bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibodies to the binding site of the receptor for luteinizing hormone-releasing hormone (LHRH): generation with a synthetic decapeptide encoded by an RNA complementary to LHRH mRNA.
Authors: Mulchahey J J , Neill J D , Dion L D , Bost K L , Blalock J E
Published: May 2006

A molecular recognition code has been hypothesized to exist in which ligands and their binding sites are encoded on complementary segments of genomic DNA. We have tested this hypothesis by generating a rabbit antibody to a synthetic decapeptide (complementary peptide) encoded by an RNA complementary to the mRNA for luteinizing hormone-releasing hormone (LHRH) and determining whether this antibody recognizes the LHRH receptor. When the antibody was used for immunoperoxidase staining of enzymatically dispersed rat anterior pituitary cells, only those that contained and secreted luteinizing hormone (i.e., the gonadotropes) were recognized. This staining could be abolished by preincubation with the complementary peptide or with an LHRH agonist, suggesting that the antibody is specific to the complementary peptide and is directed at the binding site of the receptor. Further evidence that the antibody recognizes the LHRH receptor was obtained in immunoblot experiments on solubilized receptors from pituitary glands. Immunoperoxidase staining with the antibody revealed two bands at 60 kDa and 51 kDa, which are values similar to those previously obtained for the LHRH receptor in photoaffinity-labeling experiments. The staining of these bands was inhibited by preincubation with the complementary peptide or an LHRH agonist. The antibody as well as the complementary peptide to LHRH also suppressed LHRH-stimulated luteinizing hormone release in a quantitative reverse hemolytic plaque assay, presumably by binding to the LHRH receptor and by binding LHRH, respectively. These findings suggest that the synthetic decapeptide whose sequence is specified by the complementary RNA to LHRH mRNA is sufficiently similar to an LHRH binding site that the peptide not only binds LHRH but was also recognized by the immune system as such a site. These findings provide strong support for the hypothesis that recognition molecules are encoded by complementary segments of genomic DNA.
Long-term modulation of Ca2+-stimulated autophosphorylation and subcellular distribution of the Ca2+/calmodulin-dependent protein kinase in the brain of Drosophila.
Authors: Willmund R , Mitschulat H , Schneider K
Published: May 2006

After prolonged visual adaptation of Drosophila, dramatic long-term changes of in vitro phosphorylation of a 50-kDa brain protein that is identical to the Ca2+/calmodulin-dependent protein kinase (EC 2.7.1.37) can be measured in isolated heads. By selective receptor cell desensitization in blue light, subcellular distribution of the 50-kDa kinase in fly brain is modified, and Ca2+-stimulated in vitro phosphorylation is increased. Concomitantly the 50-kDa kinase is translocated by in vitro phosphorylation from the membrane-cytoskeleton complex into the cytoplasm. After adaptation, association of the enzyme to the membrane shows long-term modification. In yellow light, which reverts receptor cell adaptation within seconds, the changes in kinase activity and distribution remain for about 2 hr, corresponding to the duration of behavioral modification induced by blue light. Reducing protein synthesis with cycloheximide inhibits the induction of behavioral modification as well as the prolonged modulation of the 50-kDa kinase by blue light. From our simple assay to measure biochemical changes induced in the intact organism by sensory stimulation, we propose that Ca2+/calmodulin-dependent kinase II is involved in long-term modulation of synaptic transmission.
A purified transcription factor (TIF-IB) binds to essential sequences of the mouse rDNA promoter.
Authors: Clos J , Buttgereit D , Grummt I
Published: May 2006

A transcription factor that is specific for mouse rDNA has been partially purified from Ehrlich ascites cells. This factor [designated transcription initiation factor (TIF)-IB] is required for accurate in vitro synthesis of mouse rRNA in addition to RNA polymerase I and another regulatory factor, TIF-IA. TIF-IB activity is present in extracts both from growing and nongrowing cells in comparable amounts. Prebinding competition experiments with wild-type and mutant templates suggest that TIF-IB interacts with the core control element of the rDNA promoter, which is located immediately upstream of the initiation site. The specific binding of TIF-IB to the RNA polymerase I promoter is demonstrated by exonuclease III protection experiments. The 3' border of the sequences protected by TIF-IB is shown to be on the coding strand at position -21 and on the noncoding strand at position -7. The results suggest that direct binding of TIF-IB to sequences in the core promoter element is the mechanism by which this factor imparts promoter selectivity to RNA polymerase I.
Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts.
Authors: Flier J S , Usher P , Moses A C
Published: May 2006

Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [3H]thymidine into DNA in these cells, the identity of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody alpha IR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125I-labeled IGF-I but not 125I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. alpha IR-3 competitively inhibits IGF-I-mediated stimulation of [3H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of alpha IR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of less than 1 microgram/ml, the effect of insulin to stimulate [3H]thymidine incorporation is not inhibited by alpha IR-3. However, the incremental effects of higher concentrations (greater than 1 microgram/ml) of insulin on [3H]thymidine incorporation are inhibited by alpha IR-3. alpha IR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself.
Rates of nuclear DNA evolution in pheasant-like birds: evidence from restriction maps.
Authors: Helm-Bychowski K M , Wilson A C
Published: May 2006

