bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet stimulation releases a cAMP-dependent protein kinase that specifically phosphorylates a plasma protein.
Authors: Korc-Grodzicki B , Tauber-Finkelstein M , Shaltiel S
Published: May 2006

Rabbit serum is shown to contain a cAMP-dependent protein kinase (biochemically characterized as type II) that specifically phosphorylates a 135-kDa endogenous protein. This endogenous phosphorylation can be reproduced with platelet-rich plasma, after stimulation with thrombin, but not with plasma devoid of platelets. Stimulation of isolated platelets ("washed" by gel filtration) with either thrombin or ADP brings about a release of this kinase. The supernatant of these stimulated platelets, which contains the kinase, does not undergo a cAMP-dependent endogenous phosphorylation because it does not contain the 135-kDa protein substrate. On the other hand, plasma devoid of platelets does not contain cAMP-dependent protein kinase. By combining the supernatant of the physiologically stimulated platelets with the plasma devoid of platelets, it is possible to reconstitute the system and to reproduce the specific endogenous phosphorylation of the 135-kDa target substrate. On the basis of the above evidence it is proposed that upon physiological stimulation of platelets, they release into the blood a cAMP-dependent protein kinase in addition to the well-known release of MgATP. This kinase specifically phosphorylates the 135-kDa plasma protein.
Amylin found in amyloid deposits in human type 2 diabetes mellitus may be a hormone that regulates glycogen metabolism in skeletal muscle.
Authors: Cooper G J , Leighton B , Dimitriadis G D , Parry-Billings M , Kowalchuk J M , Howland K , Rothbard J B , Willis A C , Reid K B
Published: May 2006

Diabetes-associated peptide has recently been isolated and characterized from the amyloid of the islets of Langerhans in type 2 (non-insulin-dependent) diabetics, and immunoreactivity with antibodies to the peptide has been demonstrated in islet B cells of both normal and type 2 diabetic subjects. In view of the evidence presented in this paper that this 37-amino acid peptide may be a hormone present in normal individuals, we now propose the name "amylin" to replace "diabetes-associated peptide." Because increased amylin, deposited as amyloid within the islets of Langerhans, is characteristic of type 2 diabetes, the study below was performed to examine the possible effects of amylin on peripheral glucose metabolism. Whole amylin was synthesized by using solid-phase techniques, with formation of the disulfide linkage by oxidation in dilute aqueous solution and recovery of the peptide by lyophilization. The effects of amylin on glucose metabolism were studied in two preparations in vitro, isolated rat soleus muscle strips and isolated rat adipocytes. In skeletal muscle exposed to 120 nM amylin for 1 hr, there was a marked decrease in both basal and submaximally insulin-stimulated rates of glycogen synthesis, which resulted in significant reduction in the rates of insulin-stimulated glucose uptake. In muscles treated with amylin there was no response at the concentration of insulin required to stimulate glucose uptake half-maximally in untreated (control) muscles. In marked contrast, amylin had no effect on either basal or insulin-stimulated rates of glucose incorporation into either CO2 or triacylglycerol in isolated adipocytes. Therefore, amylin may be a factor in the etiology of the insulin resistance in type 2 diabetes mellitus, as both deposition of the peptide in islet amyloid and decreased rates of glucose uptake and glycogen synthesis in skeletal muscle are characteristic of this condition.
Hemagglutinin polymorphism as the basis for low- and high-yield phenotypes of swine influenza virus.
Authors: Kilbourne E D , Taylor A H , Whitaker C W , Sahai R , Caton A J
Published: May 2006

Single amino acid substitutions at the rim of the receptor binding site of the hemagglutinin molecule of swine influenza virus markedly influence the replicative capacity of the virus in chicken embryos, Madin-Darby canine kidney cells (MDCK), and swine as well as its antigenic phenotype. Mutants of low-yield (L) phenotype replicate poorly in chicken embryos and produce small plaques in MDCK cells but are highly infective for swine. Such mutants have lysine at position 153 and glycine at position 155 of the hemagglutinin (residues 156 and 158 in the H3 model). High-yield (H) mutants have the converse replicative characteristics and can be antigenically distinguished from L mutants (and from each other) based on their differential reactivity with two monoclonal antibodies, 9C8 and Sa-13. H mutants differ from L mutants in that the H mutants express glutamic acid at either position 153 or 155. L and H mutants act in an allelic fashion in effecting predictable one-step adaptation to different hosts. Selection for replication (e.g., high-yielding) phenotype results in concordant pleiotropic change in antigenic phenotype and in genotype. Conversely, immunoselection leads to change in replicative phenotype. Although the mechanism by which these mutations affect viral replication has not yet been defined, they may reflect differences in the affinity of each mutant for different host receptors.
Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.
Authors: Zanger U M , Hauri H P , Loeper J , Homberg J C , Meyer U A
Published: May 2006

In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative.
Unified structure theory of icosahedral quasicrystals: Evidence from neutron powder diffraction patterns that AlCrFeMnSi, AlCuLiMg, and TiNiFeSi icosahedral quasicrystals are twins of cubic crystals containing about 820 or 1012 atoms in a primitive unit cube
Authors: Pauling Linus
Published: May 2006

