bluuebunny/crossref_metadata_2025 huggingface dataset:
Solubilization and characterization of high-affinity [3H]serotonin binding sites from bovine cortical membranes.
Authors: VandenBerg S R , Allgren R L , Todd R D , Ciaranello R D
Published: May 2006

High-affinity [3H]serotonin binding activity has been solubilized from bovine cerebral cortical membranes by using Triton X-100, Tween-80, and octyl-beta-D-glucopyranoside. This mixture of detergents solubilizes the high-affinity [3H]serotonin binding activity present in crude membrane preparations with retention of 75-90% specific binding. The detergent mixture was chosen because it can easily be removed from the solubilized fraction by dialysis and polystyrene bead adsorption, thus permitting further purification and isolation of the binding sites. Saturation analysis reveals multiple components of high-affinity [3H]serotonin binding. In crude bovine cortical membranes, at least two binding components are present. A higher-affinity binding component, as defined from curvilinear Scatchard plots, has a Kd for [3H]serotonin of 1-3 nM, whereas a lower-affinity component has a Kd of 10-20 nM. In the solubilized preparation, only a single class of binding sites is apparent, with a Kd of 50-100 nM. Removal of detergents by dialysis and polystyrene bead adsorption results in restoration of the curvilinear Scatchard plot with apparent Kds similar to those observed in crude membrane preparations and with increased Bmax values for each component. [3H]Serotonin binding activity in the solubilized preparation is stable to Sephacryl S-300 column chromatography and to glycerol gradient sedimentation. Saturation analysis of the peak binding fractions from both these procedures once again yields curvilinear Scatchard plots, indicating that the multiple high-affinity binding components are preserved and migrate together. The molecular weight, Stokes radius, and frictional coefficient of the binding site(s) have been calculated. After detergent removal the solubilized material shows many of the characteristics usually attributed to S1 receptors, such as high affinity for [3H]serotonin and its analogs and low affinity for serotonin antagonists.
Plasticity in the central nervous system: do synapses divide?
Authors: Carlin R K , Siekevitz P
Published: May 2006

Changes in the proportion of synapses containing postsynaptic densities with perforations during periods of increased synapse formation have led us to propose a hypothesis describing a possible division of preexisting synapses. Relevant features of this model are that various types of stimulation result in the following sequence of events: (i) the synaptic junction increases in area; (ii) a perforation forms in the enlarging synaptic junction; (iii) a synaptic spinule appears apposed to the perforation in the postsynaptic density; (iv) the perforation in the synaptic junction increases in size until the synaptic junction splits into two separate synaptic junctions within the same synaptic terminal; and (v) the dendritic spine divides into two, each containing a synaptic junction. Physiological responses in which synapse division may possibly play a role include hormone-induced neuronal changes, reinnervation of dendrites after lesions, and learning and memory.
Isolation of a nicotine binding site from rat brain by affinity chromatography.
Authors: Abood L G , Latham W , Grassi S
Published: May 2006

With the use of affinity chromatography, a [3H]-nicotine binding site was purified almost 1,000-fold from a Triton X-100-solubilized extract of rat brain neural membranes. The affinity column was prepared by conjugation of (R,S)-6-(2-hydroxyethyl)nicotine to epoxy-activated Sepharose. Further purification of the material from the affinity column was resolved by using another column of the same affinity gel, resulting in the isolation of a major protein (about 95% purity) that had a Mr of 56,000, as determined by NaDodSO4/polyacrylamide gel electrophoresis, with very minor components ranging in Mr from 47,000 to 83,000. With the use of various nicotine analogues, it was shown that the purified material exhibited nearly identical binding characteristics to rat brain membrane preparations, including stereoselectivity for the nicotine enantiomers. The Kd of the purified site, 3.5 x 10(-9) M, was similar to that observed with membrane and Triton X-100-soluble preparations, whereas the binding capacity was greater than 25 pmol/mg of protein, as compared to 0.07 pmol/mg of protein in the starting material. The results are discussed in relation to the purified nicotinic cholinergic receptor from electroplax. It was concluded that the nicotine site in rat brain was different from the cholinergic receptor of electroplax or calf skeletal muscle.
Isolation of a cDNA clone for the type I regulatory subunit of bovine cAMP-dependent protein kinase.
Authors: Lee D C , Carmichael D F , Krebs E G , McKnight G S
Published: May 2006

