bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific inhibition of the T-cell response to myelin basic protein by the synthetic copolymer Cop 1.
Authors: Teitelbaum D , Aharoni R , Arnon R , Sela M
Published: May 2006

Cop 1 is a synthetic basic random copolymer of L-alanine, L-glutamic acid, L-lysine, and L-tyrosine in a residue molar ratio of 6.0:1.9:4.7:1.0 and with a molecular weight of 21,000 which proved to be effective in specific suppression of experimental allergic encephalomyelitis and has been proposed as a candidate drug against multiple sclerosis. In the present study we further investigated the mechanism of Cop 1 suppressive activity and tested whether Cop 1 could inhibit the specific T-cell response to myelin basic protein (BP). Eight BP-specific T-cell lines and clones with various H-2 restrictions and antigenic specificities were used. The responses of all these lines and clones to BP, as followed by both cell proliferation and interleukin 2 secretion assays, were affected by Cop 1. For one line, a direct cross proliferation with Cop 1 was observed, whereas in the other seven lines and clones, Cop 1 specifically inhibited the responses to BP in a competitive dose-dependent manner. The inhibition of the response to BP is specific to Cop 1, as D-Cop 1 and another random acidic polymer, poly(Tyr,Glu,Ala) (TGA), both of which were previously demonstrated to be ineffective in suppression of experimental allergic encephalomyelitis, did not inhibit the response to BP. Furthermore, Cop 1 specifically inhibited only the response of the T-cell lines and clones to BP. It did not inhibit their response to the mitogen Con A, nor did it inhibit the responses of the purified protein derivative-specific T-cell line and clone. These results suggest that Cop 1 may be effective in suppression of experimental allergic encephalomyelitis, not only because of the selective stimulation of suppressor T cells, as we have previously demonstrated, but also by specific inhibition of BP-specific effector T cells.
Synthesis of phosphatidylethanol--a potential marker for adult males at risk for alcoholism.
Authors: Mueller G C , Fleming M F , LeMahieu M A , Lybrand G S , Barry K J
Published: May 2006

Phosphatidylethanol, whose synthesis is catalyzed by a phospholipase D in a transphosphatidylation reaction, is a unique metabolite of ethanol. Phorbol 12-tetradecanoate 13-acetate, a tumor-promoting phorbol ester and stimulator of protein kinase C, activates this enzyme in peripheral blood lymphocytes. This system has been developed into an assay for measuring the potential of this pathway in human subjects. A pilot study of phosphatidylethanol synthesis in lymphocytes of adult males who have both an alcohol dependency and a family history of alcoholism has revealed that the average potential for phosphatidylethanol synthesis in this population is significantly elevated over that of control subjects.
Endothelial cell growth factor and ionophore A23187 stimulation of production of inositol phosphates in porcine aorta endothelial cells.
Authors: Moscat J , Moreno F , Herrero C , López C , García-Barreno P
Published: May 2006

The existence of a bovine brain-derived endothelial cell growth factor has recently been reported, but its mode of action is unknown. We show that the endothelial cell growth factor is a potent stimulant of inositol monophosphate release in porcine aorta endothelial cells. Although the activation of phospholipase C by this factor does not appear to be dependent on Ca2+, the Ca2+ ionophore A23187 stimulates release of inositol phosphates. It is suggested that the inositol 1,4,5-trisphosphate 3-kinase/5-phosphomonoesterase pathway could account for the ionophore-induced changes in inositol 1,3,4-triphosphate.
Complete amino acid sequence of human plasma Zn-alpha 2-glycoprotein and its homology to histocompatibility antigens.
Authors: Araki T , Gejyo F , Takagaki K , Haupt H , Schwick H G , Bürgi W , Marti T , Schaller J , Rickli E , Brossmer R
Published: May 2006

In the present study the complete amino acid sequence of human plasma Zn-alpha 2-glycoprotein was determined. This protein whose biological function is unknown consists of a single polypeptide chain of 276 amino acid residues including 8 tryptophan residues and has a pyroglutamyl residue at the amino terminus. The location of the two disulfide bonds in the polypeptide chain was also established. The three glycans, whose structure was elucidated with the aid of 500 MHz 1H NMR spectroscopy, were sialylated N-biantennas. The molecular weight calculated from the polypeptide and carbohydrate structure is 38,478, which is close to the reported value of approximately equal to 41,000 based on physicochemical measurements. The predicted secondary structure appeared to be comprised of 23% alpha-helix, 27% beta-sheet, and 22% beta-turns. The three N-glycans were found to be located in beta-turn regions. An unexpected finding was made by computer analysis of the sequence data; this revealed that Zn-alpha 2-glycoprotein is closely related to antigens of the major histocompatibility complex in amino acid sequence and in domain structure. There was an unusually high degree of sequence homology with the alpha chains of class I histocompatibility antigens. Moreover, this plasma protein was shown to be a member of the immunoglobulin gene superfamily. Zn-alpha 2-glycoprotein appears to be a truncated secretory major histocompatibility complex-related molecule, and it may have a role in the expression of the immune response.
Applications of the twist difference to DNA structural analysis.
Authors: White J H , Bauer W R
Published: May 2006

