bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the expression of the beta nerve growth factor (NGF) gene in the central nervous system: level and regional distribution of NGF mRNA suggest that NGF functions as a trophic factor for several distinct populations of neurons.
Authors: Shelton D L , Reichardt L F
Published: May 2006

Beta nerve growth factor (NGF), a target-derived protein necessary for survival and development of sympathetic and sensory neurons, can also affect subpopulations of neurons in the central nervous system (CNS). Using a blot hybridization assay capable of detecting 10 fg of mRNA, we measured the levels of NGF mRNA in the major brain regions, including those innervated by NGF-responsive neurons. NGF mRNA was detected unambiguously in each major region of the CNS. The levels were comparable to those in sympathetic effector organs. Discrete areas contained very different amounts of NGF mRNA. Up to 40-fold differences were seen, a range comparable to the differences between richly and sparsely innervated sympathetic effector organs. The highest concentrations of NGF mRNA were found in the cortex and hippocampus, which are the major targets of the NGF-responsive cholinergic neurons of the basal forebrain nuclei. Significant amounts of NGF mRNA were also found in areas that contain the central processes of NGF-responsive sensory neurons, such as the pons, medulla, and spinal cord. The presence of NGF mRNA in these areas suggests that brain NGF may act as a target-derived trophic factor for both populations of neurons. NGF mRNA was also found in the striatum, suggesting that locally derived NGF may act there as a trophic factor for a recently identified population of NGF-responsive cholinergic local circuit neurons. However, high levels of NGF mRNA were also found in some regions, such as the diencephalon, that have no relation to any identified population of NGF-responsive neurons. This suggests that there may be additional populations of NGF-responsive neurons in the CNS that have not yet been discovered.
Inhibitors of protein and RNA synthesis cause a rapid block in prostaglandin production at the prostaglandin synthase step.
Authors: Fagan J M , Goldberg A L
Published: May 2006

Inhibitors of protein or RNA synthesis prevented prostaglandin (PG) production in isolated skeletal muscles, brain, and spleen. Incubation of rat muscles with cycloheximide prevented the stimulation of PGE2 production induced in vitro by the Ca2+ ionophore A23187 and in vivo by injection of endotoxin. Cycloheximide also inhibited the stimulation by arachidonic acid of PGE2, PGF2 alpha, and prostacyclin. These observations suggest that the block in prostanoid production results from a loss of PG synthase activity (EC 1.14.99.1). These effects were detectable within 10 min after exposure of the muscle to cycloheximide. The degree of inhibition of PG production correlated with the degree of inhibition of protein synthesis. Other inhibitors of protein synthesis, puromycin and emetine, also prevented conversion of arachidonate into PGE2 in these tissues, but they did not inhibit purified PG synthase. Exposure of muscles to actinomycin D for 20 min also reduced PGE2 production from arachidonate by 90%. Thus, both the PG synthase and its mRNA appear to be inactivated rapidly (t1/2 less than 10 min) in muscle and other mammalian tissues. The block in PG production induced by inhibitors of protein and RNA synthesis may account for their antipyrogenic actions and certain of their other physiological effects.
Spreading of human neutrophils is immediately preceded by a large increase in cytoplasmic free calcium.
Authors: Kruskal B A , Shak S , Maxfield F R
Published: May 2006

When human polymorphonuclear leukocytes (PMN) are placed on various surfaces, they attach and spread rapidly, increasing their diameter severalfold. The spreading is associated with extensive changes in the cytoskeleton. Since many cytoskeletal events are regulated by Ca2+, we measured the cytosolic free calcium concentration ([Ca2+]i) in individual human PMN as they spread. [Ca2+]i was measured in single cells by microspectrofluorometry using the fluorescent Ca2+-sensitive dye fura-2. Immediately before spreading, PMN exhibit a rapid increase in [Ca2+]i, from 69 +/- 51 nM to 547 +/- 190 nM (mean +/- SD, n = 12). [Ca2+]i returns to near resting levels during the next minute, as the cells spread. Neither the spreading nor the [Ca2+]i spike is blocked by removal of extracellular calcium, by verapamil, by calmodulin antagonists, or by mitochondrial or microtubule poisons. Spreading, but not the [Ca2+]i increase, is blocked by the microfilament inhibitor cytochalasin B. Both spreading and the [Ca2+]i spike are blocked by ATP depletion and reversibly blocked by placing the cells in medium containing hypertonic sucrose or sodium chloride. These data strongly suggest that an increase in [Ca2+]i, derived from nonmitochondrial intracellular pools, plays an important role in the microfilament-mediated process of PMN spreading.
Purification and characterization of peptides with corticotropin-releasing factor activity from porcine hypothalami.
Authors: Patthy M , Schlesinger D H , Horvath J , Mason-Garcia M , Szoke B , Schally A V
Published: May 2006