A unified structure theory of icosahedral quasicrystals, combining the twinned-cubic-crystal theory and the Penrose-tiling-six-dimensional-projection theory, is described. Values of the primitive-cubic lattice constant for several quasicrystals are evaluated from x-ray and neutron diffraction data. The fact that the low-angle diffraction maxima can be indexed with cubic unit cells provides additional support for the twinned-cubic-crystal theory of icosahedral quasicrystals.
Amplification and increased expression of alpha class glutathione S-transferase-encoding genes associated with resistance to nitrogen mustards.
Authors: Lewis A D , Hickson I D , Robson C N , Harris A L , Hayes J D , Griffiths S A , Manson M M , Hall A E , Moss J E , Wolf C R
Published: May 2006

Glutathione-dependent enzymes play a central role in the protection of cells from cytotoxic chemicals and have been implicated in the intrinsic and acquired resistance of tumors to cytotoxic drugs. We have generated a Chinese hamster ovary line resistant to bifunctional nitrogen mustards and in this report have characterized and isolated the protein that represents the major observable phenotypic difference between the drug-sensitive and drug-resistant cell lines. This purified protein is shown to be an alpha class glutathione S-transferase comprising YcYc subunits and possessing a pI value of approximately 8.0. The intracellular level of the Yc subunit is elevated greater than 40-fold in the drug-resistant cell line, which could account for the increase in glutathione S-transferase (RX:glutathione R-transferase; EC 2.5.1.18) activity toward both 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Other glutathione S-transferase subunits within this gene family are also elevated. These changes are accompanied by a significant elevation in alpha class mRNA levels. Southern analysis indicates that the genes coding for these proteins are amplified 4- to 8-fold in the drug-resistant cell line. In addition, gamma-glutamyl transpeptidase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase; EC 2.3.2.2] activity is increased 3.6-fold in the drug-resistant Chinese hamster ovary cell line, which may explain the increase in cellular glutathione level. In this case no gene amplification was seen. These data indicate that gene amplification may be important in drug resistance toward alkylating agents and also that other enzymes in glutathione homeostasis are involved.
Rhizobium meliloti nodA
and
nodB
genes are involved in generating compounds that stimulate mitosis of plant cells
Authors: Schmidt Jürgen , Wingender Ruth , John Michael , Wieneke Ursula , Schell Jeff
Published: May 2006

The
nodB
gene of
Rhizobium meliloti
encodes a 23.8-kDa protein that is conserved in several
Rhizobium
species. Monospecific polyclonal antibodies against NodB were used to localize this protein in the cytosol of
R. meliloti
and
Escherichia coli
cells containing
nodABC
genes. In comparison to the NodA and NodC proteins, NodB is synthesized in a disproportionately low amount. The NodA and NodB proteins are involved in generating small, heat-stable compounds that stimulate the mitosis of various plant protoplasts. Our experiments suggest that NodC is not involved in the synthesis of the factors. On the basis of their properties, we speculate that the factors are cytokinin-like substances.
Honeybee retinal glial cells transform glucose and supply the neurons with metabolic substrate.
Authors: Tsacopoulos M , Evêquoz-Mercier V , Perrottet P , Buchner E
Published: May 2006

The retina of the honeybee drone is a nervous tissue in which glial cells and photoreceptor cells (sensory neurons) constitute two distinct metabolic compartments. Retinal slices incubated with 2-deoxy[3H]glucose convert this glucose analogue to 2-deoxy[3H]glucose 6-phosphate, but this conversion is made only in the glial cells. Hence, glycolysis occurs only in glial cells. In contrast, the neurons consume O2 and this consumption is sustained by the hydrolysis of glycogen, which is contained in large amounts in the glia. During photostimulation the increased oxidative metabolism of the neurons is sustained by a higher supply of carbohydrates from the glia. This clear case of metabolic interaction between neurons and glial cells supports Golgi's original hypothesis, proposed nearly 100 years ago, about the nutritive function of glial cells in the nervous system.
Energy metabolism of the untrained muscle of elite runners as observed by 31P magnetic resonance spectroscopy: evidence suggesting a genetic endowment for endurance exercise.
Authors: Park J H , Brown R L , Park C R , Cohn M , Chance B
Published: May 2006

The purpose of this study was to investigate whether genetically determined properties of muscle metabolism contribute to the exceptional physical endurance of world-class distance runners. ATP, phosphocreatine, inorganic phosphate, and pH were quantitatively determined by 31P nuclear magnetic resonance spectroscopy in the wrist flexor muscles of elite long-distance runners and sedentary control subjects. These muscles had not been exposed to any specific program of exercise training in either group of subjects. The "untrained" muscles were examined at rest, during two cycles of three grades of exercise, and in recovery. The flexor muscles of the athletes had higher concentrations of phosphocreatine and ATP than did those of the control subjects at rest and during exercise. The athletes' muscles possessed a higher capacity for generation of ATP by oxidative metabolism than did control subjects' muscles according to the following criteria: (i) high force output, 60% of maximum voluntary contraction, was more easily reached and better maintained in both exercise cycles; (ii) the ratio of inorganic phosphate to phosphocreatine rose less during exercise and recovered faster in the postexercise period; (iii) there was no loss of adenine nucleotides or total phosphate from the athletes' muscles but significant losses from the control subjects' muscles; and (iv) the pH decreased no more than 0.1 unit in the athletes' muscles during exercise, attesting to a relatively slow glycolysis and/or a rapid oxidation of lactate. In the muscles of the control subjects, on the other hand, the pH decreased nearly 0.4 unit early in the first exercise cycle, indicating a relatively fast glycolysis and/or slower oxidation of lactate. In the second exercise cycle, the pH returned to near normal in the control subjects' muscles, reflecting diminished lactate formation because of glycogen depletion and lactate washout by the high blood flow induced by exercise. By the end of the exercise program, the maximum voluntary contractile force for the control subjects had declined to less than 60% of the initial value. This decline could be explained best by exhaustion of the glycolytic contribution to muscle contraction. Therefore, the residual maximum strength provided a measure of the oxidative capacity to support contraction, as is discussed. In conclusion, we suggest that a greater oxidative capacity relative to glycolytic capacity for support of contraction in untrained muscle of world-class runners reflects a genetic endowment for physical endurance. Additional systemic effects of training cannot be completely excluded. 31P magnetic resonance spectroscopy provides a noninvasive method for assessing this endowment.
NF-kappa B protein purification from bovine spleen: nucleotide stimulation and binding site specificity.
Authors: Lenardo M J , Kuang A , Gifford A , Baltimore D
Published: May 2006