To examine the tempo of genomic evolution in birds, we mapped 161 restriction sites in the nuclear DNA of seven species of birds belonging to the pheasant superfamily Phasianoidea. The three regions mapped lie on different chromosomes and bear eight genes, coding for lysozyme c, three "alpha-like" globins, and four "beta-like" globins. Together, the three regions span about 56 kilobases, most of which is presumably noncoding. The maps differed from one another at a minimum of 77 sites and by 9 length mutations. The extent of sequence divergence due to base substitutions was inferred to be similar for all three regions, even though the three coding regions differ by 5-fold from one another in mean rate of evolution at the amino acid level. A tree relating the maps differs in branching order from that implied by the traditional classification of phasianoid birds and is supported by published protein comparisons. Five of the nodes in the tree were associated with fossil evidence and historical biogeographic information, allowing us to estimate the mean rate of DNA divergence to be 0.34-0.40% per million years. This rate is similar to that estimated for the globin gene regions of higher primates, which validates the concept of an evolutionary clock at the DNA level. Our fossil-based calibration of DNA evolution differs by a factor of almost 2 from that proposed by others on the basis of biogeography. In consequence, published estimates of divergence times for birds and primates that are based on a biogeographically calibrated DNA clock may be too long.
A complete library of point substitution mutations in the glucocorticoid response element of mouse mammary tumor virus.
Authors: Hutchison C A , Nordeen S K , Vogt K , Edgell M H
Published: May 2006

The glucocorticoid response element (GRE) of mouse mammary tumor virus (MMTV) was chemically synthesized as two complementary DNA strands bearing cohesive termini. During automated synthesis, random mutations were introduced into the DNA by "doping" each of the four nucleoside phosphoramidites (A, G, C, and T) with a low level of the other three. These preparations were annealed and cloned into an M13 phage vector to produce a library of GRE mutants. Mutations within the synthesized region were identified by sequencing phage isolates at random. All of the chemically distinct classes of transition and transversion mutations have been observed. Statistical considerations indicate that the library contains all of the possible 90 point substitution mutations within a 30-nucleotide mutagenic target. So far 88 of these substitutions have been isolated, 74 as single mutants. At least two of the three possible single mutants at each of the 30 positions have been identified.
High-efficiency genetic transformation of maize by a mixture of pollen and exogenous DNA
Authors: Ohta Yasuo
Published: May 2006

High-efficiency genetic transformation was induced in a genetic stock of maize,
Zea mays
Linnaeus, by self-pollination of the recipient plants along with DNA of the donor. The highest frequency of transformed endosperm per ear was 9.29%. DNA was applied onto silks in a pollen/DNA pasty mixture. The exogenous DNA transferred into endosperm expressed itself in endosperm formation. It is not known, however, whether the exogenous DNA segment in endosperm had been incorporated or whether it was present in the nucleus additionally, fragmentally, or in some other state. It was revealed that all of four possible cases had occurred as follows: exogenous DNA was transferred into (
i
) both embryo and endosperm of the same kernel, (
ii
) embryo only, (
iii
) endosperm only, and (
iv
) neither embryo nor endosperm. It was also revealed that exogenous DNA that had entered into the embryo had been maintained through embryo formation, germination, vegetative growth and differentiation, and reproductive growth, and finally was manifested in embryo and/or endosperm of the following generation and acted in endosperm formation. The frequency of transformed endosperm in this generation, however, was rather low. It may be that exogenous DNA taken into the embryo was unstable.
Induction of NAD(P)H:quinone reductase in murine hepatoma cells by phenolic antioxidants, azo dyes, and other chemoprotectors: a model system for the study of anticarcinogens.
Authors: De Long M J , Prochaska H J , Talalay P
Published: May 2006

Exposure of murine hepatoma (Hepa 1c1c7) cells to a variety of chemical agents known to protect animals against the neoplastic, mutagenic, and other toxic effects of chemical carcinogens results in dose- and time-dependent inductions of NAD(P)H:quinone reductase (EC 1.6.99.2). This enzyme protects against quinone toxicity by promoting obligatory two-electron reductions that divert quinones from oxidative cycling or direct interactions with critical nucleophiles. Quinone reductase levels are stable in culture, are easily measured, and are useful markers for the inductive effects of chemoprotective agents. The Hepa 1c1c7 system responds to chemoprotective compounds such as phenolic antioxidants (e.g., BHA [3(2)-tert-butyl-4-hydroxyanisole], BHT (3,5-ditert-butyl-4-hydroxytoluene), and tert-butylhydroquinone), lipophilic azo dyes belonging to the 1,1'-azonaphthalene, Sudan I (1-phenylazo-2-naphthol), and Sudan III [1-(4-phenylazophenylazo)-2-naphthol] families, polycyclic aromatic hydrocarbons, coumarin and various other lactones, flavonoids, and certain sulfur compounds (e.g., benzylisothiocyanate, dithiolthiones, and dithiocarbamates), all of which are recognized enzyme inducers and chemoprotectors in vivo. Quinone reductase induction in Hepa 1c1c7 cells therefore provides a simple, versatile, and reliable system for the evaluation of the potency, kinetics, and mechanism of action of anticarcinogens.
Monoclonal antibody MB19 detects genetic polymorphism in human apolipoprotein B.
Authors: Young S G , Bertics S J , Curtiss L K , Casal D C , Witztum J L
Published: May 2006

