bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of translocated c-myc genes transfected into plasmacytoma cells.
Authors: Feo S , Harvey R , Showe L , Croce C M
Published: May 2006

We have transfected two translocated c-myc oncogene clones, derived from two human lymphomas carrying the t(8;14) chromosome translocation, into mouse plasmacytoma cells to study the regulation of their expression. In one case, the transfected clone contained the two coding exons of the c-myc oncogene translocated to an immunoglobulin heavy-chain switch region; in the other case, the two coding exons were translocated 5' of the enhancer element located between the heavy-chain joining region (JH) and the switch region S mu. Nuclease S1 protection experiments indicate that only the c-myc translocated 5' of the enhancer element is transcribed in the plasmacytoma cells. Thus, 5'-truncation of the c-myc gene per se does not lead to c-myc deregulation. Further, since the level of c-myc transcripts in the parental human lymphoma cells was 3- to 4-fold higher than in the transfectants, it seems likely that additional elements within the heavy-chain locus may play a role in the enhancement of c-myc gene transcription in lymphoma cells.
Influence of a genetic difference confined to mutation of H-2K on the incidence of pregnancy block in mice.
Authors: Yamazaki K , Beauchamp G K , Matsuzaki O , Kupniewski D , Bard J , Thomas L , Boyse E A
Published: May 2006

In mice, pregnancy is often terminated when the stud male is removed and the pregnant female is exposed to the presence or scent of an unfamiliar male. It is reported here that the incidence of such blocking of pregnancy was higher when the stud and unfamiliar male differed by mutation of the H-2K class I gene of the major histocompatibility complex than when the stud and unfamiliar males were genetically identical. Thus, the olfactory distinction of mice differing by mutation of the H-2K gene, previously demonstrated after training of mice in a Y maze, can spontaneously influence neuroendocrine communications affecting reproduction.
Small basic proteins of myelin from central and peripheral nervous systems are encoded by the same gene.
Authors: Mentaberry A , Adesnik M , Atchison M , Norgård E M , Alvarez F , Sabatini D D , Colman D R
Published: May 2006

Peripheral nervous system (PNS) and central nervous system (CNS) rodent myelins, which are produced by different cell types, share common morphological and functional characteristics although their major integral membrane proteins are completely different. Both types of myelin however, contain sets of four myelin basic proteins (MBPs), which share similar immunochemical and electrophoretic properties. We have isolated and characterized cDNA clones corresponding to the rat mRNAs encoding the small MBPs (SMBPs) found in both CNS and PNS myelin. Sequence analysis of these clones indicate that SMBPs in both divisions of the nervous system are encoded by the same nucleotide sequences, which suggests that they are the products of the same gene expressed in both oligodendrocyte and Schwann cells. In dot-blot hybridization experiments with the CNS SMBP cDNA as a probe, it was shown that there is a 20-fold higher level of MBP mRNA in a CNS myelin fraction than in total brainstem mRNA. It also was found that in optic and sciatic nerves, which contain oligodendrocytes and Schwann cells respectively, there are higher levels (4-fold and 2-fold, respectively) of MBP mRNA than in brainstem. Blot-hybridization experiments showed that a probe derived from the coding region of the rat SMBP cDNA hybridizes to an homologous mRNA (approximately equal to 2.6 kilobases) present in human optic nerve, which is not detectable with a probe derived from the 3' untranslated region. This conservation of coding-region sequences is in accord with the highly homologous amino acid sequences reported for the MBPs in the two species.
Glucocorticoid sensitivity of vasopressin mRNA levels in the paraventricular nucleus of the rat.
Authors: Davis L G , Arentzen R , Reid J M , Manning R W , Wolfson B , Lawrence K L , Baldino F
Published: May 2006

Immunocytochemical studies have shown that adrenalectomy produces changes in the content and distribution of [arginine-8]vasopressin (AVP) immunoreactivity in the paraventricular nucleus of the hypothalamus. The purpose of this study was to determine whether manipulation of adrenal hormones affects the levels of AVP mRNA. In situ hybridization assays with highly specific synthetic oligodeoxyribonucleotide probes and immunocytochemistry were used to detect the distribution of AVP mRNA and AVP-immunoreactive perikarya. AVP mRNA is codistributed with AVP immunoreactivity in the posterior magnocellular subdivision of the paraventricular nucleus and its accessory nuclei, the supraoptic nucleus and the suprachiasmatic nucleus. In adrenalectomized rats, the density and distribution of the hybridization signal were increased in the paraventricular nucleus; a 2-fold increase in the area comprising the signal was observed. At the cellular level, silver grains were detected in corticotropin-releasing-factor-immunoreactive neurons throughout the medial parvocellular subdivision of the paraventricular nucleus. No changes were seen in the distribution of AVP mRNA in the supraoptic or suprachiasmatic nuclei. Treatment with dexamethasone prevented the increase in AVP mRNA produced by adrenalectomy. In contrast, adrenalectomy did not alter the hybridization signal obtained with a probe for alpha-tubulin mRNA. These results suggest, at the cellular level, that adrenalectomy induces a glucocorticoid-sensitive stimulation of AVP mRNA synthesis in the central nervous system. Thus, considerable plasticity in gene expression is retained in the hypothalamus of the adult rat.
Purification and characterization of a membrane-associated 3,3',5-triiodo-L-thyronine binding protein from a human carcinoma cell line.
Authors: Cheng S Y , Hasumura S , Willingham M C , Pastan I
Published: May 2006