A cDNA clone for the type I regulatory subunit (RI) of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was isolated from bovine testis by a differential screening method. mRNA coding for RI was enriched 50- to 100-fold by polysome immunoadsorption chromatography with affinity-purified rabbit anti-RI and protein A-Sepharose. Poly(A)+ RNA from these polysomes was utilized to construct a cDNA library in pBR322, and this library was screened for hybridization to 32P-labeled cDNAs synthesized from either total or RI-enriched poly(A)+ RNA. Plasmids isolated from colonies showing preferential hybridization to the latter probe were further characterized by hybrid selection and DNA sequence analysis. One of these plasmids (designated 62C12) contains a 1,350-nucleotide insert that hybridized to RI mRNA; partial nucleotide sequence analysis confirmed its identity and indicated that it may contain the entire RI coding region. We also have identified a recombinant plasmid with a 1,550-nucleotide insert that selected through hybridization a mRNA coding for a 55,000-dalton protein that crossreacts with anti-RI antibodies. The function of this latter protein is unknown.
Subfragments of the large terminal repeat cause glucocorticoid-responsive expression of mouse mammary tumor virus and of an adjacent gene.
Authors: Hynes N , van Ooyen A J , Kennedy N , Herrlich P , Ponta H , Groner B
Published: May 2006

After transfection of mouse mammary tumor virus (MMTV) proviral DNA into cultured cells, the DNA is transcribed in a glucocorticoid-sensitive fashion. The large terminal repeat (LTR) region of MMTV is 1,328 nucleotides long and contains the regulatory information necessary for the hormonal response. We have constructed a MMTV LTR-thymidine kinase (tk) chimeric gene and have tested the biological activity of molecules containing various deletions in the LTR after transformation of LTK- APRT- mouse cells. In the TK+ transformants, both a LTR- tk chimeric RNA and an authentic tk RNA are correctly initiated and transcribed. The synthesis of the chimeric RNA as well as that of the tk RNA is hormonally regulated. A plasmid containing 202 nucleotides of LTR DNA 5' to the RNA initiation site is fully sensitive to glucocorticoids; 50 nucleotides still cause a residual inducibility.
Isolation and characterization of the gene coding for cytosolic phosphoenolpyruvate carboxykinase (GTP) from the rat.
Authors: Yoo-Warren H , Monahan J E , Short J , Short H , Bruzel A , Wynshaw-Boris A , Meisner H M , Samols D , Hanson R W
Published: May 2006

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from the rat was isolated from a recombinant library containing the rat genome in phage lambda Charon 4A. The isolated clone, lambda PCK1, contains the complete gene for phosphoenolpyruvate carboxykinase and approximately equal to 7 kilobases (kb) of flanking sequence at the 5' end and 1 kb at the 3' terminus. Restriction endonuclease mapping, R-loop mapping, and partial DNA sequence assay indicate that the gene is approximately equal to 6.0 kb in length (coding for a mRNA of 2.8 kb) and contains eight introns. Southern blotting of rat DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of lambda PCK1. A control region at the 5' end of the gene contained in a 1.2-kb restriction fragment was isolated and subcloned into pBR322. This segment of the gene contains the usual transcription start sequences and a 24-base sequence virtually identical to the sequence found in the 5'-flanking region of the human proopiomelonocortin gene, which is known to be regulated by glucocorticoids. The 1.2-kb fragment of the phosphoenolpyruvate carboxykinase gene can be transcribed into a unique RNA fragment of predicted size by an in vitro transcription assay.
Structure-function relationships for a voltage-dependent ion channel: properties of COOH-terminal fragments of colicin E1.
Authors: Cleveland M V , Slatin S , Finkelstein A , Levinthal C
Published: May 2006