Using a specific monoclonal antibody (MB19) against human apolipoprotein B (apo B), we have detected a genetic polymorphism in human low density lipoprotein (LDL). MB19 bound to LDL from different individuals in one of three distinct patterns of immunoreactivity: strong, weak, and intermediate. Scatchard analysis revealed that LDLs with strong and with weak binding patterns differed 10-fold in their affinity for MB19, but both bound the same total amount of antibody (about one mole of MB19 per mole of apo B). LDL showing the intermediate binding pattern yielded a curvilinear Scatchard plot that could be resolved into two distinct components with affinities similar to those of LDLs exhibiting only the high- or only the low-affinity binding of MB19. LDL chemical composition was similar for all three MB19 binding patterns, and the polymorphism remained after removal of LDL lipid or carbohydrate. Analysis of plasmas from 77 unrelated individuals indicated that 40% of them bound MB19 with low affinity, 23% with high affinity, and 36% with intermediate or "hybrid" affinity. Family studies showed that the three MB19 binding patterns result from codominant transmission of two common apo B alleles, each coding for an allotype with different affinity for MB19, conditionally designated here MB19(1) (high affinity) and MB19(2) (low affinity).
Facilitating and nonfacilitating synapses on pyramidal cells: a correlation between physiology and morphology.
Authors: Bower J M , Haberly L B
Published: May 2006

Pyramidal cells in piriform cortex receive excitatory inputs from two different sources that are segregated onto adjacent segments of their apical dendrites. The present studies show that excitatory postsynaptic potentials (EPSPs) evoked by primary olfactory tract afferents that terminate on distal apical segments display paired shock facilitation whereas ESPSs evoked by intrinsic association fibers that terminate on proximal apical segments do not. An ultrastructural comparison of the presynaptic elements of these two fiber systems has revealed that the facilitating olfactory tract afferent synapses have a much lower packing density of synaptic vesicles than do the nonfacilitating association fiber synapses. Further, a search of the literature has revealed that where both morphological and physiological data are available for the same synapses, this same correlation appears to apply. We propose a hypothesis to account for this correlation based on synaptic vesicles to buffer internal calcium and the biochemical characteristics of preterminal calcium-dependent mechanisms affecting the number of vesicles available for release.
Bradykinin-activated transmembrane signals are coupled via No or Ni to production of inositol 1,4,5-trisphosphate, a second messenger in NG108-15 neuroblastoma-glioma hybrid cells.
Authors: Higashida H , Streaty R A , Klee W , Nirenberg M
Published: May 2006

The addition of bradykinin to NG108-15 cells results in a transient hyperpolarization followed by prolonged cell depolarization. Injection of inositol 1,4,5-trisphosphate or Ca2+ into the cytoplasm of NG108-15 cells also elicits cell hyperpolarization followed by depolarization. Tetraethylammonium ions inhibit the hyperpolarizing response of cells to bradykinin or inositol 1,4,5-trisphosphate. Thus, the hyperpolarizing phase of the cell response may be due to inositol 1,4,5-trisphosphate-dependent release of stored Ca2+ into the cytoplasm, which activates Ca2+-dependent K+ channels. The depolarizing phase of the cell response to bradykinin is due largely to inhibition of M channels, thereby decreasing the rate of K+ efflux from cells and, to a lesser extent, to activation of Ca2+-dependent ion channels and Ca2+ channels. In contrast, injection of inositol 1,4,5-trisphosphate or Ca2+ into the cytosol did not alter M channel activity. Incubation of NG108-15 cells with pertussis toxin inhibits bradykinin-dependent cell hyperpolarization and depolarization. Bradykinin stimulates low Km GTPase activity and inhibits adenylate cyclase in NG108-15 membrane preparations but not in membranes prepared from cells treated with pertussis toxin. Reconstitution of NG108-15 membranes from cells treated with pertussis toxin with nanomolar concentrations of a mixture of highly purified No and Ni [guanine nucleotide-binding proteins that have no known function (No) or inhibit adenylate cyclase (Ni)] restores bradykinin-dependent activation of GTPase and inhibition of adenylate cyclase. These results show that [bradykinin . receptor] complexes interact with No or Ni and suggest that No and/or Ni mediate the transduction of signals from bradykinin receptors to phospholipase C and adenylate cyclase.
Cloning of the Escherichia coli gene for primosomal protein i: the relationship to dnaT, essential for chromosomal DNA replication.
Authors: Masai H , Bond M W , Arai K
Published: May 2006