A membrane-associated binding protein for 3,3',5-triiodo-L-thyronine (T3) was purified to apparent homogeneity from A431 human epidermoid carcinoma cells. A431 cells were specifically labeled with the N-bromoacetyl derivative of T3 labeled with 125I at the 3' position (BrAc[125I]T3) and were extracted with 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent. The solubilized BrAc[125I]T3-labeled protein was successively purified by chromatography on Sephadex G-200 and QAE-Sephadex followed by NaDod-SO4/PAGE. Approximately 0.2 mg of purified protein was obtained from 2.5 X 10(9) cells, which represents a 3000-fold purification. The membrane-associated T3 binding protein is an acidic protein with a pI of 5.1 and an apparent molecular mass of 55,000 daltons determined by NaDodSO4/PAGE. Polyclonal antibodies against the 55-kDa protein were prepared and used in indirect immunofluorescence to show that the 55-kDa protein was mainly found in the nuclear envelope and endoplasmic reticulum.
Fate of reversing factor during restoration of protein synthesis by hemin or GTP in heme-deficient reticulocyte lysates.
Authors: Matts R L , Levin D H , London I M
Published: May 2006

The inhibition of protein synthesis in hemedeficient reticulocyte lysates is reversed by the addition of hemin (20 microM) or MgGTP (2 mM). The rate of recovery is rapid and approaches control kinetics within a few minutes after the addition of either component. The restoration of protein synthesis is dependent upon the availability of functional reversing factor (RF). The fate of RF was monitored during recovery by using a method that measures RF activity in the lysate under physiological conditions. In the fully inhibited lysate, RF is sequestered in a nondissociable 15S [RF . eIF-2(alpha P)] complex (where eIF-2 indicates eukaryotic initiation factor 2) in which RF activity is not functional and cannot be assayed. The first step in the rescue of protein synthesis in inhibited lysates by hemin or MgGTP is the inhibition of heme-regulated eIF-2 alpha kinase, which enables endogenous phosphatase to dephosphorylate eIF-2(alpha P) and [RF . eIF-2(alpha P)]. The release of approximately 50% of the sequestered RF activity is sufficient to support optimal kinetics of recovery. Hemin and MgGTP both reverse inhibition by blocking the activation and/or activity of heme-regulated eIF-2 alpha kinase in the lysate. The conclusion that MgGTP exerts its effect on eIF-2 alpha kinase is supported by several in vitro findings: (i) 2 mM MgGTP inhibits the autophosphorylation of purified heme-regulated eIF-2 alpha kinase and abolishes its ability to phosphorylate eIF-2 alpha; (ii) 2 mM MgGTP cannot displace GDP in the binary complexes [eIF-2 . GDP] or [eIF-2(alpha P) . GDP] by mass action; and (iii) RF in the [RF . eIF-2(alpha P)] complex is not dissociated by 2 mM MgGTP.
Isolation of cDNA clones coding for the catalytic subunit of mouse cAMP-dependent protein kinase.
Authors: Uhler M D , Carmichael D F , Lee D C , Chrivia J C , Krebs E G , McKnight G S
Published: May 2006

mRNA coding for the catalytic (C) subunit of cAMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was partially purified from bovine testis by polysome immunoadsorption and oligo(dT)-chromatography. This enriched mRNA preparation was used to prepare and differentially screen a cDNA library. One of the selected cDNA clones was shown to hybrid-select mRNA coding for a 40-kDa protein that was specifically precipitated with antibodies to the C subunit. This bovine cDNA clone was then used to isolate a series of mouse cDNA clones that are complementary to the entire mouse C subunit mRNA. The mouse clones code for a protein of 351 amino acids that shows 98% homology to the bovine C subunit and hybridize to a single mRNA of 2.4 kilobases in mouse heart and brain. Southern blot analysis of total genomic DNA suggests that there is a single mouse gene coding for the C subunit. mRNA levels for both the C subunit and the type I regulatory subunit in various mouse tissues and cell lines were quantitated and compared by using single-stranded RNA probes prepared with SP6 polymerase.
Heat shock causes destabilization of specific mRNAs and destruction of endoplasmic reticulum in barley aleurone cells.
Authors: Belanger F C , Brodl M R , Ho T H
Published: May 2006

In response to a phytohormone, gibberellic acid, the aleurone layers of barley seeds synthesize and secrete alpha-amylases, which are coded by a set of stable mRNAs. When aleurone layers are subjected to heat shock treatment, the synthesis of alpha-amylase is suppressed while heat shock proteins are induced. The suppression of alpha-amylase synthesis is not the result of translational control as reported in several other systems. Rather, the sequences of alpha-amylase mRNA are rapidly degraded during heat shock as shown by in vitro translation and dot blot hybridization with a cDNA probe. Upon recovery from heat shock, the tissue resumes the synthesis of alpha-amylase in 2-4 hr. However, in the presence of a transcription inhibitor, cordycepin, the resumption of synthesis of alpha-amylase does not take place, indicating that new transcription of alpha-amylase genes is necessary for this recovery process. The degradation of alpha-amylase mRNAs correlates with the rapid destruction of endoplasmic reticulum as observed by electron microscopy, a phenomenon that has not been reported previously as a heat shock response. Since alpha-amylase mRNA is associated with the endoplasmic reticulum via membrane-bound polyribosomes, we suggest that the destruction of the endoplasmic reticulum during heat shock causes the destabilization and the eventual degradation of alpha-amylase mRNA.
Neuroanatomy influences speciation rates among anurans
Authors: Ryan Michael J.
Published: May 2006