The activity of the enhancer for the kappa immunoglobulin light chain gene critically depends on the presence in the nucleus of the NF-kappa B protein. We purified NF-kappa B over 50,000-fold and identified two protein species, 42 and 44 kDa, that could be eluted and renatured from a sodium dodecyl sulfate/polyacrylamide gel to give specific DNA-binding activity. Binding of the purified bovine NF-kappa B as well as that from human and murine B- or T-lymphoid cell extracts was dramatically stimulated by nucleoside triphosphates. This effect distinguished NF-kappa B from a related factor, H2-TF1. Purified NF-kappa B interacted efficiently with regulatory sequences that function during either B- or T-lymphocyte activation, including the human immunodeficiency virus enhancer and a NF-kappa B binding site we detected in the interleukin 2 enhancer.
Identification of phosphorylated 422(aP2) protein as pp15, the 15-kilodalton target of the insulin receptor tyrosine kinase in 3T3-L1 adipocytes.
Authors: Hresko R C , Bernier M , Hoffman R D , Flores-Riveros J R , Liao K , Laird D M , Lane M D
Published: May 2006

[32P]pp15, the [32P]phosphorylated form of a specific cytosolic substrate of the insulin receptor tyrosine kinase, was purified to homogeneity from mouse 3T3-L1 adipocytes incubated with 32Pi. Evidence presented here and previously indicates that pp15 contains a single phosphotyrosine residue. Alkylated [32P]pp15 was subjected to limited digestion with trypsin, after which three incompletely digested tryptic [32P]phosphopeptides were purified for analysis. Amino acid and radiochemical sequence analysis of the [32P]phosphopeptides revealed that pp15 is the phosphorylation product of 422(aP2) protein, a 15-kDa adipocyte protein previously sequenced in this laboratory from the corresponding cDNA.
Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity.
Authors: Morrison D K , Kaplan D R , Rapp U , Roberts T M
Published: May 2006

We have examined the phosphorylation and the serine/threonine-specific kinase activity of the protooncogene product Raf-1 (formerly c-raf) in response to oncogenic transformation or growth-factor treatment of mouse 3T3 cells. Expression of the membrane-bound oncogene products encoded by v-fms, v-src, v-sis, polyoma virus middle-sized tumor antigen, and Ha-ras increased the apparent molecular weight and phosphorylation of the Raf-1 protein, while expression of the nuclear oncogene and protooncogene products encoded by v-fos and c-myc did not. Changes in electrophoretic mobility and phosphorylation occurred rapidly in response to treatment of cells with platelet-derived growth factor, acidic fibroblast growth factor, epidermal growth factor, and the protein kinase C activator phorbol 12-myristate 13-acetate, but not insulin. The phosphorylation of the Raf-1 protein occurred primarily on serine and threonine residues. However, a subpopulation of Raf-1 molecules was phosphorylated on tyrosine residues in cells transformed by v-src or stimulated with platelet-derived growth factor. Transformation by v-src, or treatment with platelet-derived growth factor or phorbol 12-myristate 13-acetate, activated the Raf-1-associated serine/kinase activity as measured in immune-complex kinase assays. These findings suggest that proliferative signals generated at the membrane result in the phosphorylation of the Raf-1 protein and the activation of its serine/threonine kinase activity. Raf-1 activation may thus serve to transduce signals from the membrane to the cytoplasm and perhaps on to the nucleus.
Ca2+ transient, Mg2+, and pH measurements in the cardiac cycle by 19F NMR.
Authors: Kirschenlohr H L , Metcalfe J C , Morris P G , Rodrigo G C , Smith G A
Published: May 2006

