bluuebunny/crossref_metadata_2025 huggingface dataset:
Cultured cells as a model for amphibian metamorphosis.
Authors: Kanamori A , Brown D D
Published: May 2006

Gene expression screens have been applied to a cultured cell line of Xenopus laevis, XL-177, to isolate genes that are up- and down-regulated in the first 8 h after thyroid hormone (TH) induction. At least 14 up-regulated genes were isolated from TH-induced cells grown in the presence or absence of cycloheximide, an inhibitor of protein synthesis. These genes respond directly to TH as demonstrated by the resistance of up-regulation to protein synthesis inhibition in the cultured cells or in tadpoles. Kinetics of mRNA accumulation after TH induction is similar for these genes, including those that are superinduced by cycloheximide. Their mRNAs start to be up-regulated several hours after TH treatment and reach maximum levels between 8 and 16 h. These genes show up-regulation in one or more tadpole organs in response to exogenous TH. Only a few minimally down-regulated genes were identified. Fourteen of the 20 genes that were found to be up-regulated by TH in tadpole tail are also up-regulated in XL-177 cells. Their up-regulation falls into the same two kinetic patterns in the cultured cells as it does in tadpole tail. Another cell line of X. laevis, XLA, is greatly reduced in its ability to up-regulate the same genes isolated from XL-177 cells and tadpole tails in response to TH. Thus these cell lines make up a model system to examine the interactions of gene expression triggered by TH during amphibian metamorphosis.
Carvedilol, a cardiovascular drug, prevents vascular smooth muscle cell proliferation, migration, and neointimal formation following vascular injury.
Authors: Ohlstein E H , Douglas S A , Sung C P , Yue T L , Louden C , Arleth A , Poste G , Ruffolo R R , Feuerstein G Z
Published: May 2006

Carvedilol is a cardiovascular drug currently used for the treatment of hypertension. Clinical studies have recently demonstrated efficacy in angina and congestive heart failure. Recently, carvedilol has been shown to attenuate oxygen free radical-initiated lipid peroxidation and to inhibit vascular smooth muscle mitogenesis induced by a wide variety of growth factors. These findings are of interest since smooth muscle proliferation and abnormal lipid metabolism are proposed to play an important role in the pathogenesis of atherosclerotic plaque formation and in development of stenotic lesions following vascular injury by balloon angioplasty and coronary artery bypass grafting. On the basis of these observations, the antiproliferative actions of carvedilol have been explored in detail. In human cultured pulmonary artery vascular smooth muscle cells, carvedilol (0.1-10 microM) produced a concentration-dependent inhibition of the mitogenesis stimulated by platelet-derived growth factor, epidermal growth factor, thrombin, and serum, with IC50 values ranging from 0.3 to 2.0 microM. Carvedilol also produced a concentration-dependent inhibition of vascular smooth muscle cell migration induced by platelet-derived growth factor, with an IC50 value of 3 microM. The extensive neointimal formation that occurs following balloon angioplasty of rat carotid arteries was markedly attenuated by carvedilol (1 mg/kg, i.p.; twice daily starting 3 days before angioplasty and continuing until 14 days after angioplasty). Quantitative image analysis demonstrated that carvedilol reduced the neointimal growth following angioplasty by 84% without altering either medial or adventitial cross-sectional areas. These observations indicate that carvedilol may also be effective in the treatment of pathological disorders principally associated with abnormal vascular smooth muscle growth, such as atherosclerosis and acute vascular wall injury induced by angioplasty or coronary artery bypass grafting.
Stimulation by nitric oxide of an NAD linkage to glyceraldehyde-3-phosphate dehydrogenase.
Authors: McDonald L J , Moss J
Published: May 2006

Nitric oxide-stimulated modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by [adenylate-32P]NAD has been interpreted in recent reports as ADP-ribosylation. Incubations of GAPDH with the NO-releasing agent sodium nitroprusside (SNP) and NAD resulted, however, in essentially equal incorporation of radiolabel from the adenine, phosphate, and nicotinamide moieties to the extent of approximately 0.02 mol of NAD.mol of GAPDH-1. Modification of GAPDH by free adenosine 5'-diphosphoribose (ADP-ribose) was only 10% of that by NAD. Exposure of GAPDH modified by NAD in the presence of SNP to HgCl2, which acts at thiol linkages, released two products. Both contained nicotinamide and adenylate but did not cochromatograph with NAD. GAPDH activity was inhibited by SNP in a dose-dependent manner in the presence of NAD. When inhibition was 80%, with 1 mM SNP and 1 mM dithiothreitol, covalent modification with NAD was < 2%. This result is consistent with the conclusion that inhibition of GAPDH activity by SNP in the presence of NAD is due primarily to active-site nitrosylation, as reported by other workers, and is not due to the minor modification with NAD. These results demonstrate that NO-stimulated modification of GAPDH with NAD is not ADP-ribosylation as previously reported but rather is covalent binding of NAD through a NO-dependent thiol intermediate, possibly providing an example of an unexpected, altered reactivity of a nitrosylated protein.
Lipid advanced glycosylation: pathway for lipid oxidation in vivo.
Authors: Bucala R , Makita Z , Koschinsky T , Cerami A , Vlassara H
Published: May 2006