The effects on planar lipid bilayer membranes of carboxyl-terminal fragments derived from the bacteriocin colicin E1 by either proteolysis or CNBr cleavage are indistinguishable from those of the voltage-dependent parent colicin molecule. An upper limit to the length of the COOH-terminal peptide required for channel formation is 152 amino acid residues from the COOH-terminal end, as indicated by the CNBr fragment. In addition, use of carboxypeptidase shows that the COOH-terminal end of the molecule remains on the side of the membrane to which it was added. COOH-terminal peptides of colicin E1 spontaneously associate with oil or hexane droplets in an aqueous system and remain at the interface between the two phases to a significantly greater degree than other colicin E1 fragments or cytochrome c. These results, together with the amino acid sequence, suggest a model wherein the colicin E1 channel is formed first by spontaneous attachment to a membrane of an alpha-helical hairpin centered at a 35-residue hydrophobic region near the COOH-terminal end. Application of a potential of the correct polarity then facilitates a major conformational change in the protein, allowing insertion of the remainder of the COOH-terminal end to form the open channel.
Neoplastic transformation by a cloned human cytomegalovirus DNA fragment uniquely homologous to one of the transforming regions of herpes simplex virus type 2.
Authors: Clanton D J , Jariwalla R J , Kress C , Rosenthal L J
Published: May 2006

Specific DNA fragments of human cytomegalovirus strain Towne exhibited sequence homology to the transforming regions of herpes simplex virus type 2 (HSV-2) when examined by nitrocellulose filter hybridization under nonstringent conditions. Cloned Towne Xba I fragments B and C were homologous to both Bgl II transforming fragments N and C of HSV-2 DNA, whereas cloned Towne Xba I fragment E was uniquely homologous to HSV-2 Bgl II fragment C. Furthermore, Towne Xba I fragment E exhibited homology to a unique fragment of cytomegalovirus strain AD169 but lacked homology to the recently identified Xba I transforming (focus-forming) fragment N. Normal diploid Syrian hamster embryo cells transfected with cloned Towne Xba I fragment E displayed colonies of refractile, rapidly dividing cells which escaped senescence to form immortal cell lines. At early passages, these lines exhibited growth in 2% serum and formed small (less than 0.1 mm) colonies in 0.3% agarose. Serial passaging resulted in the appearance of large (greater than 0.25 mm) colonies in agarose, indicating the involvement of more than one step in Towne Xba I fragment E-induced transformation of the diploid hamster embryo cells. NIH 3T3 cells transfected with Towne Xba I fragment E rapidly displayed large colonies in agarose and tumors in vivo.
In vivo and in vitro observations on laminin production by Schwann cells.
Authors: Cornbrooks C J , Carey D J , McDonald J A , Timpl R , Bunge R P
Published: May 2006

Recent reports on the dystrophic mouse mutant suggest that the prominent extracellular matrix component of peripheral nerve tissues plays an important role in peripheral nerve development. We have examined the disposition of two prominent extracellular matrix components, fibronectin and laminin, both in mature peripheral nerve in vivo and in an in vitro system that allows study of Schwann cells in various functional states. In frozen sections of whole nerve, staining with antibodies to fibronectin and laminin shows that fibronectin stains throughout the endoneurium while laminin staining is restricted to regions known to contain basal lamina, particularly the basal lamina of each ensheathing Schwann cell. Tissue culture studies indicate that fibronectin staining at the light microscopic level is a reliable marker for fibroblasts (and not Schwann cells) in culture; conversely, antibodies to laminin stain components related to the Schwann cell surface but not components related to fibroblasts. Unexpectedly, Schwann cells in culture produce laminin at all stages in development, whether in contact with axons or not. As Schwann cells in culture begin to ensheathe axons, punctate regions of laminin on their surfaces become confluent. After ensheathment is completed, a continuous line of staining is found in the region of the Schwann cell basal lamina. It has been established that Schwann cells produce a basal lamina only when in contact with axons. Therefore, the production of laminin appears to be necessary but not sufficient for basal lamina formation. The constancy of laminin production by Schwann cells provides a reliable basis for distinguishing between Schwann cells and fibroblasts in tissue culture.
Phencyclidine in nanomolar concentrations binds to synaptosomes and blocks certain potassium channels.
Authors: Blaustein M P , Ickowicz R K
Published: May 2006