The twist is a fundamental geometric property of nucleic acids. Calculation of the twist in the most general case requires detailed specification of the three-dimensional path of each strand, but many important cases may be analyzed by considering only the twist difference. If C1, C2, and C3 are three distinct space curves, the twist difference about C1 is defined as Tw(C3, C1) - Tw(C2, C1). We show here that this difference measures the rotation of the correspondence surface joining C1 to C2 about the correspondence surface joining C1 to C3. This result has application to DNA containing local nonuniformities, such as denatured regions, cruciforms, and other altered structures. It also facilitates the calculation of twist for three-stranded structures, including D-loops in mitochondrial DNA and replication and transcription intermediates. The twist difference may also be used to simplify greatly the analysis of twist changes in duplex DNA due to winding on surfaces, such as histones and certain enzymes. In such cases the strand-axis twist of DNA divides into two independent terms. The first term arises from the twist of the local reference frame, and the second arises from the rotation of either strand about the duplex axis as measured in the local reference frame. Twist changes consequent to nucleosome winding, for example, arise from the twist of the nucleosome axis, a straight line, about the DNA axis plus the rotation of either strand of the DNA about its axis in the reference frame of the cylinder.
Protein comparison and classification: a differential geometric approach.
Authors: Rackovsky S , Goldstein D A
Published: May 2006

A method is proposed for rapidly, quantitatively comparing protein structures of arbitrary sizes, based on the differential geometric representation. The method is applied to a group of 22 protein x-ray structures, and the resulting network of closest relationships is delineated. Several well-known fold types are automatically detected as groupings of related structures, even when the constituent proteins are of different lengths. A complete gradation of types is shown to be detected, ranging from all-helical to all-beta-structure proteins. A relationship among functionally similar proteins is shown in several cases, even where their three-dimensional structures differ. It is suggested that the positions of proteins within the network of relationships correspond with their folding mechanisms.
cDNA cloning and primary structure of a white-face hornet venom allergen, antigen 5.
Authors: Fang K S , Vitale M , Fehlner P , King T P
Published: May 2006

A major allergen of white-face hornet (Dolichovespula maculata) venom is antigen 5 (also designated Dol m V). We have determined the primary structures of two forms of this protein by cDNA and protein sequencings. These two forms with 204 and 205 amino acid residues differ in 23% of their sequences but they are antigenically similar. Both forms have sequence similarity with a pathogenesis-related protein of tobacco leaf. In a 130-residue overlap of these proteins, 35-39 residues were identical. Hornet antigen 5 cDNAs were isolated from an expression library in lambda gt11 phage using antibody probes. Several of the cDNAs were not full length, but the fusion fragments expressed were immunoreactive. These results suggest that antigenic determinants of the sequential type are distributed throughout the entire molecule of antigen 5. After subcloning, antigen 5 was also expressed in pKK233-2 plasmid.
Synaptic transmission between rat cerebellar granule and Purkinje cells in dissociated cell culture: effects of excitatory-amino acid transmitter antagonists.
Authors: Hirano T , Hagiwara S
Published: May 2006

Monosynaptic excitatory connections between cerebellar granule and Purkinje cells were studied in dissociated cell cultures, and identification of the transmitter and the postsynaptic receptor at this synapse was pharmacologically investigated. The presynaptic granule cell and the postsynaptic Purkinje cell were voltage- or current-clamped simultaneously, and the excitatory postsynaptic current induced by the granule cell was examined. The neurons and monosynaptic excitatory connections were identified as in our earlier study. Several pairs of granule and Purkinje cells were stained with Lucifer yellow and propidium iodide, respectively, and their morphology was examined after electrophysiological recording. The monosynaptic excitatory postsynaptic current was suppressed by 1 mM kynurenate, an antagonist for excitatory-amino acid receptors, but was little affected by 0.2 mM DL-2-amino-5-phosphonovalerate, a selective antagonist of N-methyl-D-aspartate receptors. Glutamate and aspartate induced inward current in the Purkinje cells. These currents were suppressed by kynurenate at 1 mM. DL-2-Amino-5-phosphonovalerate at 0.2 mM suppressed the inward current induced by 100 microM aspartate but did not affect the inward current induced by 10 microM glutamate. These results are consistent with the idea that glutamate, or a glutamate-like substance, but not aspartate is the transmitter released at the synapse between granule and Purkinje cells and that non-N-methyl-D-aspartate receptor channels are functioning in the postsynaptic membrane.
Somatostatin depresses excitability in neurons of the solitary tract complex through hyperpolarization and augmentation of IM, a non-inactivating voltage-dependent outward current blocked by muscarinic agonists.
Authors: Jacquin T , Champagnat J , Madamba S , Denavit-Saubié M , Siggins G R
Published: May 2006

The synaptic function of somatostatin-containing fibers in the nervous system is controversial. Therefore, we used a slice preparation of the rat brain stem to test the electrophysiological effects of prosomatostatin-derived peptides on neurons of the solitary tract complex, which contains an abundance of somatostatin-containing fibers and cell bodies. Superfusion of both somatostatin-14 and somatostatin-28 (the precursor for somatostatin-14), but not somatostatin-28-(1-12) or -(1-10), predominantly inhibited spontaneous spike and subthreshold (probably synaptic) activity. In intracellular recordings, somatostatin-14 and -28 hyperpolarized most neurons in association with a slight (10-35%) but reproducible decrease in input resistance. These hyperpolarizing responses were augmented in depolarized cells and persisted in cells in which spontaneous inhibitory postsynaptic potentials became depolarizing after Cl- injection. These data suggest that somatostatin receptors regulate a K+ conductance. In voltage-clamp studies, somatostatin-28 and -14 induced a steady outward current and augmented the voltage-dependent, nonactivating outward K+ conductance (IM) shown to be blocked by activation of muscarinic cholinergic receptors. These results suggest (i) that somatostatin-containing elements in the solitary tract complex play an inhibitory role through the activation of postsynaptic permeability to potassium ions and (ii) that the same ion channel type may be coregulated by two neurotransmitter candidates, somatostatin and acetylcholine, through a reciprocal control mechanism.
Insulin and phorbol ester stimulate conductive Na+ transport through a common pathway.
Authors: Civan M M , Peterson-Yantorno K , O'Brien T G
Published: May 2006