To address potential mechanisms for oxidative modification of lipids in vivo, we investigated the possibility that phospholipids react directly with glucose to form advanced glycosylation end products (AGEs) that then initiate lipid oxidation. Phospholipid-linked AGEs formed readily in vitro, mimicking the absorbance, fluorescence, and immunochemical properties of AGEs that result from advanced glycosylation of proteins. Oxidation of unsaturated fatty acid residues, as assessed by reactive aldehyde formation, occurred at a rate that paralleled the rate of lipid advanced glycosylation. Aminoguanidine, an agent that prevents protein advanced glycosylation, inhibited both lipid advanced glycosylation and oxidative modification. Incubation of low density lipoprotein (LDL) with glucose produced AGE moieties that were attached to both the lipid and the apoprotein components. Oxidized LDL formed concomitantly with AGE-modified LDL. Of significance, AGE ELISA analysis of LDL specimens isolated from diabetic individuals revealed increased levels of both apoprotein- and lipid-linked AGEs when compared to specimens obtained from normal, nondiabetic controls. Circulating levels of oxidized LDL were elevated in diabetic patients and correlated significantly with lipid AGE levels. These data support the concept that AGE oxidation plays an important and perhaps primary role in initiating lipid oxidation in vivo.
Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag.
Authors: Chen Y T , Holcomb C , Moore H P
Published: May 2006

Small GTP-binding proteins of the YPT/SEC4/Rab family have been shown to play an essential role in intracellular membrane trafficking. In mammals, Rab8 and Rab10 are the two small GTP-binding proteins identified so far that are closest to SEC4, an essential gene product involved in post-Golgi constitutive secretion in the yeast Saccharomyces cerevisiae. To study the localization of Rab proteins, we have expressed the cDNAs with an influenza virus hemagglutinin (HA) epitope tag at the N terminus. The feasibility of this method was tested by using yeast SEC4. HA-tagged SEC4 functionally complemented a temperature-sensitive sec4 mutant similarly to wild-type SEC4, indicating that the modified protein retained functional integrity. Monoclonal antibody 12CA5, raised against the HA tag, was used to determine the expression and localization of HA-tagged proteins after transfection. In stably transfected CHO and Swiss 3T3 cells, HA-tagged Rab8 was localized to the cell periphery, with the highest concentration in the ruffling areas. In contrast, epitope-tagged Rab10 expressed in CHO and BHK cells was concentrated on membranes in the perinuclear region. By light microscopy, the staining partially overlapped with that of a Golgi marker, beta-COP. Thus, despite the high degree homology of Rab8 and Rab10 (66% identity), the two proteins are localized to distinct cellular compartments. This approach should provide a general tool for the analyses of other members of the YPT/SEC4/rab gene family.
Disease risk as a cost of outbreeding in the termite Zootermopsis angusticollis.
Authors: Rosengaus R B , Traniello J F
Published: May 2006

The effect of the sibship of primary reproductives on mate mortality and the survivorship and growth of incipient colonies was studied in the dampwood termite Zootermopsis angusticollis. Males and females paired with nonsibling mates had higher mortality during the first 10-40 days after pairing, although male and female reproductives showed similar patterns of mortality after colony establishment. The source of mortality appeared to be fungal and/or bacterial pathogens. There were no overall differences in the number of eggs and larvae produced by sibling and nonsibling pairs, and no differences in colony size and biomass 4 years after colony establishment. We therefore could not identify any negative effect of inbreeding in the early phases of colony development. Our results suggest that the risk of exposure to pathogens and the ability of termites to locally adapt to disease could influence the genetic identity of primary reproductives and the extent of inbreeding in termite populations.
An unusual RNA tertiary interaction has a role for the specific aminoacylation of a transfer RNA.
Authors: Hou Y M , Westhof E , Giegé R
Published: May 2006

The nucleotides in a tRNA that specifically interact with the cognate aminoacyl-tRNA synthetase have been found largely located in the helical stems, the anticodon, or the discriminator base, where they vary from one tRNA to another. The conserved and semiconserved nucleotides that are responsible for the tRNA tertiary structure have been shown to have little role in synthetase recognition. Here we report that aminoacylation of Escherichia coli tRNA(Cys) depends on the anticodon, the discriminator base, and a tertiary interaction between the semiconserved nucleotides at positions 15 and 48. While all other tRNAs contain a purine at position 15 and a complementary pyrimidine at position 48 that establish the tertiary interaction known as the Levitt pair, E. coli tRNA(Cys) has guanosine -15 and -48. Replacement of guanosine -15 or -48 with cytidine virtually eliminates aminoacylation. Structural analyses with chemical probes suggest that guanosine -15 and -48 interact through hydrogen bonds between the exocyclic N-2 and ring N-3 to stabilize the joining of the two long helical stems of the tRNA. This tertiary interaction is different from the traditional base pairing scheme in the Levitt pair, where hydrogen bonds would form between N-1 and O-6. Our results provide evidence for a role of RNA tertiary structure in synthetase recognition.
Cloning and characterization of cyclophilin C-associated protein: a candidate natural cellular ligand for cyclophilin C.
Authors: Friedman J , Trahey M , Weissman I
Published: May 2006