Ten polypeptides that stimulated the release of corticotropin from superfused rat pituitary cells and that are structurally related to porcine corticotropin-releasing factor were isolated from porcine hypothalami. The purification was carried out by gel filtration followed by reversed-phase HPLC using trifluoroacetic acid or heptafluorobutyric acid as the ion-pairing agent in water/acetonitrile solvent systems. The purified peptides were homogeneous by chromatography and by sequence analysis. One major polypeptide was characterized. Its structure is -H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Gl u-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2 [Patthy, M., Horvath, J., Mason-Garcia, M., Szoke, B., Schlesinger, D. H. & Schally, A. V. (1985) Proc. Natl. Acad. Sci. USA 82, 8762-8766]. This 41-amino acid sequence is thought to represent porcine corticotropin-releasing factor. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptides, amino acid analysis, and carboxypeptidase Y digestion, the other nine polypeptides were found to be structurally similar to this 41-amino acid sequence. Modifications of this structure include deamidation of glutamine at position 26 or 29, oxidation of methionine at positions 21 and/or 38, a blocked N terminus, and deletion of phenylalanine amide at the C terminus. Eight of these nine modified peptides retained significant corticotropin-releasing factor activity as shown by the stimulation of corticotropin release from superfused rat and pig pituitary cells. Some of these peptides may be present in pig hypothalami, while the others could have been produced during the isolation.
Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.
Authors: Kelner K L , Morita K , Rossen J S , Pollard H B
Published: May 2006

Tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and phenylethanolamine N-methyltransferase, EC 2.1.1.28) are involved in catecholamine biosynthesis and are considered soluble proteins. However, they may actually be localized on the surface of the chromaffin granule. We have used the detergent digitonin to permeabilize the plasma membrane of cultured adrenal chromaffin cells to investigate the subcellular localization of TyrOHase and PMTase. A digitonin titration of the release of proteins and catecholamines revealed the existence of at least three subcellular compartments that are distinguished by their digitonin sensitivity: (i) soluble proteins, which were released upon treatment of the cells with low digitonin concentrations (5 microM), (ii) a "digitonin-sensitive" cytoplasmic protein pool, which required higher concentrations of digitonin for release (10 microM) and included TyrOHase and PMTase, and (iii) the chromaffin granule, which was insensitive to digitonin. Analysis of the rates of release of all of these proteins revealed that the rate of TyrOHase and PMTase release was slower at 10 microM than at 40 microM digitonin, while the rates of release of the other proteins were similar at both concentrations and varied in proportion to their respective sizes. Treatment with cytoskeletal disrupting agents had no effect on TyrOHase or PMTase efflux. These data suggest that TyrOHase and PMTase are in a detergent-labile association in the cell. This is consistent with the concept that TyrOHase and PMTase may be localized on the surface of the chromaffin granule.
Endogenous retroviruses lead to the expression of a histocompatibility antigen detectable by skin graft rejection.
Authors: Colombo M P , Jaenisch R , Wettstein P J
Published: May 2006

Mov mouse strains differ from their respective, coisogenic partner strains by the embryonic, germ-line introduction of Moloney murine leukemia virus genomes. The possibility that retroviral insertions into the mouse genome resulted in gain or loss mutations at non-H-2 histocompatibility loci was investigated by reciprocal skin grafting between Mov mice and mice from coisogenic, background strains. Two B6-derived and eight 129-derived Mov strains were analyzed. B6 mice rejected skin from the viremic Mov-3 and Mov-14 strains, indicating that these mice had new histocompatibility antigens. No rejections were observed with reciprocal skin grafts exchanged between mice of the 129 background strain and 129-derived Mov strains, one of which (Mov-9) is viremic. To investigate the potential viral origin of the new histocompatibility antigen in Mov-14, lymphocytes from B6 mice primed in vivo with Mov-14 cells or skin were restimulated in vitro with Mov-14 spleen cells and with two retroviral-induced B6 lymphomas, MBL-2 and RBL-5. All three cell types stimulated cytotoxic lymphocytes that lysed Mov-14 Con A lymphoblasts, MBL-2 and RBL-5. The same cytotoxic lymphocytes lysed only lymphoblasts from the viremic Mov-9 strain when tested on cells from 129 and 129 Mov mice. Thus the insertion and expression of exogenous Moloney murine leukemia virus results in the appearance of a new histocompatibility antigen as defined by its stimulation of skin-graft rejection and cytotoxic effector T-cell generation. The non-H-2 histocompatibility antigen identified in this study has been designated H-43 and is encoded by genes mapping to different loci in different Mov strains. These observations suggest that at least a subgroup of non-H-2 histocompatibility antigens is encoded by endogenous retroviruses; the implications of these results for understanding the origin and the identity of non-H-2 histocompatibility antigens are discussed.
Shared active sites in oligomeric enzymes: model studies with defective mutants of aspartate transcarbamoylase produced by site-directed mutagenesis.
Authors: Wente S R , Schachman H K
Published: May 2006