Insulin stimulates Na+ transport across frog skin, toad urinary bladder, and the distal renal nephron. This stimulation reflects an increase in apical membrane Na+ permeability and a stimulation of the basolateral membrane Na,K-exchange pump. Considerable indirect evidence has suggested that the apical natriferic effect of insulin is mediated by activation of protein kinase C. However, no direct information has been available documenting that insulin and protein kinase C indeed share a common pathway in stimulating Na+ transport across frog skin. In the present work, we have studied the interaction of insulin and phorbol 12-myristate 13-acetate (PMA), a documented activator of protein kinase C. Preincubation of skins with 1,2-dioctanoylglycerol, another activator of protein kinase C, increases baseline Na+ transport and reduces the subsequent natriferic response to PMA. Preincubation with PMA markedly reduces the subsequent natriferic action of insulin. This effect does not appear to primarily reflect PMA-induced internalization of insulin receptors. The insulin receptors are localized on the basolateral surface of frog skin, but the application of PMA to this surface is much less effective than mucosal treatment in reducing the response to insulin. Preincubation with D-sphingosine, an inhibitor of protein kinase C, also reduces the natriferic action of insulin. The current results provide documentation that insulin and protein kinase C share a common pathway in stimulating Na+ transport across frog skin. The data are consistent with the concept that the natriferic effect of insulin on frog skin is, at least in part, mediated by activation of protein kinase C.
Assignment of groups responsible for the "opsin shift" and light absorptions of rhodopsin and red, green, and blue iodopsins (cone pigments).
Authors: Kosower E M
Published: May 2006

A modified structural model of rhodopsin is presented. Seven (alpha-helical) segments of 24 largely hydrophobic amino acid residues are assembled with exobilayer connecting strands into an aligned set, using the sequences of human red, green, and blue iodopsins (cone pigments) and human and bovine rod rhodopsins. (Aligned set numbering is used in this article). The inner region of the heptahelical hydrophobic domain includes one His-Glu (Asp) ion pair (red, green rod) near the retinylidene moiety in addition to an iminium ion Asp-99 pair. The negative charges posited in the "point-charge model" to cause the shift of the retinylidene iminium ion light absorption to longer wavelengths in the protein ("opsin shift") are Asp-99 (red, green rod), Glu-102 (red, green), and Glu-138 (rod). Blue iodopsin lacks both an ion pair and a counter charge to the iminium ion in the inner region, a fact that explains its absorption relative to rod rhodopsin. The spectroscopic difference between rod rhodopsin and the red/green iodopsins is due to the influence of Glu-102 in the latter. The red-green difference is due to the net effect of seven OH groups around the chromophore, all such groups being found within one helix turn of the retinylidene location. The tryptophan, which rotates as the retinylidene group isomerizes, may be Trp-142 or Trp-177. The geometric change (the rhodopsin "photoswitch") resulting from cis-trans isomerization in the first excited electronic state (S1), ultimately leads to RX (photoactivated rhodopsin, metarhodopsin II) and changes the activity of exobilayer groups, possibly causing dissociation of Lys-83 and Arg-85 from the carboxylate groups at positions 263 and 265.
Genetic analysis of "transvection" effects involving contrabithorax mutations in Drosophila melanogaster.
Authors: Micol J L , García-Bellido A
Published: May 2006

Contrabithorax (Cbx) alleles are dominant mutations in the Ultrabithorax (Ubx) gene of Drosophila that cause its ectopic expression in the mesothorax. We have studied the role of the homologous chromosome in the mesothoracic phenotype in several Cbx heterozygotes. None of the Cbx alleles studied shows variations in phenotype with extra doses of the Ubx gene. Only Cbx2 and CbxIRM (a revertant of Cbx1) show synapsis-dependent gene expression ("transvection"). The mesothoracic phenotypes of CbxIRM and Cbx2 heterozygotes are strongly modified when the homologous chromosome carries breakpoints proximal to or at the Ubx locus or null alleles of this gene. These lesions in the homologous chromosome enhance the Cbx2 mutant phenotype and reduce that of CbxIRM one. The genetic analysis of these transvection effects suggests that the transcription of the CbxIRM and Cbx2 alleles depends on RNAs of short radius of action from the homologous Ubx gene.
Clonal dominance among T-lymphocyte infiltrates in arthritis.
Authors: Stamenkovic I , Stegagno M , Wright K A , Krane S M , Amento E P , Colvin R B , Duquesnoy R J , Kurnick J T
Published: May 2006