We report the protein purification and the cloning and characterization of a cDNA encoding the proteins that bind with high affinity to cyclophilin C (Cyp-C) in the absence of cyclosporin A. Transfection of this cDNA into COS cells directs the production of a glycoprotein of 77 kDa that binds to Cyp-C in the absence, but not the presence, of cyclosporin A. Homology comparisons reveal that this protein and gene, termed CyCAP for Cyp-C-associated protein, possess a cysteine-rich domain (scavenger receptor cysteine-rich domain) found in a variety of cell-surface molecules; the rest of the sequence is apparently specific. This result raises the possibility that Cyp-C serves as a mediator or regulator of an as-yet-unidentified signal or cellular process initiated via the Cyp-C-associated protein.
The 64-kDa protein that associates with the platelet-derived growth factor receptor beta subunit via Tyr-1009 is the SH2-containing phosphotyrosine phosphatase Syp.
Authors: Kazlauskas A , Feng G S , Pawson T , Valius M
Published: May 2006

Ligand-stimulated autophosphorylation of the platelet-derived growth factor receptor (PDGFR) beta subunit creates a number of binding sites for SH2-containing proteins. One of the PDGFR-associated proteins is a 64-kDa protein of unknown identity and function. We present data indicating that the 64-kDa protein that associates with the activated PDGFR is Syp (also called SH-PTP2, PTP-1D, or SH-PTP3), the ubiquitously expressed 64-kDa SH2-containing protein-tyrosine phosphatase. Phosphorylation of Tyr-1009 in the C terminus of the PDGFR is required for the stable association of Syp, suggesting that phosphorylation of this residue creates a binding site for the Syp SH2 domains. Although Syp stably associates with the PDGFR, this event is not required for PDGF-stimulated tyrosine phosphorylation of Syp. These data raise the interesting possibility that protein-tyrosine phosphatases contribute to the intracellular relay of biological signals originating from receptor tyrosine kinases such as the PDGFR.
Noninactivating, tetrodotoxin-sensitive Na+ conductance in rat optic nerve axons.
Authors: Stys P K , Sontheimer H , Ransom B R , Waxman S G
Published: May 2006

The ionic current underlying the upstroke of axonal action potentials is carried by rapidly activating, voltage-dependent Na+ channels. Termination of the action potential is mediated in part by the rapid inactivation of these Na+ channels. We previously demonstrated that an influx of Na+ plays a critical role in the cascade leading to irreversible anoxic injury in central nervous system white matter. We speculated that a noninactivating Na+ conductance mediates this pathological Na+ influx and persists at depolarized membrane potentials as seen in anoxic axons. In the present study we measured the resting compound membrane potential of rat optic nerves using a modified "grease-gap" technique. Application of tetrodotoxin (2 microM) to resting nerves ([K+]o = 3 mM) or to nerves depolarized by 15 or 40 mM K+ resulted in hyperpolarizing shifts of membrane potential. We interpret these shifts as evidence for a persistent, noninactivating Na+ conductance. This conductance is present at rest and persists in nerves depolarized sufficiently to abolish classical transient Na+ currents. PK/PNa ratios were estimated at 35.5, 23.2, and 88 in 3 mM, 15 mM, and 40 mM K+, respectively. We suggest that this noninactivating Na+ conductance may provide an inward pathway for Na+ ions, necessary for the operation of Na+, K(+)-ATPase. Under pathological conditions, such as anoxia, this conductance is the likely route of Na+ influx, which causes damaging Ca2+ entry through reverse operation of the Na(+)-Ca2+ exchanger. The presence of this conductance in white matter axons may provide a therapeutic opportunity for diseases such as stroke and spinal cord injury.
Identification of four murine cDNAs encoding putative protein kinases from primitive embryonic stem cells differentiated in vitro.
Authors: Biesecker L G , Gottschalk L R , Emerson S G
Published: May 2006

Protein kinases transduce signals from extracellular ligands in the hematopoietic and other systems through direct phosphorylation of tyrosine, serine, or threonine residues. Little is known about the ligands and receptors that are important in the earliest stages of development--i.e., stem cell self-renewal and lineage commitment. We have made use of the lineage differentiation potential of the murine embryonic stem cell system to clone partial cDNAs encoding four putative protein kinases. Three of the four genes contain the highly conserved residues Asp-Phe-Gly in domain VII of the protein kinase family. These genes are candidates for receptors or downstream effectors of cytokines that regulate self-renewal and lineage commitment in embryogenesis.
A dominant negative retinoic acid receptor blocks neutrophil differentiation at the promyelocyte stage.
Authors: Tsai S , Collins S J
Published: May 2006

We have investigated the roles of retinoic acid receptors in the development of neutrophils by using an interleukin 3-dependent multipotent hematopoietic cell line (FDCP mix A4) as well as normal mouse bone marrow cells. Treatment of the FDCP mix A4 cells with murine granulocyte/macrophage-colony-stimulating factor (GM-CSF) induced these cells to differentiate into neutrophils and macrophages. When the endogenous retinoic acid receptor activity in FDCP mix A4 cells was suppressed by a dominant negative retinoic acid receptor construct, this GM-CSF-induced neutrophil differentiation was blocked at the promyelocyte stage. The blocked promyelocytes proliferated continuously as a GM-CSF-dependent cell line but could be induced to terminally differentiate into neutrophils with supraphysiological concentrations of all-trans-retinoic acid (1-10 microM). The ability of the dominant negative retinoic acid receptor to block neutrophil differentiation at the promyelocyte stage was also demonstrated in normal, primary mouse bone marrow cells. Our results indicate that retinoic acid receptors in conjunction with hematopoietic growth factors play a crucial role in the terminal differentiation of normal neutrophil precursors. The system described here may also serve as a model for studying the pathogenesis of human acute promyelocytic leukemia.
A perspective into ligand-receptor affinities using complex numbers.
Authors: Klotz I M
Published: May 2006