Phencyclidine [1-(phenylcyclohexyl)piperidine; PCP], in low dose (approximately equal to 0.1-0.2 mg/kg of body weight), induces a schizophrenia-like behavioral syndrome in man; this effect has been attributed to block of neuronal K channels. We used a K-stimulated 86Rb efflux assay to demonstrate that low concentrations of PCP (10-50 nM) block a class of depolarization-activated K channels in rat brain synaptosomes--pinched-off presynaptic nerve terminals. The dose-response curve is biphasic, and much higher PCP concentrations (greater than 10 microM) are required to block the remainder of the K-stimulated 86Rb efflux. The [3H]PCP binding curve for synaptosomes is also biphasic: PCP binds to some components with high affinity (Kd approximately equal to 6.0 X 10(-8) M), and to other components with much lower affinity (Kd approximately equal to 1.15 X 10(4) M). PCP can be photoactivated with UV light to form covalent bonds: after UV irradiation, previously-bound [3H]PCP is no longer displaceable by a large excess of unlabeled PCP. Preliminary data from NaDodSO4/polyacrylamide gel electrophoresis studies after covalent binding of [3H]PCP to synaptosomes, suggest that the high-affinity binding site may be on a large protein (Mr approximately equal to 220,000). We conclude that the high-affinity PCP binding protein is associated with the K channels that are blocked by nanomolar concentrations of PCP. Block of these channels could, by prolonging action-potential duration in presynaptic nerve terminals, enhance calcium entry and neurotransmitter release, thereby altering transmission at central synapses involved in behavioral expression.
Complete nucleotide sequence of the gene for the specific glycoprotein (gp55) of Friend spleen focus-forming virus.
Authors: Amanuma H , Katori A , Obata M , Sagata N , Ikawa Y
Published: May 2006

The complete nucleotide sequence of the gene for the specific glycoprotein (gp55) of the polycythemic strain of Friend spleen focus-forming virus (SFFV) was derived from the cloned SFFV DNA intermediate. The gp55 gene is present within 1.4 kilobases of the 5' side of the 3'long terminal repeat sequence. The open reading frame predicts the primary translation product has a total of 409 amino acids with a Mr of 44,752. Comparisons of the deduced amino acid sequence of gp55 with those of the envelope (env) gene products of murine leukemia viruses (MuLVs) revealed that gp55 is composed of three distinct regions. The amino-terminal 80% of the molecule has a high degree of sequence homology with the amino-terminal portion of the gp70 of the Moloney mink cell focus-forming virus (BALB/Mo-MCFV). This portion of the BALB/Mo-MCFV gp70 is known to be coded for by the acquired xenotropic env-like sequence. The sequence of the following 66 amino acids of gp55 is highly homologous to that of the middle portion of the p15E of Moloney MuLV (Mo-MuLV). The sequence of the Carboxyl-terminal 12 amino acids is specific to gp55 and a comparison of the nucleotide sequence showed that this specific amino acid sequence is due to the presence of seven extra nucleotides compared with the sequence of the Mo-MuLV.
Modulation of proline-rich protein biosynthesis in rat parotid glands by sorghums with high tannin levels.
Authors: Mehansho H , Hagerman A , Clements S , Butler L , Rogler J , Carlson D M
Published: May 2006

Feeding of sorghum with a high level of tannin (high-tannin sorghum) to rats caused changes in gene expression in parotid glands similar to isoproterenol treatment. Within 3 days the parotid glands were enlarged about 3-fold and a series of proline-rich proteins were increased about 12-fold. Unlike isoproterenol treatment, no changes were observed in the submandibular glands, and a Mr 220,000 glycoprotein in parotid glands was not induced. Amino acid analyses, electrophoretic patterns, and cell-free translations of mRNAs all confirmed that the proline-rich proteins induced by feeding high-tannin sorghum were identical to those induced by isoproterenol treatment. Binding curves for proline-rich proteins to tannins showed affinities 10-fold greater than bovine serum albumin and tannins.
Serum spreading factor (vitronectin) is present at the cell surface and in tissues.
Authors: Hayman E G , Pierschbacher M D , Ohgren Y , Ruoslahti E
Published: May 2006