Synovial membranes in patients with rheumatoid arthritis as well as other types of chronic destructive inflammatory arthritis contain infiltrates of activated T lymphocytes that probably contribute to the pathogenesis of the disease. In an effort to elucidate the nature of these infiltrates, interleukin 2 (IL-2)-responsive T lymphocytes were grown out of synovial fragments from 14 patients undergoing surgery for advanced destructive inflammatory joint disease. Eleven of the samples examined were from patients with classical rheumatoid arthritis, while three others were obtained from individuals with clinical osteoarthritis. Southern blot analysis of T-cell receptor (TCR) beta-chain genes in 13 of 14 cultures showed distinct rearrangements, indicating that each culture was characterized by the predominance of a limited number of clones. T-cell populations from peripheral blood stimulated with a variety of activators and expanded with IL-2 did not demonstrate evidence of similar clonality in long-term culture. These results suggest that a limited number of activated T-cell clones predominate at the site of tissue injury in rheumatoid synovial membranes as well as in other types of destructive inflammatory joint disease. Further characterization of these T-cell clones may aid our understanding of the pathogenesis of these rheumatic disorders.
Activation of cAMP-dependent protein kinase is required for heterologous desensitization of adenylyl cyclase in S49 wild-type lymphoma cells.
Authors: Clark R B , Kunkel M W , Friedman J , Goka T J , Johnson J A
Published: May 2006

We report here that, contrary to previously reported findings, treatment of S49 wild-type (WT) lymphoma cells with 0-50 nM epinephrine resulted in a heterologous desensitization of adenylyl cyclase (EC 4.6.1.1)--that is, epinephrine and prostaglandin E1 (PGE1) stimulations of adenylyl cyclase were reduced. Observation of this heterologous desensitization required the assay of adenylyl cyclase with submillimolar concentrations of Mg2+ and low concentrations of epinephrine. Also, whereas previously there had been no evidence for any role of cAMP-dependent protein kinase in the desensitization of the WT beta-adrenergic receptor, our data comparing the characteristics of the desensitization in WT, kin-, and cyc- lymphoma cells [where kin- and cyc- refer to variants of S49 WT cells lacking cAMP-dependent protein kinase activity (kin-) and the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein (cyc-)] now suggest that cAMP-dependent protein kinase mediates the heterologous desensitization of adenylyl cyclase. Specifically, we found that only the WT cells exhibited epinephrine-induced heterologous desensitization. The kin- and cyc- cells exhibited only homologous desensitization, and much higher concentrations of epinephrine were required to elicit the homologous desensitization in the variants relative to the heterologous desensitization of the WT. Treatment of WT and cyc- cells with dibutyryl cAMP or treatment of WT with forskolin or PGE1 caused the heterologous desensitization of adenylyl cyclase, indicating that neither receptor occupancy nor activation of adenylyl cyclase was necessary for the heterologous desensitization.
Overlapping positive and negative regulatory domains of the human beta-interferon gene.
Authors: Goodbourn S , Maniatis T
Published: May 2006

Virus or poly(I).poly(C) induction of human beta-interferon gene expression requires a 40-base-pair DNA sequence designated the interferon gene regulatory element (IRE). Previous studies have shown that the IRE contains both positive and negative regulatory DNA sequences. To localize these sequences and study their interactions, we have examined the effects of a large number of single-base mutations within the IRE on beta-interferon gene regulation. We find that the IRE consists of two genetically separable positive regulatory domains and an overlapping negative control sequence. We propose that the beta-interferon gene is switched off in uninduced cells by a repressor that blocks the interaction between one of the two positive regulatory sequences and a specific transcription factor. Induction would then lead to inactivation or displacement of the repressor and binding of transcription factors to both positive regulatory domains.
Binding of host-cell factors to DNA sequences in the long terminal repeat of human T-cell leukemia virus type I: implications for viral gene expression.
Authors: Nyborg J K , Dynan W S , Chen I S , Wachsman W
Published: May 2006

Efficient expression of human T-cell leukemia virus type I (HTLV-I) genes requires both host and viral proteins and is dependent on DNA sequences in the proviral long terminal repeats (LTRs). We have used DNase I-protection assays (footprinting) to construct a map of protein-DNA interactions over a 250-nucleotide region of the LTR upstream of the start site for viral RNA synthesis. We find that a host factor (host expression factor 1, or HEF-1) binds to the imperfect 21-nucleotide repeats that have previously been implicated in HTLV-I gene expression. HEF-1 binding activity is present in preparations from both lymphoid and nonlymphoid cell lines. However, the boundaries of the protected regions and the presence of a flanking DNase-hypersensitive site vary with cell type. Several regions of binding are detected in addition to the HEF-1 sites, including a complex group of sites 40-90 nucleotides upstream of the RNA start site. A comparison of HTLV-I-transformed T lymphocytes that do and do not express the viral trans-activating protein p40xI shows that none of the observed features of the DNase I footprint pattern correlate directly with the presence of this protein in the extract. These results suggest (i) that the primary recognition of promoter elements in the HTLV-I LTR involves specific interactions with host-cell proteins and (ii) that p40xI influences the activity of one or more of these proteins, rather than interacting directly with the DNA.
Platelet-derived growth factor A chain: gene structure, chromosomal location, and basis for alternative mRNA splicing.
Authors: Bonthron D T , Morton C C , Orkin S H , Collins T
Published: May 2006