The binding of ligands by a receptor can be expressed in an equation derived by a thermodynamic stoichiometric approach in which the stoichiometric equilibrium constants are all positive and real. An alternative binding equation can be derived algebraically in which the equilibrium constants can have values that are complex numbers. Some consequences of this situation are explored, and numerical values of the ghost equilibrium constants are evaluated for a number of ligand-receptor complexes.
Engineering of stable and fast-folding sequences of model proteins.
Authors: Shakhnovich E I , Gutin A M
Published: May 2006

The statistical mechanics of protein folding implies that the best-folding proteins are those that have the native conformation as a pronounced energy minimum. We show that this can be obtained by proper selection of protein sequences and suggest a simple practical way to find these sequences. The statistical mechanics of these proteins with optimized native structure is discussed. These concepts are tested with a simple lattice model of a protein with full enumeration of compact conformations. Selected sequences are shown to have a native state that is very stable and kinetically accessible.
Integration of gene maps: chromosome 21.
Authors: Lawrence S , Collins A , Keats B J , Hulten M , Morton N E
Published: May 2006

Physical, cytogenetic, and genetic data including microsatellite markers and a covering sequence-tagged site (STS) map have been entered into a location database and integrated into a summary map that subsumes a composite physical location, sex-specific genetic locations, cytogenetic and regional assignments, mouse homology, rank, and references. With the omission of 52 loci whose location is known only from cytogenetic assignment to an interval greater than 10 megabases, there are 198 loci in the covering STS map and an additional 145 loci. The physical length is consistent with 11 megabases for 21p and 39 megabases for 21q. With error filtration and allowance for high interference, the genetic length in males corresponds to the chiasma map (54.7 centimorgans), and the genetic length in females is 76.5 centimorgans. The relation between map integration and the STS paradigm is illustrated and discussed.
Protein oxidative damage is associated with life expectancy of houseflies.
Authors: Sohal R S , Agarwal S , Dubey A , Orr W C
Published: May 2006

The objective of this study was to test some of the predictions of the oxidative-stress hypothesis of aging, which postulates that aging is causally associated with the molecular damage inflicted by reactive oxygen species. Protein carbonyl content was used as an index of molecular oxidative modifications. The carbonyl content was found to be associated with the physiological age or life expectancy of flies rather than with their chronological age. Exposure of flies to sublethal hyperoxia (100% oxygen) irreversibly enhanced the carbonyl content of the flies and decreased their rate of oxygen consumption. Results of this study indicate that protein carbonyl content may be a biomarker of aging and support the general concept that oxidative stress may be a causal factor in the aging process.
Rearrangement of sapA homologs with conserved and variable regions in Campylobacter fetus.
Authors: Tummuru M K , Blaser M J
Published: May 2006

The Campylobacter fetus surface-layer (S-layer) proteins mediate both complement resistance and antigenic variation in mammalian hosts. Wild-type strain 23D possesses the sapA gene, which encodes a 97-kDa S-layer protein, and several sapA homologs are present in both wild-type and mutant strains. Here we report that a cloned silent gene (sapA1) in C. fetus can express a functional full-length S-layer protein in Escherichia coli. Analysis of sapA and sapA1 and partial analysis of sapA2 indicate that a block of approximately 600 bp beginning upstream and continuing into the open reading frames is completely conserved, and then the sequences diverge completely, but immediately downstream of each gene is another conserved 50-bp sequence. Conservation of sapA1 among strains, the presence of a putative Chi (RecBCD recognition) site upstream of sapA, sapA1, and sapA2, and the sequence identities of the sapA genes suggest a system for homologous recombination. Comparison of the wild-type strain (23D) with a phenotypic variant (23D-11) indicates that variation is associated with removal of the divergent region of sapA from the expression locus and exchange with a corresponding region from a sapA homolog. We propose that site-specific reciprocal recombination between sapA homologs leads to expression of divergent S-layer proteins as one of the mechanisms that C. fetus uses for antigenic variation.
Distribution of the aquaporin CHIP in secretory and resorptive epithelia and capillary endothelia.
Authors: Nielsen S , Smith B L , Christensen E I , Agre P
Published: May 2006