Many oligomeric enzymes are functional only in the assembled form, and it is often difficult to determine unambiguously why monomers are inactive. In some cases individual monomers cannot fold into stable correct ("native") conformations without contributions from interchain interactions. For other oligomers, catalysis requires the contributions of amino acid residues at the interface between adjacent polypeptide chains, and monomers are inactive because they cannot form complete active sites. A test for the presence of shared sites was devised that is based on the formation of active hybrid oligomers from appropriate inactive parental mutants produced by site-directed mutagenesis. This approach was applied in a study of the catalytic trimer of aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, using three mutants, in which Ser-52 was replaced by His, Lys-84 by Gln, or His-134 by Ala. Hybrid trimers formed from the virtually inactive Ser and Lys mutants were 10(5) more active than the parental proteins, and the specific activities of each hybrid were about 33% that of the wild-type trimer, as expected for the scheme based on shared sites. Hybrids from the His and Lys mutants had comparable specific activities. Moreover, one hybrid with approximately 33% activity had one high-affinity binding site for a bisubstrate analog as compared to about three for wild-type trimer. As a further test, hybrids were also formed from wild-type and double-mutant (Lys-84----Gln and His-134----Ala) trimers. The hybrid containing two chains with the double mutation and one wild-type chain had very little activity, and that composed of one double mutant and two wild-type chains had 32% the specific activity of wild-type trimers. This negative complementation experiment is in quantitative accord with the scheme based on shared sites at or near the interfaces between adjacent chains. The techniques used to demonstrate shared active sites in the catalytic subunits of aspartate transcarbamoylase can be applied generally to various types of oligomers (dimers, tetramers, etc.) to determine whether the participation of amino acid residues from adjoining chains is essential for forming active sites in oligomeric enzymes.
Heat shock stabilizes highly unstable transcripts of the Xenopus ribosomal gene spacer.
Authors: Labhart P , Reeder R H
Published: May 2006

We have shown recently that, in Xenopus laevis oocytes, the 3' end of the longest detectable ribosomal precursor RNA is not formed by transcription termination but by RNA processing and that RNA polymerase I continues to transcribe through the intergenic spacer region. In oocytes, these spacer transcripts are turned over rapidly, and the only apparent transcription termination site is located 215 base pairs upstream of the 5' end of the next transcription unit. In this paper we show that, at heat shock temperature (34 degrees C), processing at the 3' end of the precursor, rapid turnover of spacer transcripts, and termination are all severely impaired. In contrast, transcription initiation and chain elongation are not significantly affected by heat shock. This results in the appearance of large RNA in the range of 10-20 kilobases and longer.
“Cluster-ion” bombardment of biological macromolecules: Morphological evidence for disassembly of single tobacco mosaic virus particles
Authors: Ledbetter M. C. , Beuhler R. J. , Friedman L.
Published: May 2006

Cluster ions containing 50, 100, or 200 water molecules and one proton are accelerated to over 275 kV and impacted on thin carbon films on which tobacco mosaic virus has been dispersed. After bombardment the films are examined with a transmission electron microscope to study induced morphological alterations with respect to the energy transfer processes that take place during and after single cluster-ion impact. Observations on tobacco mosaic virus indicate that a single cluster impact results in the removal of a cylindrical segment of the virus. No fragments from the excised TMV segment were observed on the carbon foil. We speculate that cluster-ion bombardment may become a useful analytical technique for structural studies of large biological macromolecular complexes.
Protein import into chloroplasts requires a chloroplast ATPase.
Authors: Pain D , Blobel G
Published: May 2006

We have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the [35S]methionine-labeled protein into the stroma of pea chloroplasts. We found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H+, K+, Na+, or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. Our data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. We have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors.
Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure.
Authors: Seed B , Aruffo A
Published: May 2006

A cDNA encoding the CD2 antigen has been isolated by a highly efficient technique based on transient expression in COS cells and adherence of cells expressing surface antigen to antibody-coated dishes. COS cells expressing a CD2 cDNA isolated by this method readily formed rosettes with sheep as well as human and other mammalian erythrocytes. Pretreatment of transfected COS cells with anti-CD2 antibody, or pretreatment of human erythrocytes with anti-LFA-3 antibody, abolished rosette formation.
Working principles in the immune system implied by the "peptidic self" model.
Authors: Kourilsky P , Chaouat G , Rabourdin-Combe C , Claverie J M
Published: May 2006

The hypothesis that self as well as foreign proteins are processed into peptides and presented by major histocompatibility complex antigens leads to a set of working principles that could govern cellular interactions in immune responses. In particular, "idiopeptides," derived from immunoglobulins and T-cell receptors and recognized by appropriate T cells, are expected to play an important regulatory role. We show here that these speculations fit into a consistent view of the immune system.
Tissue distribution and clearance kinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis.
Authors: Craven C M , Alexander J , Eldridge M , Kushner J P , Bernstein S , Kaplan J
Published: May 2006

Genetically hypotransferrinemic mice accumulate iron in the liver and pancreas. A similar pattern of tissue iron accumulation occurs in humans with hereditary hemochromatosis. In both disorders, there is a decreased plasma concentration of apotransferrin. To test the hypothesis that nontransferrin-bound iron exists and is cleared by the parenchymal tissues, the tissue distribution of 59Fe was studied in animals lacking apotransferrin. Two groups of animals were used: normal rats and mice whose transferrin had been saturated by an intravenous injection of nonradiolabeled iron, and mice with congenital hypotransferrinemia. In control animals, injected 59Fe was found primarily in the bone marrow and spleen. In the transferrin iron-saturated animals, injected 59Fe accumulated in the liver and pancreas. Gastrointestinally absorbed iron in hypotransferrinemic or transferrin iron-saturated mice was deposited in the liver. This indicates that newly absorbed iron is released from mucosal cells not bound to transferrin. Clearance studies demonstrated that transferrin-bound 59Fe was removed from the circulation of rats with a half-time of 50 min. In transferrin iron-saturated animals, injected 59Fe was removed with a half-time of less than 30 s. Analysis of the distribution of 59Fe in serum samples by polyacrylamide gel electrophoresis demonstrated the presence of 59Fe not bound to transferrin. These results demonstrate the existence of and an uptake system for non-transferrin-bound iron. These observations support the hypothesis that parenchymal iron overload is a consequence of reduced concentrations of apotransferrin.
Identification and purification of an irreversible presynaptic neurotoxin from the venom of the spider Hololena curta.
Authors: Bowers C W , Phillips H S , Lee P , Jan Y N , Jan L Y
Published: May 2006