Genomic clones encoding the A chain of platelet-derived growth factor (PDGF) have been isolated. The gene contains seven exons spanning about 24 kilobases of DNA. The positions of intervening sequences closely match those of the related B-chain (c-sis) gene on chromosome 22. In situ hybridization was used to localize the PDGF A-chain gene to the distal portion of the short arm of chromosome 7 (7p21-p22). Within the (G + C)-rich 5' region, a single transcriptional start site was identified approximately equal to 36 base pairs downstream of a TATAA consensus promoter element. The three size classes of A-chain mRNA probably arise by selection of alternative poly(A) sites in exon 7, but only a single consensus AATAAA signal was identified in this region. Two functionally different A-chain precursors, which differ by the presence or absence of a basic C terminus, are generated as a result of alternative mRNA splicing events, which include or exclude exon 6. This and other structural features of the A-chain gene suggest that PDGF expression may be modulated at transcriptional and post-transcriptional levels.
Aggregation of IgE receptors induces degranulation in rat basophilic leukemia cells permeabilized with alpha-toxin from Staphylococcus aureus.
Authors: Hohman R J
Published: May 2006

A method has been developed by which rat basophilic leukemia (RBL) cells can be permeabilized to small molecules while maintaining their ability to degranulate in response to aggregation of IgE receptors. alpha-Toxin was isolated from culture supernatants of Staphylococcus aureus by precipitation with (NH4)2SO4 and chromatography on phenyl-Sepharose. The isolated toxin binds to the plasma membrane of RBL cells and polymerizes to form a transmembrane pore that allows small molecules (Mr less than 1000), but not macromolecules, to diffuse freely across the membrane. There was no spontaneous release of the contents of RBL cell secretory granules during permeabilization or subsequent incubations. Substantial IgE receptor-mediated exocytosis occurred in the absence of Ca2+, and degranulation was maximal at 0.1-1.0 microM Ca2+, the physiologically important range of Ca2+ concentrations. Using these permeabilized cells, small molecules (i.e., substrates and inhibitors of various enzymes) normally excluded by the plasma membrane can be introduced into the cell. Moreover, the intracellular concentration of ions (such as Ca2+) can be precisely controlled. This method will allow a detailed examination of the individual biochemical events involved in degranulation of mast cells.
Expression of the firefly luciferase gene in vaccinia virus: a highly sensitive gene marker to follow virus dissemination in tissues of infected animals.
Authors: Rodriguez J F , Rodriguez D , Rodriguez J R , McGowan E B , Esteban M
Published: May 2006

We have introduced the firefly luciferase gene of Photinus pyralis into the vaccinia virus genome. This gene is expressed in a coordinate fashion during virus infection. Luminescence produced by the action of luciferase [Photinus-luciferin:oxygen 4-oxidoreductase(decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] was easily detectable in infected cells in culture as well as in cells of tissues of infected mice. The limits of detection were about one infected cell in a background of a million noninfected cells. The luciferase assay was about 1000-fold more sensitive than that of beta-galactosidase. Our findings show that the luciferase assay can be conveniently used to follow viral gene expression and virus dissemination both in cell cultures and in tissues of infected animals.
An endogenous activator of the Ca2+-dependent proteinase of human neutrophils that increases its affinity for Ca2+.
Authors: Pontremoli S , Melloni E , Michetti M , Salamino F , Sparatore B , Horecker B L
Published: May 2006

An endogenous activator of the Ca2+-dependent proteinase (calpain) has been identified in human neutrophils. In the presence of the activator, the affinity of calpain for Ca2+ is increased by greater than 100-fold and maximum catalytic activity is observed with Ca2+ concentration below 1 microM. The activator is a heat-stable protein having an apparent molecular mass of approximately equal to 40 kDa. It appears to be associated with the cytoskeletal fraction of human neutrophils. Neutrophils also contain an endogenous cytosolic calpain inhibitor (calpastatin), which is readily separated from the activator by size-exclusion chromatography. The effects of the activator and inhibitor appear to be antagonistic and may constitute a physiological mechanism for modulating intracellular calpain activity.
Complex foraging polymorphism in bluegill sunfish
Authors: Ehlinger Timothy John , Wilson David Sloan
Published: May 2006

The bluegill sunfish (
Lepomis macrochirus
) is considered a generalist predator, adept at feeding in both the littoral and open-water habitats of North American freshwater lakes. We demonstrate adaptive intraspecific variation in morphology and foraging behaviors within single lakes. This variation appears to make individual fish specialized for feeding in either the littoral or open-water habitat. Discovery of a complex polymorphism in such a well-studied species suggests that adaptive variation may be more common than is currently perceived.
Identification of specific apical membrane polypeptides associated with the antidiuretic hormone-elicited water permeability increase in the toad urinary bladder.
Authors: Harris H W , Wade J B , Handler J S
Published: May 2006

Antidiuretic hormone (ADH) increases the water permeability of the toad urinary bladder. The increase occurs in the apical plasma membrane of granular cells that line the urinary surface of the bladder and is produced by the insertion of water permeability units that have been identified by freeze-fracture electron microscopy as intramembrane particle aggregates. Under water-impermeable conditions, particle aggregates reside in intracellular vesicles called "aggrephores." In response to ADH, the aggrephores fuse with the apical plasma membrane and render it water permeable. When ADH is removed, intramembrane particle aggregates and aggrephores are retrieved from the apical membrane, and it returns to a water-impermeable state. To identify proteins involved in the water permeability response, we used lactoperoxidase/glucose oxidase to 125I-label external apical membrane proteins to compare control and ADH-treated bladders. Several polypeptides were consistently labeled in ADH-treated bladders and not in paired controls. After demonstrating that lactoperoxidase behaves as a fluid-phase marker and is sequestered in aggrephore-like vesicles when ADH is withdrawn, we used the technique of Mellman et al. [Mellman, I.S., Steinman, R. M., Unkeless, J. C. & Cohn, Z. A. (1980) J. Cell Biol. 86, 712-722] to label proteins endocytosed when water permeability declines after ADH is withdrawn to test whether the membrane proteins labeled in ADH-treated bladders behaved like particle aggregates. The internalized membranes contained polypeptides of the same molecular weights (55,000, 17,000-14,000, and 7,000) as those labeled on the apical surface of ADH-treated but not control bladders. These polypeptides are evidently involved in the ADH-stimulated water permeability response and may be components of particle aggregates.
Human ovarian theca cells are a source of renin.
Authors: Do Y S , Sherrod A , Lobo R A , Paulson R J , Shinagawa T , Chen S W , Kjos S , Hsueh W A
Published: May 2006