The existence of water-selective channels has been postulated to explain the high water permeability of erythrocytes and certain epithelial cells. The aquaporin CHIP (channel-forming integral membrane protein of 28 kDa), a molecular water channel, is abundant in erythrocytes and water-permeable segments of the nephron. To determine whether CHIP may mediate transmembrane water movement in other water-permeable epithelia, membranes of multiple organs were studied by immunoblotting, immunohistochemistry, and immunoelectron microscopy using affinity-purified anti-CHIP IgG. The apical membrane of the choroid plexus epithelium was densely stained, implying a role for CHIP in the secretion of cerebrospinal fluid. In the eye, CHIP was abundant in apical and basolateral domains of ciliary epithelium, the site of aqueous humor secretion, and also in lens epithelium and corneal endothelium. CHIP was detected in membranes of hepatic bile ducts and water-resorptive epithelium of gall bladder, suggesting a role in bile secretion and concentration. CHIP was not detected in glandular epithelium of mammary, salivary, or lacrimal glands, suggesting the existence of other water-channel isoforms. CHIP was also not detected within the epithelium of the gastrointestinal mucosa. CHIP was abundant in membranes of intestinal lacteals and continuous capillaries in diverse tissues, including cardiac and skeletal muscle, thus providing a molecular explanation for the known water permeability of certain lymphatics and capillary beds. These studies underscore the hypothesis that CHIP plays a major role in transcellular water movement throughout the body.
All-trans-retinol is a ligand for the retinoic acid receptors.
Authors: Repa J J , Hanson K K , Clagett-Dame M
Published: May 2006

Competition of all-trans-retinol and all-trans-retinaldehyde with 3H-labeled all-trans-retinoic acid (RA) for binding to retinoic acid receptors (RARs) was examined in human neuroblastoma cell nuclear extracts. All-trans-retinol was 35-fold less potent than all-trans-RA, whereas all-trans-retinaldehyde was 500-fold less active in binding to the nuclear receptors. To confirm that all-trans-retinol binds to RARs, experiments were carried out with RARs alpha, beta, and gamma expressed as bacterial fusion proteins. All-trans-retinol was only 4- to 7-fold less potent than all-trans-RA in binding to all three RAR subtypes. The all-trans-retinol binding observed was not the result of metabolism of retinol to RA or some other active compound during the binding experiment. Retinyl acetate was virtually inactive in competition binding experiments, while very slight activity was observed with 13-cis-RA and all-trans-retinaldehyde. Significant competition occurred with 4-hydroxy-RA and 4-keto-RA, which were 15- to 40-fold less potent than all-trans-RA. The 9-cis isomer of RA was equipotent with all-trans-retinol in these studies. These results suggest that all-trans-retinol cannot be excluded as a physiologically significant ligand for RAR-mediated gene expression.
Thyroid hormone-dependent transcriptional regulation of exogenous genes transferred into Xenopus tadpole muscle in vivo.
Authors: de Luze A , Sachs L , Demeneix B
Published: May 2006

Metamorphosis in amphibians is marked by dramatic thyroid hormone-induced changes that include tail regression. To examine thyroid hormone effects on gene transcription during the early stages of tail resorption, we injected exogenous genes directly into the caudal skeletal muscle of Xenopus tadpoles and followed their expression in vivo. Gene expression was both strong and reproducible, and it correlated with the amount of DNA injected. Moreover, expression continued as long as the animals were blocked in prometamorphosis by antithyroid drugs (for up to 4 months). Thyroid hormone-dependent effects on transcription were examined by using a palindromic thyroid hormone response element linked to a chloramphenicol acetyltransferase reporter gene. Reporter gene expressions were normalized for transfection efficiency by using a constitutively expressed luciferase construct. Physiological concentrations of 3,5,3' triiodo-L-thyronine (1 nM), applied for 120 hr, produced a 5-fold increase in transcription (P < 0.05) from the thyroid hormone response element but did not modify transcription from constitutive viral promoters. This study thus demonstrates that by directly expressing genes in Xenopus tadpole muscle in vivo, one can exploit the powerful experimental advantages of gene transfer systems in an intact, physiologically normal animal.
Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a.
Authors: Alexander D , Goodman R M , Gut-Rella M , Glascock C , Weymann K , Friedrich L , Maddox D , Ahl-Goy P , Luntz T , Ward E
Published: May 2006

Expression of pathogenesis-related protein 1a (PR-1a), a protein of unknown biochemical function, is induced to high levels in tobacco in response to pathogen infection. The induction of PR-1a expression is tightly correlated with the onset of systemic acquired resistance (SAR), a defense response effective against a variety of fungal, viral, and bacterial pathogens. While PR-1a has been postulated to be involved in SAR, and is the most highly expressed of the PR proteins, evidence for its role is lacking. In this report, we demonstrate that constitutive high-level expression of PR-1a in transgenic tobacco results in tolerance to infection by two oomycete pathogens, Peronospora tabacina and Phytophthora parasitica var. nicotianae.
Change of apparent stoichiometry of proximal-tubule Na(+)-HCO3- cotransport upon experimental reversal of its orientation.
Authors: Planelles G , Thomas S R , Anagnostopoulos T
Published: May 2006

Electrogenic cotransport of Na+ with HCO3- has been reported in numerous tissues. It has always been shown with a net transfer of negative charge, but in some situations achieves net outward transport of both species with a stoichiometry of at least three HCO3- ions per Na+ ion (3:1), and in other situations achieves net inward transport of both species and has a stoichiometry of at most two HCO3- ions per Na+ ion (2:1). This suggests either that there may be more than one protein responsible for Na(+)-HCO3- cotransport in different tissues or that if there is a single protein, its stoichiometry may differ depending on the orientation of net transport. The present study, using conventional or double-barreled ion-selective microelectrodes to follow basolateral membrane potential and intracellular pH or Na+ activity in Necturus proximal convoluted tubule in vivo, shows that the orientation of the basolateral Na(+)-HCO3- cotransporter can be reversed upon switching from a perfusate simulating normal acid-base conditions to one that imposes peritubular isohydric hypercapnia. Moreover, accompanying the reversal of orientation is a change of apparent stoichiometry from 3:1 to 2:1. Given that the observed change of orientation and accompanying change of apparent stoichiometry occur within seconds and in the same preparation, these results suggest that a single transport protein is responsible for both types of behavior.
Peripheral blood progenitors as a target for genetic correction of p47phox-deficient chronic granulomatous disease.
Authors: Sekhsaria S , Gallin J I , Linton G F , Mallory R M , Mulligan R C , Malech H L
Published: May 2006