19F NMR indicators have been used to measure the free cytosolic cation concentrations ([Mn+]i, where M is the atomic symbol and n is the value of the charge) of Ca2+, H+, and Mg2+ in perfused ferret hearts. The [Ca2+]i transient, cytosolic pH (pHi), and [Mg2+]i have also been followed at 16 phases in the cardiac cycle in hearts paced at 1.25 Hz at 30 degrees C. The initial [Ca2+]i rose rapidly after a 50-ms delay, was maximal at greater than 1.5 microM after 150 ms, and declined thereafter to the initial concentration. In contrast, no significant changes in pHi (pH 7.03 +/- 0.08) or [Mg2+]i (1.2 +/- 0.1 mM) were detected in the cycle. A decrease in developed pressure when the [Ca2+]i indicator (but not the pHi or [Mg2+]i indicator) was loaded into hearts was substantially reversed by the addition of 50 microM ZnCl2 to the perfusion medium. The Zn2+ was taken up into the myoplasm and displaced Ca2+ bound to the indicator, a symmetrically substituted difluoro derivative of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), as evidenced by the appearance of the Zn-5FBAPTA resonance. The decrease in developed pressure caused by 5FBAPTA, therefore, may be due to its Ca2+ buffering effect on the myoplasm. By coloading hearts with the [Ca2+]i and pHi indicators, simultaneous measurement of several [Mn+]i was demonstrated, which should provide a useful addition to the methods available to monitor cardiac function and pharmacology.
Tandemly repeated tRNA pseudogenes in photobacterium.
Authors: Giroux S , Cedergren R
Published: May 2006

A region distal to three tRNA genes in Photobacterium phosphoreum, a Gram-negative eubacterium, unexpectedly contains a high number of repeated DNA segments that are closely related to the adjacent tRNAPro gene. The 5' to 3' order of this cluster is tRNAPro-tRNAHis-tRNAPro followed by eight tRNAPro-like structures interspersed by rho-independent terminators. The two tRNAPro genes, which are identical, and the tRNAHis gene have 86% and 87% positional identity, respectively, to their counterparts in the argT operon of Escherichia coli. The facts that these tRNA-like structures are not transcribed, in contrast to the tRNA retropseudogenes of eukaryotes, and that these structures are clustered near their progenitor suggest they are an unusual class of tRNA pseudogenes that arose by tandem duplication.
Synthesis of an opine-like compound, a rhizopine, in alfalfa nodules is symbiotically regulated.
Authors: Murphy P J , Heycke N , Trenz S P , Ratet P , de Bruijn F J , Schell J
Published: May 2006

We show that the promoter of the mos locus, which encodes genes required for the synthesis of a nodule-specific, opine-like compound, a rhizopine, in alfalfa nodules is regulated by the symbiotic nitrogen-fixation regulatory gene nifA. The 5'-regulatory region and amino-terminal end of the first open reading frame of the mos locus are highly homologous to the 5'-regulatory region and amino-terminal portion of the Rhizobium meliloti nifH gene. The coordinate regulation of mos and nif genes suggests that the mos locus plays a symbiotic role. We propose that the rhizopine enhances the survival of the bacterial partner in the symbiosis.
Microtubule-dependent effect of phorbol ester on the contractility of cytoskeleton of cultured fibroblasts.
Authors: Lyass L A , Bershadsky A D , Vasiliev J M , Gelfand I M
Published: May 2006

The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) upon the contractility of permeabilized cell models (cytoskeletons) of mouse fibroblasts was examined. Contraction was induced by incubation of cell models in a solution containing ATP and was assessed quantitatively by measuring alterations of the area of cell model projection on the substrate. Immunofluorescence microscopy was used to assess alterations of cytoskeleton morphology in the course of permeabilization and contraction. It was found that contractility of cell models from PMA-treated fibroblasts was considerably diminished as compared with the models from control fibroblasts. ATP induced only local contraction of certain zones of actin cortex in models from PMA-treated fibroblasts; it did not induce general contraction, characteristic of control models. Normal high contractility was characteristic of the models from the cells preincubated with PMA in combination with Colcemid. PMA is a specific activator of protein kinase C, one of the key enzymes of the membrane signal-transduction pathway. It is suggested that protein kinase C regulates contractility of actin cortex and that the pathway of this regulation has a microtubule-dependent stage blocked by Colcemid.
Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine.
Authors: D'Onofrio M , Starr C M , Park M K , Holt G D , Haltiwanger R S , Hart G W , Hanover J A
Published: May 2006

The nuclear pore complex contains a family of proteins ranging in molecular mass from 35 to 220 kDa that are glycosylated with O-linked N-acetylglucosamine (GlcNAc) residues. We sought to determine the primary sequence of a nuclear pore protein modified by O-linked GlcNAc. The major (62 kDa) nuclear pore glycoprotein (np62) was purified from rat liver nuclear envelopes by immunoaffinity chromatography and preparative gel electrophoresis. After CNBr fragmentation, a glycopeptide was isolated and microsequenced. An oligonucleotide probe based on this sequence information was used to screen a lambda gt11 cDNA library constructed from poly(A) mRNA of the rat thyroid cell line FRTL-5. A clone (B5) was isolated and shown to hybridize to a single 2.5-kilobase species in poly(A) mRNA from rat liver and FRTL-5. This insert was sequenced and found to contain a 691-base-pair cDNA encoding a 155-amino acid open reading frame. This open reading frame contained a CNBr fragment identical to the original glycopeptide sequence and a second CNBr fragment corresponding to a nonglycosylated peptide that was also isolated from the purified pore glycoprotein. The B5 cDNA produced a beta-galactosidase fusion protein of the size predicted by the open reading frame. Analysis of the residues making up a presumptive glycosylation site suggests that the sequence is unlike any known sites for enzymatic N- or O-linked glycosylation. The partial sequence of the 62-kDa nuclear pore glycoprotein shows little similarity to other characterized proteins and elucidates structural features of a member of the family of nuclear pore glycoproteins.
Sex change in cytochrome P-450 phenotype by growth hormone treatment of adult rat hepatocytes maintained in a culture system on matrigel.
Authors: Guzelian P S , Li D , Schuetz E G , Thomas P , Levin W , Mode A , Gustafsson J A
Published: May 2006