Human ovarian follicular fluid contains renin-like activity. In normal women, circulating levels of prorenin, the biosynthetic precursor of renin (EC 3.4.23.15), change in parallel with changes in progesterone during the menstrual cycle. Therefore, the ovary has been implicated as a source of plasma prorenin. In the present studies, we report the finding of high concentrations of prorenin in human ovarian follicular fluid (3000 ng.ml-1.hr-1 vs. 10-40 ng.ml-1.hr-1 in normal human plasma) obtained from follicles of women prepared for in vitro fertilization. The inactive renin-like enzyme was identified as prorenin by its activation characteristics, its molecular weight of 47,000, which is the same as that for recombinant prorenin, and its cross-reactivity with human renal renin antibodies. Culture of isolated human theca cells and isolated granulosa cells indicated that prorenin is secreted by theca cells but not by granulosa cells. Prorenin production by theca cells peaked during the first 10 days of culture and gradually decreased by 17 days. Active renin levels were 10% or less of the prorenin levels. Prorenin was barely detectable in medium from granulosa cells cultured for 24 days. Immunohistochemical staining of human ovaries (n = 5) with anti-human renin antibody demonstrated the presence of renin primarily in theca cells. These studies suggest that the theca cell is the source of the large quantities of prorenin in human ovarian follicular fluid.
Rapid reorganization of adult rat motor cortex somatic representation patterns after motor nerve injury.
Authors: Sanes J N , Suner S , Lando J F , Donoghue J P
Published: May 2006

The potential for peripheral nerve injury to reorganize motor cortical representations was investigated in adult rats. Maps reflecting functional connections between the motor cortex and somatic musculature were generated with intracortical electrical stimulation techniques. Comparison of cortical somatotopic maps obtained in normal rats with maps generated from rats with a facial nerve lesion indicated that the forelimb and eye/eyelid representations expanded into the normal vibrissa area. Repeated testing from an electrode placed chronically in the motor cortex showed a shift from vibrissa to forelimb within hours after facial nerve transection. These comparatively quick changes in motor cortex representation pattern suggest that synaptic relations between motor cortex and somatic musculature are continually reshaped in adult mammals.
Locally asymptotically rank-based procedures for testing autoregressive moving average dependence
Authors: Hallin Marc , Puri Madan L.
Published: May 2006

The problem of testing a given autoregressive moving average (ARMA) model (in which the density of the generating white noise is unspecified) against other ARMA models is considered. A distribution-free asymptotically most powerful test, based on a
generalized linear serial rank statistic
, is provided against contiguous ARMA alternatives with specified coefficients. In the case in which the ARMA model in the alternative has unspecified coefficients, the asymptotic sufficiency (in the sense of Hájek) of a finite-dimensional vector of rank statistics is established. This asymptotic sufficiency is used to derive an asymptotically maximin most powerful test, based on a
generalized quadratic serial rank statistic
. The asymptotically maximin optimal test statistic can be interpreted as a rank-based, weighted version of the classical Box-Pierce portmanteau statistic, to which it reduces, in some particular problems, under gaussian assumptions.
Cytosine methylation does not affect binding of transcription factor Sp1.
Authors: Harrington M A , Jones P A , Imagawa M , Karin M
Published: May 2006

DNA methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. We used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor Sp1 to its target sequence (a G + C-rich sequence known as a "GC box"). Concatemers of double-stranded 14-mers containing a GC box successfully competed with the human metallothionein IIA promoter for binding to Sp1 in DNase I protection experiments. The presence of 5-methylcytosine in the CpG sequence of the GC box did not influence Sp1 binding. The result was confirmed using double-stranded 20-mers containing 16 base pairs of complementary sequence. Electrophoretic gel retardation analysis of annealed 28-mers containing a GC box incubated with an Sp1-containing HeLa cell nuclear extract demonstrated the formation of DNA-protein complexes; formation of these complexes was not inhibited when an oligomer without a GC box was used as a competitor. Once again, the presence of a 5-methylcytosine residue in the GC box did not influence the binding of the protein to DNA. The results therefore preclude a direct effect of cytosine methylation on Sp1-DNA interactions.
Splicing mutation in human hereditary analbuminemia.
Authors: Ruffner D E , Dugaiczyk A
Published: May 2006