Peripheral blood contains hematopoietic progenitors (PBHPs), which can be harvested in clinically relevant amounts by apheresis. PBHPs have been used as a source of progenitors alternative to marrow for autologous transplantation following intensive chemotherapy. We have determined culture conditions for growth and differentiation of PBHPs to the mature myeloid phenotype, which in the present study are employed to demonstrate the functional correction of an inherited disorder of myeloid cells in retrovirus-transduced human primary hematopoietic progenitors. Patients with chronic granulomatous disease (CGD) suffer from recurrent life-threatening infections because blood phagocytes fail to produce microbicidal superoxide (O2-.). One-third of the cases of CGD result from defects in the gene encoding p47phox, a cytoplasmic oxidase component required for O2-. generation. In the present study, a replication-defective retrovirus encoding p47phox was used to transduce PBHPs from patients with p47phox-deficient CGD, which resulted in significant correction of O2-. generation when PBHPs were differentiated to mature neutrophils and monocytes. This study provides a model for use of PBHPs in development of gene therapy for diseases affecting bone marrow.
Molecular mechanics calculations of the structures of polyamide nucleic acid DNA duplexes and triple helical hybrids.
Authors: Almarsson O , Bruice T C , Kerr J , Zuckermann R N
Published: May 2006

Polyamide nucleic acids (PNAs) have emerged as useful agents for recognition of single- and double-stranded nucleic acids. Interresidue hydrogen bonds between the amide carbonyl nearest the nucleobase and chain NH moieties provide inherent stability to the helical conformation of PNA 1. Moving the amide carbonyl away from the nucleobase to the backbone, and replacing it with a methylene group, results in 2 lacking the stabilizing hydrogen bond. Oligomers of 2 do not interact with DNA. Modeling suggests that 2 displays a more extended conformation than 1, and nucleobase orientation is disrupted in 2 in the absence of a complementary DNA strand. This is in contrast to 1, which retains a centrosymmetric arrangement of nucleobases. Structures for 1-T10.DNA and (1-T10)2.DNA species spanned by a pyrimidine strand (D-loop) were constructed. In the triple helical (1-T10)2.DNA structure, the two PNA strands form the complementary Watson-Crick paired strand and the Hoogsteen base-paired strand in the major groove of the 1.DNA duplex. The PNA strands are proposed to bind antiparallel to one another in (1-T10)2.DNA structure. The factors suggested to account for the stability of this 2:1 complex are (i) a hydrophobic attraction between two PNA backbones and (ii) a favorable electrostatic effect resulting from replacement of a phosphodiester backbone by a neutral peptide backbone.
Molecular cloning and expression of a 90-kDa diacylglycerol kinase that predominantly localizes in neurons.
Authors: Goto K , Kondo H
Published: May 2006

A diacylglycerol kinase cDNA was isolated from a rat brain cDNA library. This cDNA encoded an 801-amino acid protein of 90,287 Da. This 90-kDa diacylglycerol kinase showed 58% identity in deduced amino acid sequence with a previously isolated rat 80-kDa diacylglycerol kinase. EF-hand motifs, cysteine-rich zinc-finger-like sequences, and putative ATP-binding sites were all conserved between the two kinase species. However, mRNA encoding the 90-kDa kinase was confined to restricted neuronal populations such as the caudate-putamen, the accumbens nucleus, and the olfactory tubercle. Further, the 90-kDa kinase was found to exhibit high phosphorylation activity for long-chain diacylglycerols and was mainly associated with the membrane fraction when the cDNA was transfected into COS-7 cells.
Replication of UV-irradiated DNA in human cell extracts: evidence for mutagenic bypass of pyrimidine dimers.
Authors: Thomas D C , Kunkel T A
Published: May 2006

We have examined the efficiency and fidelity of simian virus 40-origin-dependent replication of UV-irradiated double-stranded DNA in extracts of human cells. Using as a mutational target the alpha-complementation domain of the Escherichia coli lacZ gene in bacteriophage M13mp2 DNA, replication of undamaged DNA in HeLa cell extracts was highly accurate, whereas replication of DNA irradiated with UV light (280-320 nm) was both less efficient and less accurate. Replication was inhibited by irradiation in a dose-dependent manner. Nonetheless, covalently closed, monomer-length circular products were generated that were resistant to digestion by Dpn I, showing that they resulted from semiconservative replication. These products were incised by T4 endonuclease V, whereas the undamaged replication products were not, suggesting that pyrimidine dimers were bypassed during replication. When replicated, UV-irradiated DNA was used to transfect an E. coli alpha-complementation host strain to score mutant M13mp2 plaques, the mutant plaque frequency was substantially higher than that obtained with either unirradiated, replicated DNA, or unreplicated, UV-irradiated DNA. Both the increased mutagenicity and the inhibition of replication associated with UV irradiation were reversed by treatment of the irradiated DNA with photolyase before replication. Sequence analysis of mutants resulting from replication of UV-irradiated DNA demonstrated that most mutants contained C-->T transition errors at dipyrimidine sites. A few mutants contained 1-nt frameshift errors or tandem double CC-->TT substitutions. The data are consistent with the interpretation that pyrimidine dimers are bypassed during replication by the multiprotein replication apparatus in human cell extracts and that this bypass is mutagenic primarily via misincorporation of dAMP opposite a cytosine (or uracil) in the dimer.
Females have a larger hippocampus than males in the brood-parasitic brown-headed cowbird.
Authors: Sherry D F , Forbes M R , Khurgel M , Ivy G O
Published: May 2006