The Escherichia coli gene encoding one of the primosomal proteins, protein i, was cloned by the use of synthetic oligonucleotide probes. Nucleotide sequence analysis revealed a coding region for protein i of 537 base pairs preceded by a possible promoter sequence. The gene is located adjacent to the dnaC locus, probably both being in a single operon. The protein i gene was shown to be closely related to the dnaT locus based on the following observations. (i) A multicopy plasmid carrying only the protein i gene suppresses the temperature-sensitive phenotype of a dnaT strain and restores the ability of the strain to carry out stable DNA replication in the absence of protein synthesis. (ii) An extract from a dnaT strain does not support replication of the plasmid pBR322 in vitro; addition of purified protein i restores its activity. These results indicate that protein i is encoded by dnaT and that it is essential for chromosomal DNA replication and is involved in the induction of stable DNA replication during the SOS response.
Structure-activity relationship in vinculin: an IR/attenuated total reflection spectroscopic and film balance study.
Authors: Fringeli U P , Leutert P , Thurnhofer H , Fringeli M , Burger M M
Published: May 2006

Surfacing and membrane-penetrating ability of vinculin and bovine serum albumin have been studied on a macroscopic level by means of a Langmuir film balance and on a molecular level by means of infrared attenuated total reflection spectroscopy. It is suggested that the driving force of the nonspontaneous process of membrane penetration by native vinculin is the spontaneous formation of rigid vinculin monolayers in the membrane. Lateral adhesion of vinculin molecules results from the formation of intermolecular pleated-sheet structures. Vinculin surface activity was found to result from an alpha-helical segment oriented approximately perpendicular to plane of the membrane. There is a conformational equilibrium between this helix and random structure. High ionic strength (110 mM) favors helix formation that leads to the greater than 100-fold enhancement of surfacing velocity relative to the velocity observed at a lower ionic strength (10 mM). Vinculin has a second helical segment oriented parallel to the plane of the membrane that is in a conformational equilibrium with the pleated-sheet structure.
Evolutionary diversity of eukaryotic small-subunit rRNA genes.
Authors: Sogin M L , Elwood H J , Gunderson J H
Published: May 2006

The small-subunit rRNA gene sequences of the flagellated protists Euglena gracilis and Trypanosoma brucei were determined and compared to those of other eukaryotes. A phylogenetic tree was constructed in which the earliest branching among the eukaryotes is represented by E. gracilis. The E. gracilis divergence far antedates a period of massive evolutionary radiation that gave rise to the plants, animals, fungi, and certain groups of protists such as ciliates and the acanthamoebae. The genetic diversity in this collection of eukaryotes is seen to exceed that displayed within either the eubacterial or the archaebacterial lines of descent.
Mutations of the Drosophila myosin heavy-chain gene: effects on transcription, myosin accumulation, and muscle function.
Authors: Mogami K , O'Donnell P T , Bernstein S I , Wright T R , Emerson C P
Published: May 2006

Mutations of the myosin heavy-chain (MHC) gene of Drosophila melanogaster were identified among a group of dominant flightless and recessive lethal mutants (map position 2-52, 36A8-B1,2). One mutation is a 0.1-kilobase deletion in the 5' region of the MHC gene and reduces MHC protein in the leg and thoracic muscles of heterozygotes to levels found in 36AC haploids. Three mutations are insertions of 8-to 10-kilobase DNA elements within the MHC gene and produce truncated MHC transcripts. Heterozygotes of these insertional mutations possess levels of MHC intermediate between those of haploids and diploids. An additional mutation has no gross alteration of the MHC gene or its RNA transcripts. Although leg and larval muscles function normally in each mutant heterozygote, indirect flight muscles are defective and possess disorganized myofibrils. Homozygous mutants die during embryonic or larval development and display abnormal muscle function prior to death. These findings provide direct genetic evidence that the MHC gene at 36B (2L) is essential for both larval and adult muscle development and function. The results are consistent with the previous molecular evidence that Drosophila, unlike other organisms, has only a single muscle MHC gene per haploid genome. Quantitative expression of both copies of the MHC gene is required for function of indirect flight muscle, whereas expression of a single MHC gene is sufficient for function of larval muscles and adult tubular muscles.
Isolation of a cDNA clone encoding the amino-terminal region of human apolipoprotein B.
Authors: Protter A A , Hardman D A , Schilling J W , Miller J , Appleby V , Chen G C , Kirsher S W , McEnroe G , Kane J P
Published: May 2006

A partial cDNA clone for the B-26 region of apolipoprotein B was isolated from an adult human liver DNA library by screening with an oligonucleotide probe derived from amino-terminal protein sequence obtained from purified B-26 peptide. Antisera against a synthetic 17-residue peptide whose amino acid sequence was encoded by the clone cross-reacts with apolipoproteins B-26, B-100, and B-48, but not with B-74. The nucleotide sequence immediately upstream from the amino terminus of B-26 codes for an apparent signal sequence, implying that the B-26 moiety is in an amino-terminal locus in the B-100 protein. That this sequence represents a 5' end region is further supported by primer extension analysis using a fragment of the cDNA clone and by S1 nuclease protection experiments using the corresponding region in a genomic clone.
Comparison of nucleotide interactions in water, proteins, and vacuum: model for DNA polymerase fidelity.
Authors: Petruska J , Sowers L C , Goodman M F
Published: May 2006