Evolutionary divergence among animal courtship signals is an important component of the speciation process. In anurans, the preferential response of females to the mating call of conspecific males often maintains reproductive isolation among populations. Much of the information in the call is initially processed in the inner ear, and there is considerable variation in the structure of this organ among lineages of frogs. This variation is responsible for differences in the frequency range to which frog species are sensitive and thus influences the frequency range over which mating calls can effectively diverge. Data suggest that this influence of neuroanatomy on mating call divergence is partly responsible for the different rates of speciation among lineages of frogs.
Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor.
Authors: Hagen D C , McCaffrey G , Sprague G F
Published: May 2006

Haploid yeast cells of the a mating type secrete a peptide pheromone, a factor, which acts on cells of the alpha mating type to prepare them for conjugation. We show that the STE3 gene, which is required for mating only by alpha cells and is transcribed only in alpha cells, likely encodes a cell-surface receptor for a factor. This view is based on three findings. First, wild-type Ste3 product is required for response to the pheromone: mutants with any one of five different ste3 mutations are unresponsive to a factor. Second, a hybrid Ste3-beta-galactosidase protein encoded by a STE3-lacZ gene fusion fractionates to the particulate fraction of yeast cell extracts, suggesting that Ste3 is a membrane protein. Finally, the DNA sequence of STE3, which we report here, encodes a protein of 470 amino acid residues that contains seven distinct hydrophobic segments of sufficient length to span a lipid bilayer.
Gated currents generate single spike activity in amacrine cells of the tiger salamander retina.
Authors: Barnes S , Werblin F
Published: May 2006

Amacrine cells form the neural networks mediating the second level of lateral interactions in the vertebrate retina. Members of a prominent class of amacrine cells, found in most vertebrates, respond at both the onset and termination of steps of illumination with a single, large transient depolarization. We show here how specific relationships between membrane currents control this single spike activity. Using whole-cell patch clamp on living retinal slices, we studied the membrane currents in amacrine cells. The currents elicited by depolarizing voltage steps could be separated into three main ionic components: a transient inward voltage-gated sodium current, a relatively small sustained inward voltage-gated calcium current, and a calcium-dependent outward current. A specific relationship between the sodium and potassium current alone appears to preclude repetitive spike activity. Potassium current is activated at potentials positive to -20 mV, but the sodium inactivation, between -60 and -20 mV, does not intersect potassium activation. Therefore, a steady depolarizing current step elicits an initial spike but then the membrane cannot be sufficiently hyperpolarized by potassium current to remove sodium inactivation and the cell remains refractory.
Isolation of structural genes for yeast RNA polymerases by immunological screening.
Authors: Riva M , Memet S , Micouin J Y , Huet J , Treich I , Dassa J , Young R , Buhler J M , Sentenac A , Fromageot P
Published: May 2006

A lambda gt11 yeast genomic library was screened with antibodies directed against yeast RNA polymerases A, B, and C. Thirty-five individual recombinant phages that expressed proteins in Escherichia coli that were antigenically related to RNA polymerases A, B, or C were isolated by using 22 distinct antisera. Thus, all 22 genes for the RNA polymerase subunits were potentially cloned. In three cases (lambda A-43, lambda A-40, and lambda A-34.5), an antigenic protein was expressed in E. coli with the same molecular weight as the corresponding subunit. When lambda A-40 DNA was used to hybrid-select yeast mRNA, the protein translated in vitro was the expected size for the A-40 subunit, further supporting our isolation of the A-40 gene. However, mRNA hybrid selected by lambda A-27 DNA did not code for a protein of the correct size. The lengths of the mRNA that hybridized to phage lambda A-190 or lambda C-160 DNA on RNA blots were in agreement with the predicted sizes of the coding regions of the corresponding genes. As predicted by our previous immunological results, yeast DNA inserts of the lambda A-190 and lambda C-160 clones cross-hybridized to the B-220 subunit gene. The cloned genes for the RNA polymerase subunits will prove to be valuable tools for the study of the function, regulation, and genetics of the yeast RNA polymerases.
Stereospecific conversion of prostaglandin D2 to (5Z,13E)-(15S)-9 alpha-11 beta,15-trihydroxyprosta-5,13-dien-1-oic acid (9 alpha,11 beta-prostaglandin F2) and of prostaglandin H2 to prostaglandin F2 alpha by bovine lung prostaglandin F synthase.
Authors: Watanabe K , Iguchi Y , Iguchi S , Arai Y , Hayaishi O , Roberts L J
Published: May 2006