A search for potent toxins that inhibit neuronal calcium channels in Drosophila melanogaster has resulted in the identification of a presynaptic neurotoxin from the venom of the hunting spider Hololena curta. Using Drosophila neuromuscular junction as an assay, presynaptic inhibitory activity was purified using gel filtration and reverse-phase HPLC. Data from gel electrophoresis indicate that the toxin is composed of two different subunits of Mr 7000 and 9000. At nanomolar concentrations the toxin produced a complete and long-lasting inhibition of synaptic transmission at the Drosophila larval neuromuscular junction without affecting the amplitudes of the spontaneously occurring miniature junction potentials. The block of transmission produced by the toxin was observed even during the direct depolarization of the motor nerve terminal. These physiological results indicate that the terminal is the site of action for the toxin. Indirect evidence using abnormally excitable Drosophila mutants suggests that the toxin is inhibiting transmitter release by altering the electrical properties of the nerve terminal rather than by interfering with nonelectrical events that may occur subsequent to calcium influx. All of the actions of the Hololena toxin can be explained by a specific and direct effect on presynaptic calcium channels in Drosophila motor neurons.
Characteristics of binding of human seminal alpha-inhibin-92 to human pituitary membranes.
Authors: Ramasharma K , Li C H
Published: May 2006

We investigated the binding of 125I-labeled alpha-inhibin-92 (a 92-residue peptide) to human pituitary membrane preparations. Unlabeled alpha-inhibin-92 competed effectively with the labeled peptide for binding to the membranes. Binding was also inhibited by both alpha-inhibin-52 and alpha-inhibin-31, but less effectively. Scatchard analysis of the alpha-inhibin-92 binding data indicated the presence of high-affinity binding sites (1.35 nM/mg of membrane protein) with an apparent Kd of 0.37 nM. When 125I-labeled alpha-inhibin-92 was covalently crosslinked to the pituitary membrane preparation with disuccinimidyl suberate and the solubilized labeled receptor complex was analyzed by NaDodSO4/PAGE under either reducing or nonreducing conditions, a single radioactive band at an apparent molecular weight of 90,000 +/- 5000 was observed. These data suggest that human pituitary has specific binding sites for alpha-inhibins.
Ubiquitin has intrinsic proteolytic activity: implications for cellular regulation.
Authors: Fried V A , Smith H T , Hildebrandt E , Weiner K
Published: May 2006

Ubiquitin is a protein of 76 amino acids found in every eukaryotic cell. Although ubiquitin is implicated in ATP-dependent nonlysosomal protein degradation and is also conjugated to specific cellular proteins, the role played by ubiquitin in cellular events has not been defined. We report that purified ubiquitin has intrinsic proteolytic activity and demonstrate that this activity is comparable to that of other well-characterized proteases. Monoclonal antibodies specific to ubiquitin inhibit proteolysis. Ubiquitin has protease activity over a broad pH range with an optimum at pH 8.0. It is stimulated by Ca2+ and is inhibited by high concentrations of phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. Ubiquitin will cleave proteins at a limited number of sites. We propose that the ubiquitination of a protein can convert that protein into an ad hoc specific protease and models are presented as to how this can play a role in regulating a variety of cellular events.
Distinct forms of the beta subunit of GTP-binding regulatory proteins identified by molecular cloning.
Authors: Fong H K , Amatruda T T , Birren B W , Simon M I
Published: May 2006

Two distinct beta subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as beta 1 and beta 2 subunits. The bovine transducin beta subunit (beta 1) has been cloned previously. We have now isolated and analyzed cDNA clones that encode the beta 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue Mr 37,329 beta 2 protein is 90% identical with beta 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine beta 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than beta 1 subunit mRNA in all tissues examined. The beta 1 and beta 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that beta 1 and beta 2 are encoded by separate genes. The amino acid sequences for the bovine and human beta 2 subunit are identical, as are the amino acid sequences for the bovine and human beta 1 subunit. This evolutionary conservation suggests that the two beta subunits have different roles in the signal transduction process.
Absence of cytotoxic antibody to human immunodeficiency virus-infected cells in humans and its induction in animals after infection or immunization with purified envelope glycoprotein gp120.
Authors: Nara P L , Robey W G , Gonda M A , Carter S G , Fischinger P J
Published: May 2006