We propose a model for DNA polymerase fidelity in which free energy differences, delta delta G, between matched and mismatched nucleotides are magnified at the enzyme's active site. Both hydrogen bonding and stacking components of the interaction energy are amplified, with the most profound effect being on the magnitude of hydrogen-bonding interactions. Magnification in delta delta G values follows from the exclusion of water around base pairs in the active site cleft of the enzyme. After showing that base-pair dissociation energies calculated from hydrogen-bonding and base-stacking interactions in vacuo are greatly reduced by water, it is proposed that water removal results in a proportional restoration of these contributions to base pairing. Assuming approximately equal to 40% exclusion of surrounding water, one predicts magnified values of delta delta G sufficient to account for polymerase insertion and proofreading fidelity, thereby avoiding the need to postulate additional active site constraints in order to select or reject nucleotides.
Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA.
Authors: Mariottini P , Chomyn A , Riley M , Cottrell B , Doolittle R F , Attardi G
Published: May 2006

In previous work, antibodies prepared against chemically synthesized peptides predicted from the DNA sequence were used to identify the polypeptides encoded in three of the eight unassigned reading frames (URFs) of human mitochondrial DNA (mtDNA). In the present study, this approach has been extended to other human mtDNA URFs. In particular, antibodies directed against the NH2-terminal octapeptide of the putative URF2 product specifically precipitated component 11 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative URF4 product reacted specifically with components 4 and 5, and antibodies against a COOH-terminal heptapeptide of the presumptive URF4L product reacted specifically with component 26. Antibodies against the NH2-terminal heptapeptide of the putative product of URF5 reacted with component 1, but only to a marginal extent; however, the results of a trypsin fingerprinting analysis of component 1 point strongly to this component as being the authentic product of URF5. The polypeptide assignments to the mtDNA URFs analyzed here are supported by the relative electrophoretic mobilities of proteins 11, 4-5, 26, and 1, which are those expected for the molecular weights predicted from the DNA sequence for the products of URF2, URF4, URF4L, and URF5, respectively. With the present assignment, seven of the eight human mtDNA URFs have been shown to be expressed in HeLa cells.
Norepinephrine-induced alteration in the coupling of alpha 1-adrenergic receptor occupancy to calcium efflux in rabbit aortic smooth muscle cells.
Authors: Colucci W S , Alexander R W
Published: May 2006

To determine whether alpha-adrenergic desensitization of vascular smooth muscle is due to an alteration in alpha 1-adrenergic receptor coupling, we determined the relationship between receptor occupancy and maximal receptor-coupled Ca2+ efflux in cultured rabbit aortic smooth muscle cells (i) under basal conditions as defined by receptor inactivation with phenoxybenzamine and (ii) after 48 hr of exposure to several concentrations of 1-norepinephrine (NE). Neither phenoxybenzamine nor NE exposure caused a change in binding affinity for [3H]prazosin or NE. Maximal [3H]prazosin binding capacity and maximal NE-stimulated 45Ca2+ efflux decreased progressively with exposure of incubated cells to increasing concentrations of phenoxybenzamine or NE. An approximately 80% decrease in maximal [3H]prazosin binding capacity caused by either phenoxybenzamine or NE resulted in complete loss of NE-stimulated 45Ca2+ efflux, indicating that under these conditions approximately 20% of alpha 1-adrenergic receptors are not coupled to the Ca2+ efflux. Under basal conditions, the relationship between maximal [3H]prazosin binding capacity and maximal NE-stimulated 45Ca2+ efflux was markedly nonlinear, so that a near maximal response could be elicited by occupancy of only approximately 40% of the receptors. In contrast, after a 48-hr incubation of cells with NE, occupancy-response coupling was considerably less efficient, so that even full occupancy of the 35% of receptors that remained after NE exposure resulted in only approximately 20% of maximal NE-stimulated 45Ca2+ efflux. Thus, an alteration in occupancy-response coupling at a step proximal to Ca2+ mobilization and/or influx, rather than a reduction in receptor number, is of primary importance in the process of agonist-induced alpha-adrenergic receptor desensitization of vascular smooth muscle cells.
Sialidosis and galactosialidosis: chromosomal assignment of two genes associated with neuraminidase-deficiency disorders.
Authors: Mueller O T , Henry W M , Haley L L , Byers M G , Eddy R L , Shows T B
Published: May 2006

The inherited human disorders sialidosis and galactosialidosis are the result of deficiencies of glycoprotein-specific alpha-neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18; sialidase) activity. Two genes were determined to be necessary for expression of neuraminidase by using human-mouse somatic cell hybrids segregating human chromosomes. A panel of mouse RAG-human hybrid cells demonstrated a single-gene requirement for human neuraminidase and allowed assignment of this gene to the (pter----q23) region of chromosome 10. A second panel of mouse thymidine kinase (TK)-deficient LM/TK- -human hybrid cells demonstrated that human neuraminidase activity required both chromosomes 10 and 20 to be present. Analysis of human neuraminidase expression in interspecific hybrid cells or polykaryocytes formed from fusion of mouse RAG (hypoxanthine/guanine phosphoribosyltransferase deficient) or LM/TK- cell lines with human sialidosis or galactosialidosis fibroblasts indicated that the RAG cell line complemented the galactosialidosis defect, but the LM/TK- cell line did not. This eliminates the requirement for this gene in RAG-human hybrid cells and explains the different chromosome requirements of these two hybrid panels. Fusion of LM/TK- cell hybrids lacking chromosome 10 or 20 (phenotype 10+,20- and 10-,20+) and neuraminidase-deficient fibroblasts confirmed by complementation analysis that the sialidosis disorder results from a mutation on chromosome 10, presumably encoding the neuraminidase structural gene. Galactosialidosis is caused by a mutation in a second gene required for neuraminidase expression located on chromosome 20.
Inhibition of the in vitro infectivity and cytopathic effect of human T-lymphotrophic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) by 2',3'-dideoxynucleosides.
Authors: Mitsuya H , Broder S
Published: May 2006