A prostaglandin F (PGF) synthase was recently purified from bovine lung that catalyzed the reduction of both PGH2 and PGD2 but at different active sites on the enzyme. In view of the recent finding that PGD2 is stereospecifically reduced to a unique biologically active compound, (5Z, 13E)-(15S)-9 alpha, 11 beta, 15-trihydroxyprosta-5,13-dien-1-oic acid (9 alpha,11 beta-PGF2 or 11-epi-PGF2 alpha), by a human liver cytosolic enzyme, detailed characterization of the products formed from PGH2 and PGD2 by the bovine lung PGF synthase was carried out. Chromatographic characteristics of the products formed and stereochemical analysis procedures using mass spectrometry indicated that the enzyme stereospecifically reduces PGH2 to PGF2 alpha, whereas PGD2 is stereospecifically converted to 9 alpha,11 beta-PGF2. The finding that this enzyme catalyzes the formation of both C-11 hydroxy epimers of PGF2, albeit from different substrates, is of interest in that these two compounds may exert different biological actions.
Bovine parathyroid cells: cultures maintained for more than 140 population doublings.
Authors: Brandi M L , Fitzpatrick L A , Coon H G , Aurbach G D
Published: May 2006

Primary cultures of bovine parathyroid cells were developed using Coon's modified Ham's F-12 medium containing low (0.3 mM) concentrations of calcium and supplements of bovine hypothalamic extract, bovine pituitary extract, epidermal growth factor, insulin, transferrin, selenous acid, hydrocortisone, triiodothyronine, retinoic acid, and galactose. These cells were cultured serially on serum-coated dishes for 140 population doublings before signs of senescence were detected. The cells were epithelioid and maintained a high degree of differentiation as evidenced by calcium regulation of both growth and secretion and by prostaglandin E1 stimulation of cAMP formation and hormone release.
ATP-dependent formation and motility of aster-like structures with isolated calf brain microtubule proteins.
Authors: Weisenberg R C , Allen R D , Inoue S
Published: May 2006

Microtubule proteins isolated from calf brain will undergo gelation-contraction in the presence of ATP. We have now examined this process by video-enhanced contrast microscopy. After ATP addition to steady-state microtubules, slow (1-5 micron/min), linear movements of particles and microtubules toward aggregation centers occur. The resulting structures resemble mitotic spindle asters. During the time when gel contraction occurs, asters move (at 1-5 micron/min) toward other nearby asters. This is accompanied by the apparent shortening of the microtubules running between the asters. This is the first example of isolated microtubules undergoing a process that has similarities to half-spindle shortening during anaphase A. Formation of aster-like structures without preformed microtubule organizing centers raises the possibility that a similar process may contribute to microtubule organization in vivo.
Bacterial δ-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/
Bradyrhizobium japonicum
symbiosis
Authors: Guerinot Mary Lou , Chelm Barry K.
Published: May 2006

Previous studies of legume nodules have indicated that formation of the heme moiety of leghemoglobin is a function of the bacterial symbiont. We now show that a
hemA
mutant of
Bradyrhizobium japonicum
that cannot carry out the first step in heme biosynthesis forms fully effective nodules on soybeans. The bacterial mutant strain was constructed by first isolating the wild-type
hemA
gene encoding δ-aminolevulinic acid synthase (EC 2.3.1.37) from a cosmid library, using a fragment of the
Rhizobium meliloti hemA
gene as a hybridization probe. A deletion of the
hemA
gene region, generated
in vitro
, then was used to construct the analogous chromosomal mutation by gene-directed mutagenesis. The mutant strain had no δ-aminolevulinic acid synthase activity and was unable to grow in minimal medium unless δ-aminolevulinic acid was added. Despite its auxotrophy, the mutant strain incited nodules that appeared normal, contained heme, and were capable of high levels of acetylene reduction. These results rule out bacterial δ-aminolevulinic acid synthase activity as the exclusive source of δ-aminolevulinic acid for heme formation in soybean nodules.
Autocrine growth inhibition of a cloned line of helper T cells.
Authors: Horowitz J B , Kaye J , Conrad P J , Katz M E , Janeway C A
Published: May 2006

The growth of T lymphocytes is dependent on the T-cell growth factor interleukin 2 (IL-2), which causes T cells bearing high-affinity receptors for IL-2 to proliferate. Most cloned helper-T-cell lines can be shown to both produce and respond to IL-2; thus, growth of such cells is by an autocrine mechanism. We report that the failure of the cloned murine T-cell line D10.G4.1 to respond to its own IL-2 results from the secretion, by the same cells, of a potent inhibitor of the IL-2-driven T-cell proliferative response. This inhibition can be overcome by increasing the number of IL-2 receptors expressed by the target cell. In the cloned T-cell line producing the inhibitory substance, this increase in IL-2 receptors is driven by the monokine interleukin-1. We propose that this inhibitor of IL-2 responses may play a role in preventing "bystander" activation of T cells by IL-2 released in vivo and could be a potent pharmacologic agent.
Fast and slow synaptic potentials produced in a mammalian sympathetic ganglion by colon distension.
Authors: Peters S , Kreulen D L
Published: May 2006