The presence of antibody-dependent complement-mediated cytotoxicity (ACC) was assessed in humans and chimpanzees, which are capable of infection with human immunodeficiency virus isolate HTLV-IIIb, and examined in the goat after immunization with the major viral glycoprotein (gp120) of HTLV-IIIb. In infected humans no antibody mediating ACC was observed regardless of the status of disease. Even healthy individuals with high-titer, broadly reactive, neutralizing antibodies had no ACC. In contrast, chimpanzees infected with HTLV-IIIb, from whom virus could be isolated, not only had neutralizing antibody but also antibodies broadly reactive in ACC, even against distantly related human immunodeficiency virus isolates, as well as against their own reisolated virus. In the goat, the gp120 of HTLV-IIIb induced a highly type-specific response as measured by both ACC and flow cytofluorometry of live infected H9 cells. Normal human cells were not subject to ACC by animal anti-HTLV-III gp120-specific sera. Induction of ACC and neutralizing antibody were closely correlated in the animal experimental models but not in humans. The presence of ACC in gp120-inoculated goats and HTLV-III-infected chimpanzees represents a qualitative difference that may be important in the quest for the elicitation of a protective immunity in humans.
Assessment of antigen-specific receptor function of surface immunoglobulin M and D with identical hapten specificity.
Authors: Tisch R , Watanabe M , Letarte M , Hozumi N
Published: May 2006

Monoclonal B-cell lines expressing antigen-specific surface IgM or IgD were established by transferring the genes encoding immunoglobulin heavy (mu or delta) and light (kappa) chains specific for the hapten 2,4,6-trinitrophenyl (TNP) into a B-cell lymphoma line. Two IgMTNP and two IgDTNP transformants were selected on the basis of similar levels of anti-TNP idiotype expression by flow microfluorimetric analysis. The IgMTNP and IgDTNP transformants were compared in quantitative assays for their ability to bind TNP-carrier and present TNP-carrier to carrier-specific T cells. Our results indicate that IgMTNP and IgDTNP transformants have an equal capacity to bind and present specific antigen. Thus surface IgM and IgD, when present in equivalent amounts, function similarly as antigen receptors.
Endogenous adenosine and adenosine receptors localized to ganglion cells of the retina.
Authors: Braas K M , Zarbin M A , Snyder S H
Published: May 2006

Using specific sensitive antisera against adenosine, we have immunocytochemically localized endogenous adenosine to specific layers of rat, guinea pig, monkey, and human retina. Highest adenosine immunoreactivity was observed in ganglion cells and their processes in the optic nerve fiber layer. Substantial staining was also found throughout the inner plexiform layer and in select cells in the inner nuclear layer. Adenosine A1 receptors, labeled with the agonists L-[3H]phenylisopropyladenosine and 125I-labeled hydroxy-phenylisopropyladenosine, were autoradiographically localized. The highest levels of binding sites occurred in the nerve fiber, ganglion cell, and inner plexiform layers of the retina in all the species examined. The distribution of adenosine A1 receptor sites closely parallels that of retinal neurons and fibers containing immunoreactive adenosine. These results suggest a role for endogenous adenosine as a coneurotransmitter in ganglion cells and their fibers in the optic nerve.
Neurons in the dorsal motor nucleus of the vagus nerve are excited by oxytocin in the rat but not in the guinea pig.
Authors: Raggenbass M , Dubois-Dauphin M , Charpak S , Dreifuss J J
Published: May 2006

Intracellular recordings were obtained from vagal neurons and their response to oxytocin was investigated in slices from the rat and the guinea pig brainstem. After recording, Lucifer yellow was injected into the cells to verify their localization within the dorsal motor nucleus of the vagus nerve (dmnX). In the rat, virtually all neurons throughout the rostrocaudal extent of the dmnX increased their rate of firing in the presence of 10-1000 nM oxytocin and their membrane depolarized in a reversible concentration-dependent manner. This excitation was probably exerted directly on the impaled cells rather than being synaptically mediated, since it persisted in a low calcium/high magnesium medium or in the presence of tetrodotoxin. These data provide evidence for a direct membrane effect of oxytocin on a defined population of neurons in the rat brain. In the guinea pig, vagal neurons were fired by glutamate but were not excited by oxytocin, even though we detected many more oxytocin-immunoreactive structures in the guinea pig dmnX than in the rat dmnX. Therefore, homologous nuclei in the brains of two closely related mammals differ markedly in the density of oxytocinergic axons they contain. Unexpectedly, the magnitude of the electrophysiological effects of oxytocin on vagal neurons appeared inversely related to the amount of oxytocin-like immunoreactivity present in dmnX.
Exact Hausdorff dimension in random recursive constructions
Authors: Mauldin R. D. , Graf S. , Williams S. C.
Published: May 2006

The exact Hausdorff dimension function is determined for sets in R

m

constructed by using a recursion that is governed by some given law of randomness.
Structure and expression of the genes,
mcrBDCGA
, which encode the subunits of component C of methyl coenzyme M reductase in
Methanococcus vannielii
Authors: Cram D. S. , Sherf B. A. , Libby R. T. , Mattaliano R. J. , Ramachandran K. L. , Reeve J. N.
Published: May 2006