Human T-lymphotropic virus type III (HTLV-III)/lymphadenopathy-associated virus (LAV) is a a newly discovered lymphotropic retrovirus that is cytopathic for helper/inducer T cells in vitro. This virus is the etiologic agent of the acquired immunodeficiency syndrome and related diseases. In the current study, we tested the capacity of purine and pyrimidine nucleoside derivatives to inhibit the infectivity and cytopathic effect of human T-lymphotropic virus type III in vitro. With the ribose moiety of the molecule in a 2',3'-dideoxy configuration, every purine (adenosine, guanosine, and inosine) and pyrimidine (cytidine and thymidine) nucleoside tested suppressed the virus, although the thymidine derivative seemed to have substantially less activity in our system than the others. In general, we observed essentially complete suppression of the virus at doses that were lower by a factor of 10 to 20 than those needed to inhibit the proliferation of the target T cells and the immune reactivity of normal T cells in vitro. An analysis of five adenosine congeners, which differed only in the sugar moiety, revealed that reduction (an absence of hydroxyl determinants) at both the 2' and 3' carbons of the ribose was necessary for an anti-viral effect, and an additional reduction at the 5' carbon nullified the anti-viral activity. These observations may be of value in developing a new class of experimental drugs for the therapy of human T-lymphotropic virus type III infections.
Excitatory amino acid recognition sites coupled with inositol phospholipid metabolism: developmental changes and interaction with alpha 1-adrenoceptors.
Authors: Nicoletti F , Iadarola M J , Wroblewski J T , Costa E
Published: May 2006

Glutamate, aspartate, ibotenate, and quisqualate activate inositol phospholipid hydrolysis in hippocampal slices prepared from brains of 6- to 8-day-old rats. The stimulation by glutamate and aspartate progressively declines during postnatal development and is negligible after the 24th day of life. In contrast, the stimulation of inositol phospholipid hydrolysis by norepinephrine is low in hippocampal slices from newborn animals and increases during development, reaching mature values after the 35th day of life. In adult hippocampal slices, the stimulation of inositol phospholipid hydrolysis elicited by norepinephrine is inhibited by glutamate in a concentration-dependent fashion. This inhibition can also be brought about by aspartate, 2-amino-4-phosphonobutanoate, and L-phosphoserine, a product of endogenous phosphatidylserine hydrolysis.
Molecular cloning of cDNA for the nuclear ribonucleoprotein particle C proteins: a conserved gene family.
Authors: Nakagawa T Y , Swanson M S , Wold B J , Dreyfuss G
Published: May 2006

The C proteins, C1 and C2, are major constituents of the heterogeneous nuclear RNA (hnRNA) ribonucleoprotein (hnRNP) complex in vertebrates. C1 and C2 are antigenically related phosphoproteins that are in contact with hnRNA in intact cells and bind to RNA tightly in vitro. A cDNA clone for the C proteins was isolated by immunological screening of a human lambda gt11 expression vector cDNA library with monoclonal antibodies. The lacZ-cDNA fusion protein is recognized by two different anti-C protein monoclonal antibodies. HeLa cell mRNA that was hybrid-selected with the cDNA clone (1.1 kilobases long) was translated in vitro and yielded both the C1 and C2 proteins (41 and 43 kDa, respectively). RNA blot analysis showed strong hybridization to two polyadenylylated transcripts, of about 1.4 kb and 1.9 kb, in human cells. Genomic DNA blot analysis showed multiple hybridizing restriction fragments in human and mouse, and homologous DNA sequences are found across eukaryotes from human to yeast. These findings suggest that the sequences encoding the hnRNP C proteins are members of a conserved gene family and they open the way for detailed molecular and genetic studies of these proteins.
Transcriptional and post-transcriptional regulation of soybean seed protein mRNA levels
Authors: Walling Linda , Drews Gary N. , Goldberg Robert B.
Published: May 2006

We investigated soybean seed protein gene transcription during development. We found that seed protein genes are transcriptionally activated and then repressed during embryogenesis and that these genes are either inactive or transcribed at low levels in the mature plant. We further observed that genes encoding mRNAs with vastly different prevalences are transcribed at similar rates. DNA gel blot studies showed that transcriptionally active and inactive seed protein genes have indistinguishable methylation patterns. We conclude that both transcriptional and posttranscriptional processes regulate seed protein mRNA levels in the absence of detectable DNA methylation changes.
Cell-type-specific and regulated expression of a human gamma 1 heavy-chain immunoglobulin gene in transgenic mice.
Authors: Yamamura K , Kudo A , Ebihara T , Kamino K , Araki K , Kumahara Y , Watanabe T
Published: May 2006