Radial distension of the large intestine produced a slow depolarization in a population of neurons in the inferior mesenteric ganglion of the guinea pig. The slow potentials often occurred simultaneously with cholinergic fast potentials [( excitatory postsynaptic potentials (EPSPs]) yet persisted in the presence of nicotinic and muscarinic cholinergic antagonists when all fast EPSPs were absent. The amplitude of the distension-induced noncholinergic slow depolarization increased with increasing distension pressure. For distensions of 1-min duration at pressures of 10-20 cm of water, the mean depolarization amplitude was 3.4 mV. The slow depolarization was associated with an increase in membrane resistance, and prolonged periods of colon distension resulted in a tachyphylaxis of the depolarization. Desensitization of ganglion cells to the peptide substance P attenuated the distension-induced slow potential by an average of 49% +/- 17%. Thus, two colonic mechanosensory afferent pathways converge on principal ganglion cells in the inferior mesenteric ganglion: one was previously described to be mediated by acetylcholine, and the other is described here, whose transmitter remains to be determined but which preliminary evidence suggests is mediated in part by substance P. The noncholinergic afferent pathway may enhance the intestinal inhibitory reflex mediated by cholinergic mechanosensory afferent input to the abdominal prevertebral sympathetic ganglia.
Interorganelle transfer and glycosylation of yeast invertase in vitro.
Authors: Haselbeck A , Schekman R
Published: May 2006

Core glycosylated proteins formed in the yeast endoplasmic reticulum (ER) are transported to the Golgi body, where oligosaccharides are elongated by addition of outer-chain carbohydrate. The transport process is blocked in a temperature-sensitive secretion mutant (sec18) of Saccharomyces cerevisiae, which accumulates core glycosylated invertase (product of SUC2; EC 3.2.1.26) in the ER. To approach the molecular mechanism of this transport process, we have devised a reaction in which core glycosylated invertase, accumulated in sec18 cells, is transferred to the Golgi body in vitro. For this purpose, membranes from sec18, SUC2 cells that are also defective in an outer chain alpha-1----3-mannosyltransferase (mnnl) are mixed with membranes from a strain that contains the transferase but is deficient in invertase (MNNl, delta SUC2). Transfer is detected by the acquisition of outer-chain alpha-1----3-linked mannose residues dependent on both donor and recipient membranes. The reaction is temperature and detergent sensitive and requires ATP, GDP-mannose, Mg2+, and Mn2+, and the product invertase remains associated with sedimentable membranes. Treatment of donor, but not acceptor, membranes with N-ethylmaleimide or trypsin inactivates transfer competence. These characteristics suggest that the ER, or a vesicle derived from the ER, contributes invertase to a chemically distinct compartment where mannosyl modification is executed.
Restoration of normal morphology and estrogen responsiveness in cultured vaginal and uterine epithelia transplanted with stroma.
Authors: Cooke P S , Uchima F D , Fujii D K , Bern H A , Cunha G R
Published: May 2006

We have investigated the capacity of vaginal and uterine epithelia (VE and UE) to reexpress normal morphology and hormone responsiveness following cell culture. VE and UE from adult ovariectomized mice were grown in a collagen gel matrix with serum-free medium for 7-10 days. Proliferation of these cells occurs in the absence of 17 beta-estradiol and is not stimulated by 17 beta-estradiol; the VE usually does not keratinize or stratify in vitro. Cultured VE and UE were recombined with homologous vaginal or uterine stroma (VS and US, respectively) and these recombinants were grown under the renal capsule of female hosts for 4 weeks. The epithelium of the VS + VE recombinants cycled, proliferated and stratified in response to estrogen and mucified normally in response to progesterone. The UE that was grown in vivo with cultured US also showed normal morphology and estrogen responsiveness. These changes in the UE and VE were not simply a result of return to the in vivo environment, as epithelia alone in collagen gels transplanted under the renal capsule did not survive. These results indicate that both VE and UE, which are not estrogen-dependent in vitro, reexpress their estrogen dependency and normal morphology when recombined with homologous stroma and grown in vivo. Thus, the changes these cells show when grown in culture are the result of altered conditions in vitro rather than an irreversible alteration in the cells themselves or the selection of specific subpopulations that are not mitogenically or morphologically responsive to estrogen under any condition.
Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs.
Authors: Fong H K , Hurley J B , Hopkins R S , Miake-Lye R , Johnson M S , Doolittle R F , Simon M I
Published: May 2006

Retinal transducin, a guanine nucleotide regulatory protein (referred to as a G protein) that activates a cGMP phosphodiesterase in photoreceptor cells, is comprised of three subunits. We have identified and analyzed cDNA clones of the bovine transducin beta subunit that may be highly conserved or identical to that in other G proteins. From the cDNA nucleotide sequence of the entire coding region, the primary structure of a 340-amino acid protein was deduced. The encoded beta subunit has a Mr of 37,375 and is comprised of repetitive homologous segments arranged in tandem. Furthermore, significant homology in primary structure and segmental sequence exists between the beta subunit and the yeast CDC4 gene product. The Mr 37,375 beta subunit polypeptide is encoded by a 2.9-kilobase (kb) mRNA. However, there exists in retina other beta-related mRNAs that are divergent from the 2.9-kb mRNA on the basis of oligonucleotide and primer-extended probe hybridizations. All mammalian tissues and clonal cell lines that have been examined contain at least two beta-related mRNAs, usually 1.8 and 2.9 kb in length. These results suggest that the mRNAs are the processed products of a small number of closely related genes or of a single highly complex beta gene.
Potassium channel blockade: A mechanism for suppressing ventricular fibrillation.
Authors: Bacaner M B , Clay J R , Shrier A , Brochu R M
Published: May 2006