Results of studies of hypophysectomized rats suggest that growth hormone serves as a final common mediator through which gonadal steroids and other modifiers of pituitary function alter the expression of gender-specific liver genes such as the sexually dimorphic pair of cytochrome P-450 isozymes, male-specific P-450h and female-specific P-450i. We tested the effects of growth hormone in a system for primary monolayer culture of adult rat hepatocytes on a laminin-rich extracellular matrix (matrigel), which permits sustained expression of both constitutive and inducible liver genes in a chemically defined medium. Cultures of freshly isolated hepatocytes prepared from untreated male rats and samples of the intact donor liver contained readily detectable quantities of immunoreactive P-450h protein (measured on immunoblots of cell microsomes) and P-450h mRNA (measured on Northern blots of cellular RNA). Neither P-450i immunoreactive protein nor P-450i mRNA were present. Addition of physiologic concentrations of human or bovine growth hormone, but not of prolactin, to culture medium lacking insulin or other hormones resulted in prompt induction of P-450i immunoreactive protein and P-450i mRNA. Induction of P-450i mRNA in male hepatocyte cultures was dependent on the concentration of growth hormone, required as little as 24 hr of exposure, and was markedly attenuated in cultures maintained on type I collagen rather than on matrigel. Growth hormone treatment also induced the level of mRNA for insulin-like growth factor I, whereas the amount of mRNA for the male-specific urinary protein alpha 2 mu-globulin was unaffected. Cultures of hepatocytes derived from untreated adult female rats retained high levels of P-450i mRNA but only if the culture medium contained growth hormone. None of the tested treatments with estrogens, androgens, glucocorticoids, or growth hormone induced P-450h mRNA or P-450h immunoreactive protein in cultures of female hepatocytes. We conclude that the somatogenic effects of growth hormone acting alone and directly on the hepatocyte in culture are sufficient to "feminize" the cytochrome P-450 phenotype. The present culture system offers a way to explore the molecular basis for hormonal control of liver gene expression.
Adaptation of antennal neurons in moths is associated with cessation of pheromone-mediated upwind flight.
Authors: Baker T C , Hansson B S , Löfstedt C , Löfqvist J
Published: May 2006

A wind-borne plume of sex pheromone from a female moth or a synthetic source has a fine, filamentous structure that creates steep and rapid fluctuations in concentration for a male moth flying up the plume's axis. The firing rates from single antennal neurons on Agrotis segetum antennae decreased to nearly zero within seconds after the antennae were placed in a pheromone plume 70 cm downwind of a high-concentration source known from previous studies to cause in-flight arrestment of upwind progress. In a separate experiment, the fluctuating output from chilled neurons on Grapholita molesta antennae became attenuated in response to repetitive, experimentally delivered pheromone pulses. The attenuation was correlated with a previously reported higher percentage of in-flight arrestment exhibited by moths flying at cooler compared to warmer temperatures. These results indicate that two peripheral processes related to excessive concentration, complete adaptation of antennal neurons, or merely the attenuation of fluctuations in burst frequency, are important determinants of when upwind progress by a moth flying in a pheromone plume stops and changes to station keeping. Also, adaptation and attenuation may affect the sensation of blend quality by preferentially affecting cells sensitive to the most abundant components in airborne pheromone blends.
Characterization and cDNA cloning of the pheromone-binding protein from the tobacco hornworm, Manduca sexta: a tissue-specific developmentally regulated protein.
Authors: Györgyi T K , Roby-Shemkovitz A J , Lerner M R
Published: May 2006

cDNA encoding pheromone-binding protein (PBP), the major soluble protein in olfactory sensilla of male moths, has been cloned from the tobacco hornworm, Manduca sexta. A study of the developmental time course of PBP reveals that it is first synthesized just prior to eclosion and that the percentage of antennal mRNA encoding PBP shifts from zero to about 20% at that time. PBP is also found in sensilla from female M. sexta antennae. No amino acid sequence homology is observed between PBP and the vertebrate odorant-binding protein.
Receptors for neuropeptides are induced by exogenous poly(A)+ RNA in oocytes from Xenopus laevis.
Authors: Meyerhof W , Morley S , Schwarz J , Richter D
Published: May 2006

Receptors for the hormones vasopressin, angiotensin II, and thyrotropin-releasing hormone have been studied electrophysiologically in Xenopus laevis oocytes previously injected with poly(A)+ RNA from the respective receptor-containing tissues. The injected oocytes responded to the hormones by demonstrating oscillations in membrane currents as recorded by the voltage-clamp method. The response was dependent on the hormone concentrations and detectable between 5 and 1000 nM concentrations. Size fractionation of poly(A)+ RNA from the respective tissues showed that the mRNAs encoding the three hormone receptors were larger than 18S rRNA, suggesting a length of at least 2 kilobases. When vasopressin was added to the oocyte bath, an inward membrane current was generated in oocytes injected with rat poly(A)+ RNA from liver but not from kidney. This suggests that the V1-type (liver), not the V2-type (kidney), vasopressin receptor can be expressed and electrophysiologically identified in the oocyte. A V1-specific, but not a V2-specific, antagonist suppressed the vasopressin-dependent effect. Application of angiotensin II to liver poly(A)+ RNA-injected oocytes elicited oscillations in membrane current, indicating that these oocytes also expressed receptors for angiotension II; the antagonist [Sar1, O-methionyl-Tyr4]angiotensin II blocked this effect. Poly(A)+ RNA from tumor-derived GH3B6 cells, known to contain receptors for thyrotropin-releasing hormone, injected into oocytes induced receptors responding to thyrotropin-releasing hormone; the drug chlordiazepoxide suppressed the thyrotropin-releasing hormone response.
Two transcription factors, NF-kappa B and H2TF1, interact with a single regulatory sequence in the class I major histocompatibility complex promoter.
Authors: Baldwin A S , Sharp P A
Published: May 2006

