K A , Vorbroker D K , Rice W R , Clark J C , Bachurski C J , Oie H K , Whitsett J A
Published: May 2006

Murine lung epithelial (MLE) cell lines representing the distal bronchiolar and alveolar epithelium were produced from lung tumors generated in transgenic mice harboring the viral oncogene simian virus 40 (SV40) large tumor antigen under transcriptional control of a promoter region from the human surfactant protein C (SP-C) gene. The cell lines exhibited rapid growth, lack of contact inhibition, and an epithelial cell morphology for 30-40 passages in culture. Microvilli, cytoplasmic multivesicular bodies, and multilamellar inclusion bodies (morphologic characteristics of alveolar type II cells) were detected in some of the MLE cell lines by electron microscopic analysis. The MLE cells also maintained functional characteristics of distal respiratory epithelial cells including the expression of surfactant proteins and mRNAs and the ability to secrete phospholipids. Expression of the exogenous SV40 large tumor antigen gene was detected in all of the generated cell lines. The SP-C/SV40 large tumor antigen transgenic mice and the MLE cell lines will be useful for the study of pulmonary surfactant production and regulation as well as lung development and tumorigenesis.
A fertility region on the Y chromosome of Drosophila melanogaster encodes a dynein microtubule motor.
Authors: Gepner J , Hays T S
Published: May 2006

A clone encoding a portion of the highly conserved ATP-binding domain of a dynein heavy-chain polypeptide was mapped to a region of the Drosophila melanogaster Y chromosome. Dyneins are large multisubunit enzymes that utilize the hydrolysis of ATP to move along microtubules. They were first identified as the motors that provide the force for flagellar and ciliary bending. Seven different dynein heavy-chain genes have been identified in D. melanogaster by PCR. In the present study, we demonstrate that one of the dynein genes, Dhc-Yh3, is located in Y chromosome region h3, which is contained within kl-5, a locus required for male fertility. The PCR clone derived from Dhc-Yh3 is 85% identical to the corresponding region of the beta heavy chain of sea urchin flagellar dynein but only 53% identical to a cytoplasmic dynein heavy chain from Drosophila. In situ hybridization to Drosophila testes shows Dhc-Yh3 is expressed in wild-type males but not in males missing the kl-5 region. These results are consistent with the hypothesis that the Y chromosome is needed for male fertility because it contains conventional genes that function during spermiogenesis.
Visual performance in behaving cats after prenatal unilateral enucleation.
Authors: Bisti S , Trimarchi C
Published: May 2006

Prenatal unilateral enucleation in mammals causes an extensive anatomical reorganization of visual pathways. The remaining eye innervates the entire extent of visual subcortical and cortical areas. Electrophysiological recordings have shown that the retino-geniculate connections are retinotopically organized and geniculate neurones have normal receptive field properties. In area 17 all neurons respond to stimulation of the remaining eye and retinotopy, orientation columns, and direction selectivity are maintained. The only detectable change is a reduction in receptive field size. Are these changes reflected in the visual behavior? We studied visual performance in cats unilaterally enucleated 3 weeks before birth (gestational age at enucleation, 39-42 days). We tested behaviorally the development of visual acuity and, in the adult, the extension of the visual field and the contrast sensitivity. We found no difference between prenatal monocularly enucleated cats and controls in their ability to orient to targets in different positions of the visual field or in their visual acuity (at any age). The major difference between enucleated and control animals was in contrast sensitivity:prenatal enucleated cats present a loss in sensitivity for gratings of low spatial frequency (below 0.5 cycle per degree) as well as a slight increase in sensitivity at middle frequencies. We conclude that prenatal unilateral enucleation causes a selective change in the spatial performance of the remaining eye. We suggest that this change is the result of a reduction in the number of neurones with large receptive fields, possibly due to a severe impairment of the Y system.
Altered phosphorylation of growth-associated protein B50/GAP-43 in Alzheimer disease with high neurofibrillary tangle density.
Authors: Martzen M R , Nagy A , Coleman P D , Zwiers H
Published: May 2006

The growth-associated phosphoprotein B50/GAP-43, associated with axonal proliferation and regeneration, was isolated from superior temporal gyrus (area 22) of seven control and eight Alzheimer disease (AD) postmortem human brains. Membrane and cytoplasmic proteins were fractionated and B50/GAP-43 was isolated by reverse-phase HPLC and gel electrophoresis. B50/GAP-43 was identified with rabbit polyclonal antibodies 4P3 (generated against the calmodulin binding domain of B50/GAP-43) and 1B5 (generated against whole bovine B50/GAP-43). B50/GAP-43 protein was further separated into phosphorylated and dephosphorylated species by calmodulin-Sepharose chromatography. The amounts of phosphorylated and dephosphorylated B50/GAP-43 forms were determined by electrophoresis, protein staining, and densitometry. Data on the relative phosphorylation of B50/GAP-43 protein in membrane and cytoplasmic fractions show a 10-fold difference in the ratio of cytoplasmic/membrane phosphorylation of B50/GAP-43 in AD brains with high neurofibrillary tangle (NFT) density compared to AD brains with low NFT density. This difference is due to a decreased percentage of phosphorylated B50/GAP-43 in the membrane fraction relative to that in the cytosolic fraction from high NFT density. No analogous relationship was found between the phosphorylation of B50/GAP-43 and the density of neuritic plaques in the brains examined. These data indicate differential distribution of phosphorylated and dephosphorylated B50/GAP-43 in normal and AD brains is related to NFT density but not to neuritic plaque density.