The suppression of ventricular fibrillation by antidysrhythmic drugs is well correlated with their ability to block potassium channels in nerve and cardiac membranes. Blockade of potassium channels reduces electrical inhomogeneities in action potential and conduction parameters that lead to ventricular fibrillation. These actions tend to effectively decrease the electrical size of the heart, which suggests a mechanism for antifibrillatory drug action. The receptor sites for antifibrillatory drug action (IK blockade) appear to be on the outside of the cardiac membrane whereas receptors for antiarrhythmic drug action (INa blockade) appear to be on the inside of the cardiac membrane.
Changes in insulin and transferrin requirements of pure brain neuronal cultures during embryonic development.
Authors: Aizenman Y , Weichsel M E , de Vellis J
Published: May 2006

A pure neuronal culture grown in a defined serum-free environment has been developed and characterized. Insulin was the only hormone found to enhance the growth of neurons obtained from embryonic chicken brains during the early proliferative stage, a time when many neurons survived without the addition of any growth factors to the culture. With increasing embryonic age, there was an increase in the number of neurons requiring transferrin. By the time neurons reached a postmitotic state in older brains, they were completely dependent on both insulin and transferrin for survival and growth. Because this culture is free of glial cells and serum, it provides an effective basis for investigating molecular mechanisms underlying neuronal development.
Pluripoietin alpha: a second human hematopoietic colony-stimulating factor produced by the human bladder carcinoma cell line 5637.
Authors: Gabrilove J L , Welte K , Harris P , Platzer E , Lu L , Levi E , Mertelsmann R , Moore M A
Published: May 2006

We have identified a factor constitutively produced by the human bladder carcinoma cell line 5637, which has the following capacities: to induce the differentiation of the human promyelocytic leukemic cell line HL-60; to induce the expression of chemotactic peptide receptors in leukemic cells as well as in normal peripheral blood granulocytes; to function as a chemoattractant for neutrophils as well as, under certain conditions, inhibiting their migration; to support the growth of eosinophil and granulocyte/macrophage progenitors, which is maximal when scored at day 14; and to support the growth of human mixed pluripotent progenitors and erythroid bursts from normal human bone marrow. This factor has a molecular size of 32 kDa by gel filtration and 16.5 kDa by NaDodSO4/PAGE. We have termed this factor pluripoietin alpha.
Localization of the human pim oncogene (PIM) to a region of chromosome 6 involved in translocations in acute leukemias.
Authors: Nagarajan L , Louie E , Tsujimoto Y , ar-Rushdi A , Huebner K , Croce C M
Published: May 2006

The human homolog, hpim, of the murine pim-1 gene, which is activated in murine T-cell lymphomas by insertion of retrovirus proviral genomes in the pim-1 region, has been molecularly cloned; the cloned probe has been used to map the hpim locus to human chromosome region 6p21 by somatic cell hybrid analysis and chromosomal in situ hybridization. The hpim gene is expressed as a 3.2-kilobase mRNA in various human cell lines of hematopoietic lineage, most dramatically in the K562 erythroleukemia cell line, which contains a cytogenetically demonstrable rearrangement in the 6p21 region. A characteristic chromosome anomaly, a reciprocal translocation t(6;9)(p21;q33), has been described in myeloid leukemias and could involve the hpim gene.
Potassium conductance of the squid giant axon is modulated by ATP.
Authors: Bezanilla F , Caputo C , DiPolo R , Rojas H
Published: May 2006

This communication reports a modulating effect of intracellular ATP on the steady-state and kinetic properties of the delayed rectifier of the giant axon of the squid. When internally dialyzed or perfused giant axons from Loligo plei or Loligo pealei are voltage clamped at -60 mV and washed free of ATP, the potassium current at 0 mV is decreased, and the time course of the potassium current is faster. Upon readmitting ATP, the potassium current for pulses to potentials more positive than -30 mV is increased by a factor of up to 2.5, while for pulses to potentials more negative than -30 mV, it is decreased. In the presence of ATP the turn-on of the time course of the potassium current is slower, but the turn-off of the time course is faster. The effect of ATP is only observed when magnesium ions are present in the internal medium; ADP or the nonhydrolyzable ATP analogue adenosine 5'-[beta, gamma-methylene]-triphosphate has no effect. When the holding potential is -70 mV, the conductance-voltage curve is shifted to more positive potentials by ATP, but the maximum conductance is only slightly increased. Most of the effects of ATP may be explained by a phosphorylation step that alters the voltage sensor of the activation and inactivation gates of the potassium channels shifting the voltage dependence of both processes to more depolarized potentials.
Synergy between phorbol esters, 1-oleyl-2-acetylglycerol, urushiol, and calcium ionophore in eliciting aggregation of marine sponge cells.
Authors: Weissmann G , Azaroff L , Davidson S , Dunham P
Published: May 2006