A sequence centered 166 nucleotides upstream of the mouse H-2Kb class I major histocompatibility gene binds a nuclear factor, H2TF1, found in many cell types. Previous studies have shown that binding of H2TF1 to this sequence stimulates class I gene expression. Furthermore, this factor binds a similar sequence in the 72-base-pair repeat enhancer element of simian virus 40. We show here that NF-kappa B, an inducible B-cell-specific factor that binds the kappa immunoglobulin light chain gene enhancer, also binds the H2TF1 regulatory sequence. Methylation-interference experiments demonstrate that NF-kappa B closely interacts with six of the eight symmetrically positioned guanines that contact H2TF1. These experiments suggest that NF-kappa B may play a role in class I major histocompatibility gene expression and that H2TF1 and NF-kappa B may be related DNA-binding proteins.
Regulation of gene expression of class I alcohol dehydrogenase by glucocorticoids.
Authors: Dong Y , Poellinger L , Okret S , Höög J O , von Bahr-Lindström H , Jörnvall H , Gustafsson J A
Published: May 2006

The effect of glucocorticoids on gene expression of rat class I alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was investigated. A cDNA clone for the beta-subunit of human ADH (ADH2) was used to analyze class I ADH mRNA levels in rat hepatoma cells, which are known to contain a functional glucocorticoid receptor. RNA gel blot analysis of total cellular RNA isolated from these cells showed hybridization of the human ADH2 cDNA probe to a single approximately equal to 1500-base RNA species. Treatment of the cells with dexamethasone (0.1 nM to 1 microM) caused a dose-dependent increase in total cellular class I ADH mRNA levels by a factor of 2-4. Maximal levels were reached within 18-24 hr of treatment. This effect was reversible following withdrawal of dexamethasone. The glucocorticoid induction of class I ADH mRNA does not seem to require ongoing protein synthesis since treatment of the cells with cycloheximide did not affect the increase in class I ADH mRNA levels by dexamethasone. The human ADH2 gene contains both upstream and within the coding region sequence motifs that display homology with response elements of genes positively regulated by glucocorticoids. These data suggest a receptor-mediated transcriptional enhancement of the ADH2 gene as the mechanism of regulation. However, analysis of RNA decay in cells treated with actinomycin D indicates that the dexamethasone-induced increase in class I ADH mRNA might, at least in part, be due to enhanced ADH mRNA stability.
Heart as a target organ in 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity: decreased beta-adrenergic responsiveness and evidence of increased intracellular calcium.
Authors: Canga L , Levi R , Rifkind A B
Published: May 2006

The heart has not been regarded as a major target organ of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity notwithstanding that lethal cardiac dysfunction can occur in the absence of histopathological changes. To assess possible TCDD cardiotoxicity, we studied the effect of TCDD five days after treatment (10 micrograms/kg of body weight; single dose given i.p. in corn oil) on the contractility of guinea pig right ventricular papillary muscle. Controls were treated with corn oil. TCDD treatment significantly decreased beta-adrenergic responsiveness. In papillary muscles from TCDD-treated guinea pigs, the positive inotropic effect of isoproterenol (0.03-0.3 microM) was decreased by a mean of 65% (P less than 0.001), and the enhancement in the velocity of relaxation was 60% less than in the controls (P less than 0.05). On the other hand, TCDD treatment did not alter the positive inotropic effect of lower concentrations of isoproterenol (0.1-10 nM). After TCDD, responsiveness to low-frequency stimulation (0.1 and 0.25 Hz) was enhanced, responsiveness to increases in extracellular Ca2+ concentration was attenuated, and isoproterenol-elicited aftercontractions in K+-depolarized preparations were increased in magnitude. Collectively, the latter findings suggest that in addition to decreasing beta-adrenergic responsiveness, TCDD increases the intracellular Ca2+ concentration in papillary muscle. Finally, slow Ca2+ channels were not blocked after TCDD treatment, inasmuch as isoproterenol restored contractility equally effectively in K+-depolarized TCDD-treated and control papillary muscles. Our findings indicate that TCDD causes a specific pattern of cardiac dysfunction in a mammalian species, selectively augmenting or decreasing different cardiac responses. The cardiac changes are consistent with reported membrane effects of TCDD; further, they suggest that the heart may be a major target organ for TCDD toxicity.
Activation by odorants of a multistate cation channel from olfactory cilia.
Authors: Labarca P , Simon S A , Anholt R R
Published: May 2006