A functionally rearranged human gamma 1 heavy-chain immunoglobulin gene was cloned from a human plasma cell leukemia cell line, ARH-77, into the phage lambda Charon 4A. The recombinant phage DNA was introduced into fertilized mouse eggs (about 200 copies of the human gene per egg). A total of 30 mice were born and were screened for the presence of the human gamma 1 gene by dot hybridization. Two of these 30 mice had integrated one or two copies of the gene. The gamma 1 mRNAs were detected only in spleen. Levels of gamma 1 mRNA and the percentage of spleen cells producing human gamma chain increased up to 50-fold after treatment with bacterial lipopolysaccharide (a B-cell mitogen) but not with concanavalin A (a T-cell mitogen), suggesting B-cell-specific and regulated expression of the human gamma 1 heavy-chain gene. Human gamma chain-producing cells were found only in the periphery of the germinal center of the white pulp in histological sections of the spleen but not in sections of other tissues. Human gamma chains appeared to be coupled with mouse light chains to form a complete IgG molecule and were secreted into the cell supernatant. The production and secretion of endogenous immunoglobulin heavy and light chains in transgenic mice appeared to be the same as in normal mice. About one-seventh of the spleen cells that produced endogenous mouse heavy chains also produced human gamma chains, but no cells that produced only human gamma chain were observed.
Three novel genes of human T-lymphotropic virus type III: immune reactivity of their products with sera from acquired immune deficiency syndrome patients.
Authors: Arya S K , Gallo R C
Published: May 2006

Human T-lymphotropic virus type III or lymphoadenopathy associated virus (HTLV-III/LAV) is the cause of acquired immune deficiency syndrome (AIDS). In addition to the conventional retroviral genes involved in virus replication, namely, gag, pol, and env genes, DNA sequence analysis of HTLV-III genome predicted two additional open reading frames termed by us short open reading frame (sor) and 3' open reading frame (3' orf). Furthermore, functional analysis revealed another gene with transactivating function, termed tat. We have now structurally identified and functionally characterized these HTLV-III specific genes by way of cDNA cloning. DNA sequence analysis of the clones shows that the tat and 3' orf genes contain three exons and their transcription into functional mRNA involves two splicing events and that the sor gene contains at least two exons. In vitro transcription and translation of the cloned spliced sequences show that the sor, tat, and 3' orf genes code for polypeptides with apparent mobility of 24-25 kDa, 14-15 kDa, and 26-28 kDa, respectively. All three polypeptides are immune reactive and are immunogenic in the natural host. The results demonstrate that the three extra open reading frames of HTLV-III, two of which are unique to HTLV-III, are in fact genes that function in vivo and further allow the identification of three new and previously unrecognized HTLV-III antigens with differential immunogenicity in individuals with acquired immune deficiency syndrome and related disorders.
Demonstration of a receptor in Torpedo synaptic vesicles for the acetylcholine storage blocker L-trans-2-(4-phenyl[3,4-3H]-piperidino) cyclohexanol.
Authors: Bahr B A , Parsons S M
Published: May 2006

Transport and storage of acetylcholine by purified Torpedo electric organ synaptic vesicles is blocked by the drug L-trans-2-(4-phenylpiperidino)cyclohexanol (AH-5183). This study sought evidence of a specific receptor for the drug. Highly tritiated L-trans-2-(4-phenyl [3,4-3H] piperidino)-cyclohexanol (L-[3H] AH5183) was synthesized. An excess of nonradioactive L-isomer competed with L-[3H]AH5183 for binding to purified Torpedo synaptic vesicles whereas nonradioactive D-isomer did so poorly. Dissociation of bound L-[3H]AH5183 was first order with a rate constant at 23 degrees C of 0.23 +/- 0.03 min-1, and association was too rapid to study. At equilibrium the amount of L-[3H]AH5183 bound at saturation varied in different vesicle preparations, but in one typical preparation specific binding of 181 +/- 15 pmol L-[3H]AH5183 per mg of synaptic vesicle protein was observed with a dissociation constant of 34 +/- 6 nM. Neither acetylcholine nor choline compete effectively with L-[3H]AH5183 for binding. The evidence suggests that about 3.7 +/- 0.3 enantioselective receptors for L-[3H]AH5183 are typically present in each cholinergic synaptic vesicle, and the L-AH5183 binding site does not recognize acetylcholine.
Continuous Stern-Gerlach effect: Principle and idealized apparatus
Authors: Dehmelt Hans
Published: May 2006

A nondestructive type of Stern-Gerlach effect for an individual electron is described that has been thoroughly demonstrated in experiments at the University of Washington. This “continuous Stern-Gerlach effect” makes use of an inhomogeneous magnetic field provided by a weak auxiliary magnetic bottle and is nondestructive in contrast to all previous versions of the effect. As in the classic Stern-Gerlach effect, changes in the spin state are detected via changes in classical particle trajectories; however, what is observed now is not a deflection of the orbit but rather a change of its frequency in the storage well. A simplified model of the continuous Stern-Gerlach effect at zero temperature is introduced to develop the relation between minimum measurement time required for determination of spin state, driven oscillation amplitude, and zero-point fluctuations in the storage well. The problem of the reduction of the wavefunction by the interaction of the electron with the apparatus is addressed following W. Pauli.
Identification of the macrophage mannose receptor as a 175-kDa membrane protein.
Authors: Wileman T E , Lennartz M R , Stahl P D
Published: May 2006