Aggregation of marine sponge cells (Microciona prolifera) resembles stimulus-response coupling of higher organisms in which activation of protein kinase C and movements of intracellular Ca provide twin signals. We now report that activators of protein kinase C (phorbol esters) and ionomycin act synergistically to aggregate sponge cells. Surprisingly--since extracellular Ca is required for integrity of the species-specific aggregation factor--synergistic aggregation proceeded in the complete absence of added extracellular Ca (2.5-20 mM EDTA). The order of activity of phorbol esters and related compounds was that of their effect on protein kinase C (phorbol myristate acetate, phorbol dibutyrate greater than phorbol diacetate much greater than phorbol, 4 alpha-phorbol). 1-Oleyl, 2-acetylglycerol a synthetic activator of protein kinase C, also showed synergy with ionomycin. Phorbol esters and 1-oleyl, 2-acetylglycerol acted in synergy with ionomycin to liberate membrane Ca as detected by decreased fluorescence of chlortetracycline in prelabeled cells. Moreover, urushiol, the toxic principle of poison ivy, but not pentadecanylcatechol, its inert analogue, showed synergy with ionomycin. Synergistic aggregation was inhibited by calmidazolium (10 microM), piroxicam (20-100 microM), and pertussis toxin (20 micrograms/ml). The data not only confirm that marine sponge cell aggregation follows the general sequence of stimulus-response coupling in the cells of higher organisms but also support, in this most ancient of multicellular creatures, the hypothesis that mobilization of intracellular Ca and activation of protein kinase C provide the twin signals for cell activation in the absence of added extracellular Ca.
Animal art: Variation in bower decorating style among male bowerbirds
Amblyornis inornatus
Authors: Diamond Jared
Published: May 2006

Courtship bowers of the bowerbird
Amblyornis inornatus
, the most elaborately decorated structures erected by an animal other than humans, vary geographically and individually. Bowers in the south Kumawa Mountains are tall towers of sticks glued together, resting on a circular mat of dead moss painted black, and decorated with dull objects such as snail shells, acorns, and stones. Bowers in the Wandamen Mountains are low woven towers covered by a stick hut with an entrance, resting on a green moss mat and decorated with colorful objects such as fruits, flowers, and butterfly wings. Young males build simpler bowers, and adult males differ among themselves. Experiments with poker chips of seven colors offered as decorations showed that individual birds prefer some colors over others, individuals and populations differ in these preferences, certain objects are placed in specific parts of the bower, and birds steal chips from neighbors. Bower style may be partly learned. Hence, geographically varying bower styles may be a culturally transmitted trait, like human art styles.
Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer.
Authors: Price J , Turner D , Cepko C
Published: May 2006

We describe a cell-lineage marking system applicable to the vertebrate nervous system. The basis of the technique is gene transfer using the retroviral vector system. We used Escherichia coli beta-galactosidase as a marker gene and demonstrate a high level of expression of this marker from the viral long terminal repeat promoter, with simultaneous expression of the Tn5 neo gene from the simian virus 40 early promoter. This expression has allowed us to detect individual infected cells histochemically. We applied this marking technique to the study of lineage relationships in the developing vertebrate nervous system, both in vivo and in culture. In the rat retina, we injected virus in vivo and histochemically identified clones of marked neural cells. In addition, we used this virus to infect cultures of rat cerebral cortex and have analyzed the clonal relationships of morphologically different neural cell types. The host range of the marking system extends to avian as well as mammalian species. Thus, this system should have broad applicability as a means of gene transfer and expression in the nervous system.
Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication.
Authors: Dean F B , Bullock P , Murakami Y , Wobbe C R , Weissbach L , Hurwitz J
Published: May 2006

The simian virus 40 (SV40) large T antigen (large tumor antigen), in conjunction with a topoisomerase, a DNA binding protein, and ATP, catalyzed the conversion of a circular duplex DNA molecule containing the SV40 origin of replication to a form with unusual electrophoretic mobility that we have named form U. Analysis of this molecule revealed it to be a highly underwound covalently closed circle. DNA unwinding was not detected with DNA containing a SV40 T-antigen binding site II mutation that renders the DNA inactive in replication. The unwinding reaction requires the action of a helicase, and SV40 T-antigen preparations contain such an activity. The T-antigen-associated ability to unwind DNA copurified with other activities intrinsic to T antigen [ability to support replication of SV40 DNA containing the SV40 origin, poly(dT)-stimulated ATPase activity, and DNA helicase]. However, in contrast to the unwinding activity, the SV40 T-antigen-associated helicase activity was not sequence-specific. A variety of labeled oligonucleotides hybridized with circular single-stranded DNA were displaced by T antigen in the presence of ATP.
Transfection with plasmid pSV2gptEJ induces chromosome rearrangements in CHEF cells.
Authors: Stenman G , Delorme E O , Lau C C , Sager R
Published: May 2006

In previous cytogenetic studies, trisomy for 3q was found to be the most frequent chromosome change associated with induced tumorigenicity by a variety of agents in Chinese hamster cells. Here we describe similar chromosome changes in 11 lines of CHEF/18 cells transfected with the mutant c-Ha-ras containing plasmid pSV2gptEJ. All 11 lines contained the transfected EJ gene and expressed increased levels of p21, the EJ gene product. Ten of the 11 lines were tumorigenic and all but 2 of these were trisomic for all or part of 3q. One line remained diploid and was nontumorigenic despite expressing elevated p21. Two tumorigenic lines from "hit-and-run" transfection with pSV2gpt were shown to express only control levels of p21, but they were trisomic for 3q. These results show that increased p21 expression is neither necessary nor sufficient for inducing tumorigenicity of CHEF cells. We propose that tumorigenicity in the transfected CHEF/18 cells of this study was induced by chromosome rearrangements, especially trisomy for 3q, that occurred at increased frequencies following transfection with pSV2gptEJ.
T-cell antigen receptors with identical variable regions but different diversity and joining region gene segments have distinct specificities but cross-reactive idiotypes.
Authors: Rupp F , Brecher J , Giedlin M A , Mosmann T , Zinkernagel R M , Hengartner H , Joho R H
Published: May 2006