Single-channel records were obtained after fusion of ciliary membranes from the olfactory epithelium of Rana catesbeiana to planar lipid bilayers, and odorant-activated cation-selective channels were identified. In addition, a 190-pS potassium-selective channel and a 40-pS cation-selective channel were found in a 0.2 M salt-containing buffer. Odorant-sensitive channels were directly and reversibly activated by nanomolar concentrations of the bell pepper odorant 3-isobutyl-2-methoxypyrazine and the citrus odorant 3,7,-dimethyl-2,6-octadienenitrile. These channels display burst kinetics, multiple conductance levels between 35 and 420 pS, and open times in the millisecond range. With increasing concentrations of odorant, the probability of populating the higher conductance levels increases. These results show that direct activation of channels by odorants may mediate excitation of the olfactory receptor cell.
Human T-cell recognition of the circumsporozoite protein of Plasmodium falciparum: immunodominant T-cell domains map to the polymorphic regions of the molecule.
Authors: Good M F , Pombo D , Quakyi I A , Riley E M , Houghten R A , Menon A , Alling D W , Berzofsky J A , Miller L H
Published: May 2006

The definition of human T-cell antigenic sites is important for subunit vaccine development of a peptide immunogen if the goal is to allow antibody boosting during infection or to stimulate antibody-independent T-cell immunity. To identify such sites on the circumsporozoite (CS) protein of Plasmodium falciparum, 29 overlapping synthetic peptides spanning the entire CS protein were made and tested for their ability to stimulate peripheral blood lymphocytes from 35 adults living in a P. falciparum malaria-endemic region of West Africa. Three immunodominant domains were located outside the repetitive region. These domains, however, occurred in the polymorphic regions of the molecule, suggesting that parasite mutation and selection has occurred in response to immune pressure from T cells. Such polymorphism may impose an obstacle for vaccine development.
Use of synthetic peptides to identify an N-terminal epitope on mouse gamma interferon that may be involved in function.
Authors: Magazine H I , Carter J M , Russell J K , Torres B A , Dunn B M , Johnson H M
Published: May 2006

We previously have assigned N-terminal specificity to three hamster monoclonal antibodies (mAbs I, II, and III) produced to mouse recombinant gamma interferon (IFN-gamma), based on the ability of the N-terminal peptide IFN-gamma-(1-39) to block binding of 125I-labeled IFN-gamma (125I-IFN-gamma) and on the ability of these antibodies to bind 125I-IFN-gamma-(1-39). Only mAb I blocked function and binding to the IFN-gamma receptor, suggesting that it may bind to a region of the molecule involved in interaction with the receptor. To further define the epitope specificities of the antibodies, a series of N-terminal peptides were synthesized and tested for their ability to block antibody binding of 125I-IFN-gamma. Peptides IFN-gamma-(1-39), IFN-gamma-(1-20), IFN-gamma-(3-20), and IFN-gamma-(5-20) inhibited binding of 125I-IFN-gamma by mAb I in order of decreasing effectiveness, while peptide IFN-gamma-(7-20) was without effect. Peptides IFN-gamma-(1-39), IFN-gamma-(1-20), and IFN-gamma-(3-20) also inhibited binding of 125I-IFN-gamma by mAb II but were less effective when compared with their inhibition of mAb I. IFN-gamma-(5-20) and IFN-gamma-(7-20) did not inhibit binding by mAb II. Peptides IFN-gamma-(1-10), IFN-gamma-(10-30), and IFN-gamma-(21-44) did not inhibit either mAb I or mAb II. While IFN-gamma-(1-39) and IFN-gamma-(10-30) inhibited binding by mAb III, neither IFN-gamma-(1-20) nor any of its truncated forms were inhibitory. All three antibodies had similar Kd values for 125I-IFN-gamma. A prediction of the secondary structure of the molecule and the peptide inhibition data suggest that the epitope (possible receptor binding region) for mAb I involves a loop in the area containing residues 12-20, with sequences N-terminal to these residues possibly stabilizing the loop conformation. Direct evidence that the N-terminal 1-39 region of IFN-gamma is important in receptor binding was the observation that IFN-gamma-(1-39), but not the C-terminal IFN-gamma-(95-133), competed with 125I-IFN-gamma for the receptor on mouse L cells. IFN-gamma-(1-39) also specifically blocked IFN-gamma antiviral activity at concentrations that blocked binding to the receptor.The fact that IFN-gamma-(1-39) was the only peptide that blocked both IFN-gamma binding to receptor and function is consistent with the antibody competition data, where it was the most effective peptide in blocking binding of 125I-IFN-gamma by the N-terminal-specific mAbs. The combination of peptide mapping of epitope specificities and receptor competition should further help define the structural basis for IFN-gamma action.
CUUCGG hairpins: extraordinarily stable RNA secondary structures associated with various biochemical processes.
Authors: Tuerk C , Gauss P , Thermes C , Groebe D R , Gayle M , Guild N , Stormo G , d'Aubenton-Carafa Y , Uhlenbeck O C , Tinoco I
Published: May 2006

The mRNA of bacteriophage T4 contains a strikingly abundant intercistronic hairpin. Within the 55 kilobases of known T4 sequence, the hexanucleotide sequence CTTCGG is found 13 times in the DNA strand equivalent to mRNA sequences. In 12 of those occurrences, the sequence is flanked by inverted repeats predictive of RNA hairpins with UUCG in the loop. Avian myeloblastosis virus reverse transcriptase, which can traverse hairpins of larger calculated stability, terminates efficiently at these CUUCGG hairpins. Thermal denaturation studies of model hairpins show that the loop sequence UUCG dramatically stabilizes RNA hairpins when compared to a control sequence. These data, when combined with previously described parameters of helix stability, suggest that T4 has utilized this loop sequence to optimize the stability of intercistronic hairpins. The stability of CUUCGG hairpins is also utilized in the RNAs of many organisms besides T4.
A low dose of recombinant interleukin 1 protects granulocytopenic mice from lethal gram-negative infection.
Authors: van der Meer J W , Barza M , Wolff S M , Dinarello C A
Published: May 2006