The genes that encode the α, β, and γ subunits of component C of
m
ethyl
c
oenzyme M
r
eductase (
mcrA, mcrB
, and
mcrG
) in
Methanococcus vannielii
have been cloned and sequenced, and their expression in
Escherichia coli
has been demonstrated. These genes are organized into a five-gene cluster,
mcrBDCGA
, which contains two genes, designated
mcrC
and
mcrD
, with unknown functions. The
mcr
genes are separated by very short intergenic regions that contain multiple translation stop codons and strong ribosomebinding sequences. Although the genome of
M. vannielii
is 69 mol% A+T, there is a very strong preference in the
mcrA, mcrB
, and
mcrG
genes for the codon with a C in the wobble position in the codon pairs AA
U
C
(asparagine), GA
U
C
(aspartic acid), CA
U
C
(histidine), AU
U
C
(isoleucine), UU
U
C
(phenylalanine), and UA
U
C
(tyrosine). The
mcrC
and
mcrD
genes do not show this codon preference and frequently have U or A in the wobble position. As the codon pairs listed above are likely to be translated by the same tRNA with a G in the first anticodon position, the presence of C in the wobble position might ensure maximum efficiency of translation of transcripts of these very highly expressed genes.
Specific requirement for ATP at an early step of in vitro transcription of human mitochondrial DNA.
Authors: Narasimhan N , Attardi G
Published: May 2006

The ATP concentrations allowing transcription of both heavy- and light-strand of human mtDNA in a HeLa cell mitochondrial lysate were found to cover a broad range, with a maximum around 2.5 mM, and with reproducible differences in the ATP response curves for the two transcription events. Direct measurements showed that nonspecific ATP degradation during the assay did not account for the high ATP requirement. 5'-Adenylyl imidodiphosphate (p[NH]ppA), an ATP analog with a nonhydrolyzable beta-gamma bond, was unable to substitute for ATP in supporting mtDNA transcription but greatly stimulated this transcription in the presence of a low concentration of exogenous ATP. Evidence was obtained indicating that p[NH]ppA did not support an early event in mtDNA transcription (formation of preinitiation complex or initiation), whereas this analog could substitute effectively for ATP in the subsequent elongation steps. These results pointed to a specific requirement for ATP at an early step of the transcription process.
Genesis of a spatial pattern in the cellular slime mold Polysphondylium pallidum.
Authors: Byrne G , Cox E C
Published: May 2006

The branches in Polysphondylium pallidum whorls are arranged in a radial pattern. We have used a pattern-specific monoclonal antibody to study branch formation and characterize the origin of this pattern. A quantitative spatial analysis of antibody staining reveals that the branching pattern arises from a random distribution. This distribution passes through a series of intermediate stages to yield a radial prepattern. The origins and evolution of this prepattern are satisfactorily accounted for by models that produce spatial patterns by short-range autocatalytic and longer-range inhibitory forces.
Monoclonal antibody to an intracellular antigen images human melanoma transplants in nu/nu mice.
Authors: Welt S , Mattes M J , Grando R , Thomson T M , Leonard R W , Zanzonico P B , Bigler R E , Yeh S , Oettgen H F , Old L J
Published: May 2006

Mouse monoclonal antibody TA99 detects a 70-kDa pigmentation-associated glycoprotein in human melanoma cell lines. The antigen cannot be detected on the cell surface by sensitive rosetting techniques or absorption studies, nor can it be detected as a secreted product in culture fluids. Contrary to expectation, 125I-labeled TA99 specifically localized to pigmented human melanoma transplants in nu/nu mice; no localization to nonpigmented melanoma or control tumors was found. Tumor imaging was initially obscured by circulating 125I-labeled TA99 during the first 6 days after antibody injection. With clearance of 125I-labeled TA99 from the blood (half-life, 4-7 days), specific tumor images could be clearly defined by day 13. Due to the persistence of 125I-labeled TA99 at the tumor site (8.9% of the injected dose at 1 week and 4.6% at 8-10 weeks), images were obtainable for up to 10 weeks. At 8-10 weeks, the tumor/blood ratio was 10(4)-10(5), and the tumor/normal tissue ratio ranged from 10(2) to 10(5). In view of these findings, antibodies detecting intracellular antigens may have a role in tumor imaging and therapy.
Functional characterization of an antigen involved in an early step of T-cell activation.
Authors: Cosulich M E , Rubartelli A , Risso A , Cozzolino F , Bargellesi A
Published: May 2006

An activation antigen, identified by the monoclonal antibody MLR3, is described that is present on activated T lymphocytes and thymocytes but not on resting T lymphocytes. Immunoprecipitation of radiolabeled membranes from an activated T-cell line showed that the MLR3-binding molecule has a molecular size of 28-34 kDa. Immunofluorescence analysis showed that the appearance of the MLR3 antigen is an early event and precedes that of the interleukin 2 receptor both in T lymphocytes and thymocytes. The proliferative response of resting T cells to OKT3-Sepharose and interleukin 1 or accessory cells, but not the interleukin 2-dependent proliferation, was inhibited by the addition of MLR3 monoclonal antibodies. Similar results wer also obtained in an interleukin 1-dependent human thymocyte proliferation assay. In addition when MLR3-positive cells were cultured with purified interleukin 1, MLR3 surface antigen expression was not observed. Thus MLR3 monoclonal antibody appears to recognize an antigen involved in an early step of T-cell activation related to interleukin 1-dependent functions and on both T lymphocytes and thymocytes.
Neuroanatomical patterns of the mu, delta, and kappa opioid receptors of rat brain as determined by quantitative in vitro autoradiography.
Authors: Tempel A , Zukin R S
Published: May 2006