The T-cell antigen receptor alpha-chain genes of an alloreactive, H-2Db-specific cytotoxic T-cell clone (3F9) are described. This study and our work on the 3F9 beta-chain genes reveal that the variable region gene segments for the alpha and beta chains expressed in 3F9 are identical to the ones used by a chicken erythrocyte-specific, I-Ab-restricted helper T-cell clone (LB2). These two clones differ, however, in the diversity and joining portions of the alpha and beta chains of their T-cell receptor molecules. The analysis of 3F9 and LB2 with monoclonal antibodies specific for the 3F9 T-cell receptor shows that these two T-cell clones share the same idiotype; however, 3F9 and LB2 do not exhibit any antigen and/or major histocompatibility complex cross-reactivity. This suggests that the diversity and joining regions of the T-cell receptor may play a key role in antigen and/or major histocompatibility complex recognition.
Molecular analysis of TCRB and ABL in a t(7;9)-containing cell line (SUP-T3) from a human T-cell leukemia.
Authors: Westbrook C A , Rubin C M , Le Beau M M , Kaminer L S , Smith S D , Rowley J D , Diaz M O
Published: May 2006

A translocation between chromosomes 7 and 9, t(7;9), has been described in cell lines derived from the malignant cells of children with acute T-cell lymphoblastic leukemia or lymphoma. Our cytogenetic analysis of one such cell line, SUP-T3, demonstrates that the breakpoints on chromosomes 7 and 9 lie within bands q36 and q34, respectively, corresponding to the location of the gene encoding the beta chain of the T-cell receptor, TCRB, and the gene homologous to the transforming gene of the Abelson murine leukemia virus, ABL. We investigated the role of these genes in the t(7;9). In situ chromosomal hybridization of TCRB and ABL probes to metaphase cells from SUP-T3 demonstrated that ABL is translocated from chromosome 9 to 7 and that all or part of TCRB is translocated from chromosome 7 to 9. Southern blot analysis revealed that both TCRB alleles were rearranged; however, it could not be determined whether the translocation breakpoint lies within this gene. Pulsed-field gel electrophoresis and Southern blot analysis were used to examine more than 500 kilobases of the ABL locus; we concluded that there are no rearrangements within 250 kb in either direction of the sequences homologous to v-abl. Additionally, no abnormal ABL protein was detected in an in vitro phosphorylation assay. These results indicate that, in SUP-T3, the breakpoint on chromosome 9 lies proximal to ABL and that the break results in no apparent alteration of the ABL protein. We therefore hypothesize that another gene on chromosome 9, at band q34, plays a role in this translocation. This study also demonstrates that pulsed-field gel electrophoresis is a powerful new tool for the analysis of human chromosomal translocations.
Differences in tyrosine hydroxylase-like immunoreactivity characterize the mesostriatal innervation of striosomes and extrastriosomal matrix at maturity.
Authors: Graybiel A M , Hirsch E C , Agid Y A
Published: May 2006

Tyrosine hydroxylase [TyrOHase, tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (2-hydroxylating), EC 1.14.16.2] is the rate-limiting enzyme in the synthetic pathway of catecholamines and is expressed by neurons containing dopamine, norepinephrine, and epinephrine. TyrOHase is present in high concentrations in the caudate nucleus and putamen, where nearly all of it is contained in axons of the dopaminergic mesostriatal pathways. We have employed three different polyclonal antibodies directed against TyrOHase, one tested here for specificity by two-dimensional gel electrophoresis, to reexamine the anatomic distribution of fibers expressing TyrOHase-like immunoreactivity in the striatum of mature human, monkey, and cat brains. The findings suggest that this distribution is distinctly inhomogeneous. The macroscopic compartments known as striosomes have low TyrOHase-like immunoreactivity relative to the surrounding extrastriosomal matrix. These observations add to evidence that dopaminergic modulation of neural processing in the mature striatum is organized in accordance with striosomal architecture and suggest that part of the mechanism for such differentiation may involve presynaptic differences in enzymatic regulation of dopamine content in and out of striosomes.
Solubilization of a membrane-bound diol dehydratase with retention of EPR g = 2.02 signal by using 2-(N-cyclohexylamino)ethanesulfonic acid buffer.
Authors: Hartmanis M G , Stadtman T C
Published: May 2006

A procedure for solubilization of the oxygen-labile, membrane-bound diol dehydratase from Clostridium glycolicum with retention of enzymatic activity is described. The procedure involves sonication of crude membrane preparations anaerobically in 0.1 M 2-(N-cyclohexylamino)ethane-sulfonic acid (CHES) buffer (pH 8.6-9.0) containing 2 mM dithiothreitol. The addition of dimethylsulfoxide (30%) and lysophosphatidylcholine (0.15 mg/ml) to the solubilization buffer resulted in a 10-fold increase in recovery of solubilized diol dehydratase activity. After ultracentrifugation, an overall recovery of 50% of the activity initially present in the crude membrane preparations was achieved. Active membrane preparations and the solubilized enzyme exhibited an EPR signal at g = 2.02. Both enzyme activity and EPR signal were sensitive to oxygen and the radical scavengers, NH2OH and hydroxy-urea.