Natural and synthetic immunomodulators that increase nonspecific resistance to infection induce interleukin 1 (IL-1) production. Therefore, we investigated the effect of the administration of IL-1 on the survival of lethally infected granulocytopenic mice. Mice with cyclophosphamide-induced granulocytopenia were injected with approximately 10(7) Pseudomonas aeruginosa in the thigh muscle at time 0; gentamicin was administered 6 hr and 23 hr later. When recombinant human IL-1 beta (one of the two forms of IL-1) was given as a single i.p. injection 24 hr before the infection, survival was increased. Using 80 ng of IL-1 beta per mouse, survival compared to control animals was 98% vs. 71% at 24 hr, 98% vs. 60% at 30 hr, 86% vs. 36% at 36 hr, and 61% vs. 11% at 48 hr (P less than 0.001) after the infection. No effect of IL-1 was observed when it was given 0.5 hr before or 6 hr after the infection. Animals not treated with gentamicin also benefited from the IL-1. Administration of the cyclooxygenase inhibitor ibuprofen did not affect the activity of IL-1. Numbers of bacteria cultured from the blood, thigh muscle, liver, spleen, and kidney were similar in IL-1-treated and control animals. Superoxide production by peritoneal macrophages was also similar in the two groups. These studies demonstrate that IL-1 pretreatment protects granulocytopenic mice against lethal pseudomonas infection and suggest that this protection occurs through a noncellular mechanism.
Distribution of precursor amyloid-beta-protein messenger RNA in human cerebral cortex: relationship to neurofibrillary tangles and neuritic plaques.
Authors: Lewis D A , Higgins G A , Young W G , Goldgaber D , Gajdusek D C , Wilson M C , Morrison J H
Published: May 2006

Neurofibrillary tangles (NFT) and neuritic plaques (NP), two neuropathological markers of Alzheimer disease, may both contain peptide fragments derived from the human amyloid beta protein. However, the nature of the relationship between NFT and NP and the source of the amyloid beta proteins found in each have remained unclear. We used in situ hybridization techniques to map the anatomical distribution of precursor amyloid-beta-protein mRNA in the neocortex of brains from three subjects with no known neurologic disease and from five patients with Alzheimer disease. In brains from control subjects, positively hybridizing neurons were present in cortical regions and layers that contain a high density of neuropathological markers in Alzheimer disease, as well as in those loci that contain NP but few NFT. Quantitative analyses of in situ hybridization patterns within layers III and V of the superior frontal cortex revealed that the presence of high numbers of NFT in Alzheimer-diseased brains was associated with a decrease in the number of positively hybridizing neurons compared to controls and Alzheimer-diseased brains with few NFT. In contrast, no correlation was found between the densities of NP and neurons containing precursor amyloid-beta-protein mRNA transcripts. These findings suggest that the expression of precursor amyloid-beta-protein mRNA may be a necessary but is clearly not a sufficient prerequisite for NFT formation. In addition, these results may indicate that the amyloid beta protein, present in NP in a given region or layer of cortex, is not derived from the resident neuronal cell bodies that express the mRNA for the precursor protein.
Molecular cloning and nucleotide sequence for the complete coding region of human UMP synthase.
Authors: Suttle D P , Bugg B Y , Winkler J K , Kanalas J J
Published: May 2006

The last two steps in the de novo biosynthesis of UMP are catalyzed by orotate phosphoribosyltransferase (OPRT; orotidine-5'-phosphate:pyrophosphate phosphoribosyltransferase; EC 2.4.2.10) and orotidine-5'-monophosphate decarboxylase (orotidine-5'-phosphate carboxy-lyase; EC 4.1.1.23). In mammals these two activities are found in a single, bifunctional protein called UMP synthase. A human T-lymphoblastic cell cDNA library constructed in lambda gt10 was screened with a UMP synthase-specific rat cDNA probe. Human UMP synthase cDNAs were isolated and then used to select UMP synthase gene fragments. The complete coding sequence of the mRNA for UMP synthase was determined by analysis of overlapping cDNA and genomic fragments. One of the cDNAs appears to have been synthesized from an incompletely or alternatively processed form of the UMP synthase mRNA. This cDNA lacks a poly(A) tail and has an extended 3'-nontranslated region that hybridizes with larger forms of the UMP synthase mRNA. The UMP synthase protein is composed of 480 amino acids with a molecular weight of 52,199. The two activities of UMP synthase reside in distinct domains encoded by the 3' and 5' halves of the mRNA. The COOH-terminal 258 amino acids of the human UMP synthase protein contain the orotidine-5'-monophosphate decarboxylase catalytic domain. This region is highly homologous to the mouse orotidine-5'-monophosphate decarboxylase sequence. The NH2-terminal 214 amino acids contain the OPRT domain. There is amino acid homology between this protein domain and specific regions of the Escherichia coli OPRT. The human OPRT domain also contains the putative catalytic site common to other human phosphoribosyltransferases.