Highly specific radioligands and quantitative autoradiography reveal strikingly different neuroanatomical patterns for the mu, delta, and kappa opioid receptors of rat brain. The mu receptors are most densely localized in patches in the striatum, layers I and III of the cortex, the pyramidal cell layer of the hippocampal formation, specific nuclei of the thalamus, the pars reticulata of the substantia nigra, the interpeduncular nucleus, and the locus coeruleus. In contrast, delta receptors are highly confined, exhibiting selective localization in layers I, II, and VIa of the neocortex, a diffuse pattern in the striatum, and moderate concentration in the pars reticulata of the substantia nigra and in the interpeduncular nucleus. delta receptors are absent in most other brain structures. This distribution is unexpected in that the enkephalins, the putative endogenous ligands of the delta receptor, occur essentially throughout the brain. The kappa receptors of rat brain exhibit a third pattern distinct from that of the mu and delta receptors. kappa receptors occur at low density in patches in the striatum and at particularly high density in the nucleus accumbens, along the pyramidal and molecular layers of the hippocampus, in the granular cell layer of the dentate gyrus, specific midline nuclei of the thalamus, and hindbrain regions. kappa receptors appear to be uniformly distributed across regions in the neocortex with the exception of layer III, which revealed only trace levels of binding. An important conclusion of the present study is that delta receptors occur at high density only in the forebrain and in two midbrain structures, whereas mu and kappa receptors exhibit discrete patterns in most major brain regions.
Syrinx 2A: an improved lambda phage vector designed for screening DNA libraries by recombination in vivo.
Authors: Lutz C T , Hollifield W C , Seed B , Davie J M , Huang H V
Published: May 2006

The Syrinx 2A phage and pi AN13 plasmid were designed for screening of DNA libraries by homologous recombination in vivo. Syrinx 2A carries multiple cloning sites and a recently identified lambda gene, rap (recombination adept with plasmid), required for efficient phage-plasmid recombination. We describe a rapid, reliable, and technically easy method to screen Syrinx 2A libraries, expand the resulting phage-plasmid cointegrates, and subclone plasmid in as little as 2 days. Recombination screening allows one specific member of a closely related multigene family to be isolated selectively.
Next-nucleotide effects in mutations driven by DNA precursor pool imbalances at the aprt locus of Chinese hamster ovary cells.
Authors: Phear G , Nalbantoglu J , Meuth M
Published: May 2006

Imbalances of the intracellular pools of the precursors of DNA synthesis, the deoxyribonucleoside triphosphates, produce marked shifts in the spectrum of mutations at the aprt locus of Chinese hamster ovary cells. Mutations induced by excess dTTP or dCTP are dominated by misincorporation of the nucleotide in excess, as determined by sequence analysis of cloned mutant genes. The shift in spectrum is also apparently influenced by the nucleotides surrounding the one altered--those 3' to the nucleotide misincorporated being present in excess in most of the mutant genes characterized. Since next-nucleotide effects are a property of DNA polymerases with "proofreading" activities, our data suggest that this function is part of the mammalian DNA replication complex.
Organization and nucleotide sequence determination of a gene cluster involved in 3-chlorocatechol degradation.
Authors: Frantz B , Chakrabarty A M
Published: May 2006

Three critical enzymes catechol oxygenase II (chlorocatechol dioxygenase), muconate cycloisomerase II, and dienelactone hydrolase, are involved in the degradation of chlorocatechols, which are obligatory intermediates in the catabolism of chlorinated aromatic compounds. The organization and complete nucleotide sequence of the genes for these enzymes have been determined on a 4.2-kilobase-pair (kbp) Bgl II fragment cloned from the plasmid pAC27, based on the agreement of open reading frame lengths with apparent mobilities of polypeptides expressed in Escherichia coli maxicells, predicted N-terminal amino acid sequences with those of the purified proteins, and predicted total amino acid compositions with those of the purified proteins. The 4.2-kbp fragment contains the three genes and ribosome binding sites for those genes but no promoter. When placed downstream of the tac promoter in the broad-host-range plasmid pMMB22, this fragment directs the synthesis of all three enzymes in both E. coli and Pseudomonas putida only on induction with isopropyl beta-D-thiogalactopyranoside, suggesting that the gene cluster is regulated as a single unit under the control of a single promoter. Endogenous transcription initiation of the gene cluster on pAC27, however, occurs from a site present within a 386-bp Bgl II fragment upstream of the 4.2-kbp fragment, and sequences 5' to that site are similar to the sequences of other positively controlled Pseudomonas promoters occurring on the TOL and NAH plasmids.
Prothymosin alpha in human blood.
Authors: Panneerselvam C , Haritos A A , Caldarella J , Horecker B L
Published: May 2006