We have identified a structural defect in the serum albumin gene in human analbuminemia. Sequence determination of 1.1 kilobases (kb) of the 5' regulatory region and of 6 kb across exonic regions revealed a single AG-to-GG mutation within the 3' splice site of intron 6 in the defective gene of an analbuminemic individual. In an in vitro assay on the RNA transcript this mutation causes a defect in splicing of the intron 6 sequence and in subsequent ligation of the exon 6-exon 7 sequences. Using polymerase-amplified genomic DNA and allele-specific oligodeoxynucleotide probes, we have also shown that the sequence of this intron 6/exon 7 splice junction is normal in a different, unrelated analbuminemic individual. Analbuminemia in humans is therefore the result of one of multiple defects in our genome.
T-cell tolerance: exposure to virus in utero does not cause a permanent deletion of specific T cells.
Authors: Jamieson B D , Ahmed R
Published: May 2006

This study documents the curing of a congenitally acquired chronic viral infection and the acquisition of T-cell competence by a previously tolerant host. Infection of mice with lymphocytic choriomeningitis virus (LCMV) is a classic model of viral persistence and antigen-specific T-cell unresponsiveness. Mice infected at birth or in utero become lifelong carriers with no detectable virus-specific cytotoxic T lymphocyte (CTL) responses. This chronic infection can be eliminated by adoptive transfer of Lyt-2+ T cells from LCMV-immune mice. To determine whether these cured carriers were capable of generating their own LCMV-specific CTL response, mice congenic at the Thy-1 locus (Thy-1.1 and Thy-1.2) were used in the adoptive transfer experiments. Host-derived T-cell responses were checked after treating the cured carriers with a monoclonal antibody to deplete the immune donor T cells. Such cured carrier mice were able to generate a host-derived virus-specific CTL response and resisted a second LCMV challenge in the absence of any donor T cells. In addition, bone marrow cells from these cured carriers could functionally reconstitute irradiated mice. Thus this report demonstrates the acquisition of LCMV-specific T-cell competence by previously unresponsive carrier mice infected in utero. These results show that exposure to a virus even during embryonic life does not cause a permanent deletion of specific T cells. These findings are of significance to the understanding of tolerance mechanisms and have implications for the treatment of chronic viral infections.
Genetic heterogeneity in type 1 Gaucher disease: multiple genotypes in Ashkenazic and non-Ashkenazic individuals.
Authors: Tsuji S , Martin B M , Barranger J A , Stubblefield B K , LaMarca M E , Ginns E I
Published: May 2006

Nucleotide sequence analysis of a genomic clone from an Ashkenazic Jewish patient with type 1 Gaucher disease revealed a single-base mutation (adenosine to guanosine transition) in exon 9 of the glucocerebrosidase gene. This change results in the amino acid substitution of serine for asparagine. Transient expression studies following oligonucleotide-directed mutagenesis of the normal cDNA confirmed that the mutation results in loss of glucocerebrosidase activity. Allele-specific hybridization with oligonucleotide probes demonstrated that this mutation was found exclusively in the type 1 phenotype. None of the 6 type 2 patients, 11 type 3 patients, or 12 normal controls had this allele. In contrast, 15 of 24 type 1 patients had one allele with this mutation, and 3 others were homozygous for the mutation. Furthermore, some of the Ashkenazic Jewish type 1 patients had only one allele with this mutation, suggesting that even in this population there is allelic heterozygosity. These findings indicate that there are multiple allelic mutations responsible for type 1 Gaucher disease in both the Jewish and non-Jewish populations. Allelic-specific hybridization demonstrating this mutation in exon 9, used in conjunction with the Nci I restriction fragment length polymorphism described as a marker for neuronopathic Gaucher disease, provides a tool for diagnosis and genetic counseling that is approximately equal to 80% informative in all Gaucher patients studied.
cGMP phosphodiesterase of retinal rods is regulated by two inhibitory subunits.
Authors: Deterre P , Bigay J , Forquet F , Robert M , Chabre M
Published: May 2006

The cGMP phosphodiesterase (PDE) of cattle retinal rod outer segments comprises three types of subunits: the two heavy catalytic ones, PDE alpha and PDE beta, each around 85 kDa, and the light inhibitory one, PDE gamma or I (11 kDa). The relative stoichiometry is usually assumed to be 1:1:1. PDE activation in the visual transduction cascade results from removal of the inhibitor by the alpha subunit of transducin (T alpha). The stoichiometric complex T alpha-I, separated from activated PDE, has been isolated and characterized. Analyzing now the activated PDE, we find that it still contains some inhibitor and is resolvable into two species, one with 50% of the inhibitor content of the native enzyme and the other totally devoid of it. The same two species are observed upon activation of PDE by very short tryptic proteolysis, which specifically degrades the inhibitor. This leads us to conclude that the composition of the native enzyme is PDE alpha beta-I2. The two inhibitory subunits are differentially bound, sequentially removable, and exchangeable between the native complex PDE alpha beta-I2 and the fully active PDE alpha beta. The possibility of this exchange precludes as yet an unambiguous estimate of the actual activity of the intermediate complex PDE alpha beta-I. The differential binding and the exchangeability of the inhibitors raises the possibility of a fast, diffusion controlled, switch-off mechanism of PDE activity after a flash, which would shortcut the inactivation resulting from the slow GTPase rate of transducin.
Erythroid differentiation factor is encoded by the same mRNA as that of the inhibin beta A chain.
Authors: Murata M , Eto Y , Shibai H , Sakai M , Muramatsu M
Published: May 2006