Mannose-lactoperoxidase, a neoglycoprotein prepared by reaction of lactoperoxidase with cyanomethyl 1-thiomannoside, bound to alveolar macrophages at 4 degrees C (Kd = 5.8 X 10(-8) M) and was rapidly internalized at 37 degrees C (K uptake = 2 X 10(-8) M). Mannose-lactoperoxidase binding and uptake were blocked by yeast mannan, and mannose-lactoperoxidase inhibited uptake of 125I-labeled mannose-BSA (bovine serum albumin). Radioiodination of cells with surface-bound mannose-lactoperoxidase was carried out in the presence of glucose and glucose oxidase. A major polypeptide (175 kDa) was radioiodinated by this procedure. Iodination of the 175-kDa polypeptide appeared to be receptor-mediated, since it was blocked by the presence of yeast mannan. Specific iodination was absent from receptor-negative cells. To demonstrate that the 175-kDa species is a ligand-binding protein, cells were iodinated by the standard lactoperoxidase method. Washed cells were then allowed to bind mannose-BSA. Receptor-ligand complexes, prepared by detergent extraction, were passed over anti-BSA IgG affinity columns. Mannose, but not mannose 6-phosphate or galactose, eluted a radioactive protein from the column that migrated with an apparent molecular mass of 175 kDa on NaDodSO4/PAGE. Detergent extracts of crude membranes prepared from macrophage-enriched whole rabbit lung were adsorbed to mannose-Sepharose; the fraction obtained by elution with mannose contained two protein components of 175 and 55 kDa. Subsequent chromatography on N-acetylglucosamine-agarose yielded a single protein of 175 kDa. The 175-kDa polypeptide was shown to bind 125I-labeled mannose-BSA in a precipitation assay. This binding could be blocked with mannan or mannose-BSA. The results indicate that the cell-surface mannose receptor is a 175-kDa protein.
A gene expressed in undifferentiated vegetative Dictyostelium is repressed by developmental pulses of cAMP and reinduced during dedifferentiation.
Authors: Kimmel A R , Carlisle B
Published: May 2006

We describe the gene M4-1, whose unique pattern of developmental expression will allow us to study the molecular mechanisms controlling expression in undifferentiated cells in addition to repression in response to cAMP during development and reinduction during dedifferentiation. M4-1 is a Dictyostelium gene expressed in the undifferentiated cell. We have shown that M4-1 continues to be expressed very early during the developmental cycle but is repressed at a later stage of development, at a time coincident with the establishment of oscillations in the cAMP pool. Studies on the expression of the M4-1 gene in shaking culture, under conditions that mimic early development, have established that pulsatile stimulation of cells with cAMP is sufficient to repress M4-1 expression. Consistent with this, cells that are exposed to high levels of cAMP are unable to respond normally to cAMP oscillations and continue to express M4-1 at vegetative levels. These data indicate that low-level oscillations of cAMP are required for the repression of M4-1 expression rather than the continuous high levels of cAMP responsible for the regulation of a different class of Dictyostelium genes. We suggest that cAMP may mediate developmental expression of the Dictyostelium genome by different mechanisms. We also show that cell-cell interaction, a developmental event that occurs subsequent to the cAMP pulse, does not normally influence the regulation of M4-1. Finally, we have shown that when cAMP-pulsed cells are induced to dedifferentiate, M4-1 RNA sequences rapidly reappear in nuclei and cytoplasm, suggesting that regulation of M4-1 expression is primarily mediated at the level of transcription.
A DNA insertion/deletion necessitates an aberrant RNA splice accounting for a mu heavy chain disease protein.
Authors: Bakhshi A , Guglielmi P , Siebenlist U , Ravetch J V , Jensen J P , Korsmeyer S J
Published: May 2006

The human heavy chain disease protein BW is an immunoglobulin mu-chain variant whose amino terminus is initiated at the fifth amino acid of the first constant region domain. We cloned and analyzed both rearranged heavy chain alleles from BW leukemic cells to determine the molecular basis for this deleted protein. The phenotypically excluded heavy-chain allele possessed two intermediate recombinations of separate variable-diversity (V-D) and diversity-joining (D-J) junctions, neither of which were expressed. The productive allele, responsible for the mu chain, had a complete V-D-J4 recombination but as a result of a single-base deletion possessed stop codons within the variable region. More important, a small DNA insertion/deletion eliminated the J4 donor splice site. This necessitated an aberrant RNA splice between the leader region and the first constant region domain creating a shortened 2.35-kilobase muRNA. A recognition sequence for signal peptidase predicted a cleavage at the fifth amino acid of the first constant region domain. These molecular events are responsible for the truncated mu chain that lacks a variable region and fails to assemble light chains.