The major cross-reacting peptide in human plasma detected with a radioimmunoassay (RIA) for thymosin alpha 1 was identified as prothymosin alpha, based on its elution properties in gel-filtration chromatography and its amino acid composition after purification by HPLC. A small quantity (less than 10%) of the total cross-reacting material was recovered in fractions corresponding to lower molecular weight thymosin alpha 1-like peptides. The total quantity of cross-reacting material detected in human blood, expressed as thymosin alpha 1 equivalents, was 11-14 pmol/ml (approximately 90% was recovered in the leukocyte fraction, approximately 10% was in the plasma fraction, and 1-2% was in the erythrocyte fraction). The peptide present in leukocytes was also identified as prothymosin alpha. After correction for the 5-times lower molar reactivity of prothymosin alpha in the thymosin alpha 1 RIA employed in these experiments, we estimate that the content of prothymosin alpha in human blood is 55-70 pmol/ml (0.6-0.8 microgram/ml). The relatively small quantities recovered in the erythrocyte and plasma fractions may be attributed to contamination of the former by leukocytes or to leakage from leukocytes into the plasma.
A single base change in the Shine-Dalgarno region of 16S rRNA of Escherichia coli affects translation of many proteins.
Authors: Jacob W F , Santer M , Dahlberg A E
Published: May 2006

A single base mutation was constructed at position 1538 of Escherichia coli 16S rRNA, changing a cytidine to a uridine. This position is in the Shine-Dalgarno region, thought to be involved in base-pairing to mRNA during initiation of protein synthesis. The mutation was constructed by using a synthetic oligodeoxynucleotide that differs in sequence by one base from the wild-type sequence of 16S rRNA. This oligonucleotide was used as a primer on single-stranded DNA of phage M13, into which was cloned a specific region of DNA encoding 16S rRNA. The mutation is lethal when expressed from the normal promoters of rRNA operons, P1 and P2, in a high-copy-number plasmid. Expression can be repressed by a temperature-sensitive repressor, cI857, in combination with the bacteriophage lambda PL promoter. Induction of transcription by temperature shift yields mutant 16S rRNA that is processed and assembled into functional ribosomal subunits. The presence of mutant ribosomes retards cell growth and dramatically alters incorporation of [35S]methionine into a large proportion of the cellular proteins. The change in level of synthesis of individual proteins correlates with the change in base-pairing between mutant rRNA and the Shine-Dalgarno region of the mRNA.
Multiple sequence elements are required for regulation of human T-cell leukemia virus gene expression.
Authors: Rosen C A , Park R , Sodroski J G , Haseltine W A
Published: May 2006

The U3 region of the long terminal repeat (LTR) of human T-cell leukemia virus type I (HTLV-I) contains sequences that respond to the trans-activating transcription (tat) factor encoded by the pX region of the provirus. Results presented here show that there are multiple tat-responsive sequences within the LTR and that a single 21-nucleotide sequence, which is repeated three times within the U3 region, is sufficient to determine the response to the trans-activator. This sequence is capable of conferring a tat-responsive phenotype upon the HTLV-I and simian virus 40 promoters, independent of orientation. Sequences required for efficient HTLV-I LTR-directed gene expression are also located 3' to the site of RNA initiation, within the R and U5 regions of the LTR.
Platinum-DNA adducts in leukocyte DNA correlate with disease response in ovarian cancer patients receiving platinum-based chemotherapy.
Authors: Reed E , Ozols R F , Tarone R , Yuspa S H , Poirier M C
Published: May 2006

Fifty-five ovarian cancer patients receiving platinum drug-based chemotherapy have been studied prospectively to determine the extent of formation of the bidentate intrastrand adducts of diammineplatinum covalently attached to the N7 positions of adenosine and/or guanosine in leukocyte DNA. Data for clinical response, obtained from medical records, were then correlated with the adduct values. Patients were treated with platinum-based single-agent or combination chemotherapy containing cis-diamminedichloroplatinum (II) or diamminecyclobutane-dicarboxylatoplatinum on approved experimental protocols. Adduct measurements were performed by ELISA, and disease response to therapy was assessed by standard oncologic criteria. This study comprises a total of 101 blood samples obtained after intravenous cis-diamminedichloroplatinum (II) or diamminecyclobutane-dicarboxylatoplatinum infusion from 55 individuals, and in each case the highest (or "peak") adduct level for each patient was chosen for statistical analysis. Values for median adduct levels in patients grouped by complete response, partial response, and no response were 212, 193, and 62 amol of adduct per microgram of DNA, respectively. Analysis of these data by Jonckheere's test (an extension of the Mann-Whitney test) shows that higher levels of adduct formation correlates with disease response with a two-sided P value of 0.030. Of eight patients on single-agent therapy whose buffy-coat samples did not have measurable adduct levels, none responded to therapy. Analysis of these data using the exact test for trend shows that the formation of adduct at a level of 160 amol/micrograms of DNA or greater correlates with disease response with a two-sided P value of 0.032. Thus in ovarian cancer patients, the formation of the intrastrand diammineplatinum adducts in leukocyte DNA is associated with favorable disease response to cis-diamminedichloroplatinum (II) or diamminecyclobutane-dicarboxylatoplatinum chemotherapy.