Females of the brood-parasitic brown-headed cowbird (Molothrus ater) search for host nests in which to lay their eggs. Females normally return to lay a single egg from one to several days after first locating a potential host nest and lay up to 40 eggs in a breeding season. Male brown-headed cowbirds do not assist females in locating nests. We predicted that the spatial abilities required to locate and return accurately to host nests may have produced a sex difference in the size of the hippocampal complex in cowbirds, in favor of females. The size of the hippocampal complex, relative to size of the telencephalon, was found to be greater in female than in male cowbirds. No sex difference was found in two closely related nonparasitic icterines, the red-winged blackbird (Agelaius phoeniceus) and the common grackle (Quiscalus quiscula). Other differences among these species in parental care, migration, foraging, and diet are unlikely to have produced the sex difference attributed to search for host nests by female cowbirds. This is one of few indications, in any species, of greater specialization for spatial ability in females and confirms that use of space, rather than sex, breeding system, or foraging behavior per se, can influence the relative size of the hippocampus.
Acute mixed-lineage leukemia t(4;11)(q21;q23) generates an MLL-AF4 fusion product.
Authors: Domer P H , Fakharzadeh S S , Chen C S , Jockel J , Johansen L , Silverman G A , Kersey J H , Korsmeyer S J
Published: May 2006

A chromosomal translocation, t(4;11)-(q21;q23), is associated with an aggressive mixed-lineage leukemia. A yeast artificial chromosome was used to clone the chromosomal breakpoint of this translocation in the RS4;11 cell line. The breakpoint sequences revealed an inverted repeat bordered by a consensus site for topoisomerase II binding and cleavage as well as chi-like elements. The der(11) chromosome encodes a fusion RNA and predicted chimeric protein between the 11q23 gene MLL and a 4q21 gene designated AF4. The sequence of the complete open reading frame for this fusion transcript reveals the MLL protein to have homology with DNA methyltransferase, the Drosophila trithorax gene product, and the "AT-hook" motif of high-mobility-group proteins. An alternative splice that deletes the AT-hook region of MLL was identified. AF4 is a serine- and proline-rich putative transcription factor with a glutamine-rich carboxyl terminus. The composition of the complete MLL-AF4 fusion product argues that it may act through either a gain-of-function or a dominant negative mechanism in leukemogenesis.
Probing the function of Drosophila melanogaster accessory glands by directed cell ablation.
Authors: Kalb J M , DiBenedetto A J , Wolfner M F
Published: May 2006

The female Drosophila melanogaster fly undergoes behavioral changes after mating, including an increase in egg laying and an avoidance of remating. Accessory-gland products elicit these changes transiently when introduced into unmated female flies. We report here the generation and phenotype of flies that lack functional accessory-gland main cells as a consequence of genetically directed delivery of diphtheria toxin subunit A to those cells. Only main-cell secretions are essential for the short-term inhibition to remating; no other products of the genital tract can replace their function. Long-term inhibition to remating depends only on the storage of sperm in the female. Both sperm and main-cell secretions have roles in the increase of egg laying by the mated female. In addition to full-strength diphtheria toxin, we used low-activity toxins to kill only those cells that express toxin at high levels. These transgenic strains that express diphtheria toxins of different strengths in accessory-gland main cells will be useful in further defining the role of these cells.
Production of unmodified human adult hemoglobin in Escherichia coli.
Authors: Shen T J , Ho N T , Simplaceanu V , Zou M , Green B N , Tam M F , Ho C
Published: May 2006

We have constructed a plasmid (pHE2) in which the synthetic human alpha- and beta-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters. The Hbs were expressed in E. coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b. Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed alpha and beta chains of component a, respectively, have the expected masses. The remaining 10% of the beta chain in component a corresponds in mass to the beta chain plus methionine. In component b, both alpha and beta chains have the correct masses without detectable N-terminal methionine (< 2%). These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the alpha and beta chains of these two recombinant Hb (rHb) samples. rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A). Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values. 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the beta chain. These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form. Thus, our E. coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.
At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide.
Authors: Bukh J , Purcell R H , Miller R H
Published: May 2006