We have isolated a protein that exhibits a potent differentiation-inducing activity toward mouse Friend erythroleukemia (MEL) cells and human K-562 cells. The protein, designated erythroid differentiation factor (EDF), was found in the culture fluid of human THP-1 cells that had been treated with phorbol 12-myristate 13-acetate. EDF is a homodimer with a Mr of 25,000; the Mr of the monomer is 15,500. cDNA clones encoding the Mr 15,500 subunit of EDF from THP-1 libraries were isolated and sequenced. Surprisingly, the sequence of EDF mRNA is identical to that for the beta A subunit of inhibin, a gonadal protein that suppresses the secretion of pituitary follicle-stimulating hormone. Southern blot analysis indicates that only one gene for EDF/inhibin beta A exists in the human genome. When the EDF subunit cDNA was linked to a simian virus 40 expression vector containing the dihydrofolate reductase gene and transfected into Chinese hamster ovary dihydrofolate reductase negative cells, the transformants began to secrete EDF, demonstrating that the cDNA actually encoded the EDF subunit.
Calcium entry into the inositol 1,4,5-trisphosphate-releasable calcium pool is mediated by a GTP-regulatory mechanism.
Authors: Mullaney J M , Yu M , Ghosh T K , Gill D L
Published: May 2006

Intracellular Ca2+ release activated by inositol 1,4,5-trisphosphate (InsP3) plays a pivotal role in Ca2+ signaling in cells. A controlling mechanism for InsP3-induced Ca2+ movements is suggested by results showing that the InsP3-releasable Ca2+ pool is directly modified by a specific and sensitive GTP-regulated Ca2+-translocating process. By using saponin-permeabilized N1E-115 neuroblastoma cells or DDT1MF-2 smooth muscle-derived cells, InsP3 releases 30-50% of Ca2+ accumulated through intracellular high-affinity ATP-dependent Ca2+-pumping activity. Oxalate-promoted Ca2+ uptake is reversed by InsP3, indicating oxalate permeability of the InsP3-releasable pool, which is consistent with this compartment being the endoplasmic reticulum. GTP (10 microM) activates release of 50-70% of accumulated Ca2+ from cells. In the presence of 5-10 mM oxalate, GTP induces a biphasic Ca2+ flux response; initially (1-2 min) GTP induces rapid Ca2+ release followed thereafter by a profound increase in Ca2+ uptake. Thus, GTP-activated Ca2+ influx and efflux compete for Ca2+ access to the oxalate-permeable Ca2+ pool. The nonadditive effects of InsP3 and GTP suggest that InsP3 releases Ca2+ from a subcompartment of the GTP-releasable pool. Most significantly, InsP3 is observed to block the GTP-activated uptake phase in the presence of oxalate, indicating that GTP induces Ca2+ entry into the pool from which InsP3 activates release. Hence, the results provide direct evidence that loading of Ca2+ into the InsP3-sensitive Ca2+ pool is controlled by a GTP-regulated Ca2+-translocating mechanism. Such a process could be significant in regulating the extent and duration of the InsP3-induced Ca2+ signal, a crucial step in the inositol phospholipid signaling pathway.
Genetically engineered immunoglobulins reveal structural features controlling segmental flexibility.
Authors: Schneider W P , Wensel T G , Stryer L , Oi V T
Published: May 2006

We have carried out nanosecond fluorescence polarization studies of genetically engineered immunoglobulins to determine the structural features controlling their segmental flexibility. The proteins studied were hybrids of a relatively rigid isotype (mouse IgG1) and a relatively flexible one (mouse IgG2a). They have identical light chains and heavy chain variable regions and have the same combining sites for epsilon-dansyl-L-lysine, a fluorescent hapten. The fluorescence of the bound dansyl chromophore was excited at 348 nm with subnanosecond laser pulses, and the emission in the nanosecond time range was measured with a single-photon-counting apparatus. The emission anisotropy kinetics of the hybrid antibodies revealed that segmental flexibility is controlled by the heavy chain constant region 1 (CH1) as well as by the hinge. In contrast, the CH2 and CH3 domains did not influence segmental flexibility. The hinge and CH1 domains must be properly matched to allow facile movement of the Fab units. Studies of hybrids of IgG1 and IgG2a within CH1 showed that the loop formed by residues 131-139 is important in controlling segmental flexibility. X-ray crystallographic studies by others of human IgG1 have shown that this loop makes several van der Waals contacts with the hinge.
In vivo double-strand breaks occur at recombinogenic G + C-rich sequences in the yeast mitochondrial genome.
Authors: Zinn A R , Pohlman J K , Perlman P S , Butow R A
Published: May 2006

An optional 46-base-pair G + C-rich element (GC cluster) in the coding region of the yeast mitochondrial var1 gene inserts preferentially in crosses into recipient alleles that lack the sequence. Unlike a similar gene conversion event involving the insertion of an optional 1143-base-pair intron, the mitochondrial 21S rRNA gene, which requires the action of a protein encoded by a gene within that intron, conversion of the var1 GC cluster does not require any protein product of the mitochondrial genome. We have detected double-strand breaks in the var1 gene in mitochondrial DNA isolated from unmated haploid rho+ and rho- strains at or near the boundaries of the optional GC cluster, as well as at a conserved copy of that sequence 160 base pairs upstream. No double-strand breaks were detected in the recipient var1 DNA molecules in the vicinity of the optional GC cluster target sequence. This contrasts with 21S rRNA-encoding DNA (rDNA) intron conversion where the recipient, but not the donor DNA, is cleaved at the element insertion site. These results suggest that although the 21S rDNA intron and the var1 GC cluster are preferentially inserted into their respective short alleles, these conversions probably occur by different mechanisms.