In a previous study we sequenced the 5' noncoding (NC) region of 44 isolates of hepatitis C virus (HCV) and identified heterogeneous domains that provided evidence for additional genetic groups of HCV not previously recognized. In this study we have determined the complete nucleotide sequence of the putative envelope 1 (E1) gene in 51 HCV isolates from around the world and found that they could be grouped into at least 12 distinct genotypes. The E1 gene sequence of 8 of these genotypes has not been reported previously. Although the genetic relatedness of HCV isolates determined by the previous analysis of the 5' NC region predicted the relationships observed in the E1 gene, analysis of the 5' NC sequence alone did not accurately predict all HCV genotypes. The nucleotide and amino acid sequence identities of the E1 gene among HCV isolates of the same genotype were in the range of 88.0-99.1% and 89.1-98.4%, respectively, whereas those of HCV isolates of different genotypes were in the range of 53.5-78.6% and 49.0-82.8%, respectively. The latter differences are similar to those found when comparing the envelope gene sequences of the various serotypes of the related flaviviruses as well as other RNA viruses. We found that some genotypes of HCV were widely distributed around the world, whereas others were identified only in discreet geographical regions. Four genotypes were identified exclusively in Africa and comprised the majority of HCV isolates on that continent. The E1 gene was exactly 576 nucleotides in length in all 51 HCV isolates with no in-frame stop codons. Analysis of the predicted E1 protein identified several conserved domains that may be important for maintaining its biological function: (i) eight invariant cysteine residues, (ii) three potential N-linked glycosylation sites, (iii) a domain of nine amino acids (GHRMAWDMM), and (iv) an amino acid doublet (GV) near the putative cleavage site at the C terminus of the protein. In conclusion, the discovery of at least 12 genotypes of HCV has important implications for HCV diagnosis and vaccine development.
Day/night and circadian rhythm control of con gene expression in Neurospora.
Authors: Lauter F R , Yanofsky C
Published: May 2006

In the filamentous fungus Neurospora crassa, several events in the process of conidiation are influenced by light. Two genes, con-6 and con-10, which were previously shown to be transcriptionally activated during conidiation and by exposure to light, were found to be unexpressed in mycelium maintained in constant darkness or in constant light. However, when mycelium was shifted from darkness to light, transcripts of both genes appeared and were abundant. Upon further illumination both transcripts disappeared--i.e., their continued production was light repressed. When dark-grown mycelium was exposed to a light pulse and reincubated in the dark, expression of con-6 and con-10 exhibited a 20-hr circadian periodicity. Both genes were photoinducible throughout the stages of the circadian cycle. In the mutant strains bd and bd;frq9, con-6 and con-10 were light inducible but were not normally light repressible. Mutant genes such as acon-2, acon-3, and fl that block developmental expression of con-6 and/or con-10 did not prevent their photoinduction.
Mechanism of protein kinase C activation during the induction and maintenance of long-term potentiation probed using a selective peptide substrate.
Authors: Klann E , Chen S J , Sweatt J D
Published: May 2006

Previous reports using various protein kinase inhibitors have suggested that protein kinase activity is necessary for both the induction and maintenance of hippocampal long-term potentiation (LTP), a cellular phenomenon likely to contribute to mammalian memory formation. We designed and characterized a selective peptide substrate for protein kinase C (PKC), corresponding to amino acids 28 to 43 of the neuronal protein neurogranin, and used the substrate to obtain direct biochemical evidence for activation of PKC in both the induction and maintenance phases of LTP. As the effect cannot be accounted for by either of two well-known mechanisms for persistent PKC activation, membrane insertion, or proteolysis, the persistent activation of PKC in the maintenance phase of LTP appears to occur via another mechanism. The maintenance phase of LTP is associated with decreased immunoreactivity of PKC, an effect that can be reversed with phosphatase treatment. Thus, PKC appears to be both phosphorylated and persistently activated in the maintenance phase of LTP.
Guanosine binding to the Tetrahymena ribozyme: thermodynamic coupling with oligonucleotide binding.
Authors: McConnell T S , Cech T R , Herschlag D
Published: May 2006

The L-21 Sca I ribozyme derived from the group I intron of Tetrahymena thermophila pre-rRNA catalyzes an endonuclease reaction analogous to the first step of self-splicing. Guanosine (G) is bound by the ribozyme, and its 3'-hydroxyl group acts as the nucleophile. Here, we provide evidence that Km for G in several single-turnover reactions is equal to the equilibrium dissociation constant for G. This evidence includes the observation that removal of the 2'-hydroxyl group at the cleavage site of the oligoribonucleotide substrate [from CCCUCUA to CCCUC(dU)A] decreases the rate of cleavage approximately 1000-fold but has no effect on either the Km for G (0.17 mM) or for guanosine 5'-monophosphate (pG) (0.09 mM). In the course of this study, it was observed that Km for G or pG was lower by a factor of 5 for reactions with the ribozyme-CCCUC(dU)A complex compared with the free ribozyme, indicating a modest amount of thermodynamic coupled binding of the two substrates. The decrease in the rate of oligonucleotide dissociation upon addition of saturating pG provides independent support for this coupling. Coupling is lost with a substrate that cannot make the normal tertiary interactions with the ribozyme, providing evidence that coupled binding requires docking of the substrate into the catalytic core. Surprisingly, the binding of product CCCUCU and G is slightly anticooperative, indicating that the cleaved pA is important for coupling with substrate. Coupled binding suggests a splicing model in which the intron binds G tightly to promote the first step of reaction, after which its binding is an order of magnitude weaker, thereby facilitating the second step.