Monoclonal antibodies were prepared against a cell attachment-promoting protein, serum spreading factor, which had been partially purified from human serum by chromatography on glass bead columns. The antibodies selected were those that reacted with polypeptides that had cell attachment-promoting activity after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunochromatography of human plasma on columns containing the monoclonal antibodies followed by affinity chromatography on heparin-Sepharose yielded material that in sodium dodecyl sulfate/polyacrylamide gel electrophoretic analysis gave polypeptides of molecular mass 65 and 75 kilodaltons. Both polypeptides bound each of three monoclonal antibodies and had cell attachment-promoting activity after transfer to nitrocellulose filters. Immunofluorescent staining of tissues with the monoclonal antibodies revealed a fibrillar pattern that was mostly associated with loose connective tissue and overlapped with fibronectin fibrils. Fetal membrane tissue, which showed strong staining with the antibodies in immunofluorescence, also gave 65- and 75-kilodalton polypeptides with cell attachment-promoting activity after chromatography on columns containing the monoclonal antibodies. One source of the tissue protein may be fibroblastic cells, because cultured human fibroblasts also stained with the monoclonal antibodies. The staining was fibrillar and appeared to be associated with the cell surface extracellular matrix. We propose the name "vitronectin" for the various forms of this protein, on the basis of its binding to glass and its adhesive properties.
Intramolecular heterogeneity of degradation in plasma membrane glycoproteins: evidence for a general characteristic.
Authors: Tauber R , Park C S , Reutter W
Published: May 2006

Five integral plasma membrane glycoproteins (60, 80, 120, 140, and 160 kilodaltons) were isolated to homogeneity from rat liver by a four-step procedure: (i) extraction of plasma membranes with lithium diiodosalicylate, (ii) solubilization of glycoproteins with Nonidet P-40, (iii) affinity chromatography on concanavalin A-Sepharose, and (iv) semipreparative NaDodSO4/polyacrylamide gel electrophoresis. The glycoproteins contained 48.5--51.5% hydrophobic amino acids. Carbohydrate moieties contained N-acetyl-D-glucosamine, D-mannose, D-galactose, L-fucose, and N-acetylneuraminic acid. N-Acetyl-D-galactosamine was not detectable. Half-lives of degradation of the carbohydrate and protein moieties of the five glycoproteins were measured by pulse-chase experiments in vivo. Protein moieties had half-lives ranging from 52 to 88 hr in the five glycoproteins, with a mean of 73 +/- 15 hr. Terminal sugars, L-fucose, and N-acetylneuraminic acid had significantly shorter half-lives, averaging 18 +/- 2 hr and 29 +/- 3 hr, respectively. The half-life of D-mannose varied between that of the terminal sugars and that of the protein moiety, depending on the type of the glycoprotein. The data show that the carbohydrate moieties are degraded faster than the protein portion of the glycoproteins. As this finding was obtained in each of the five glycoproteins, intramolecular heterogeneity of breakdown may be a general characteristic of plasma membrane glycoproteins in liver.
Expression and regulation of a human metallothionein gene carried on an autonomously replicating shuttle vector.
Authors: Karin M , Cathala G , Nguyen-Huu M C
Published: May 2006

A human metallothionein (MT) gene was inserted into a bovine papillomavirus (BPV) vector. The chimeric vector (pMTII-BPV) transforms rodent fibroblasts to a cadmium-resistant phenotype. The resistance is due to the high level of expression of human MT-II in those cells. The vector is maintained in the cells as a free replicating plasmid, present at about 10--15 copies per cell. Transcription of the episomal human MT-IIA gene is initiated from its authentic start sites and is regulated by the level of cadmium in the growth medium. The presence of the human MT-IIA gene allows the BPV replicon to function even though it is ligated to an intact copy of pBR322. Due to the presence of plasmid origins of replication and dominantly acting selective markers functional in both Escherichia coli and mammalian cells, pMTII-BPV can be used as a shuttle vector.
Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.
Authors: Leary J J , Brigati D J , Ward D C
Published: May 2006

Biotin-labelled DNA probes, prepared by nick-translation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2--5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal alkaline phosphatase. The filters are then incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, which results in the deposition of a purple precipitate at the sites of hybridization. This procedure will detect target sequences in the 1- to 10-pg range after enzyme incubation periods of 1 hr or less. The incubation period can be extended up to 24 hr, if required, to increase the color intensity of the hybridization signal. Furthermore, at high probe concentrations (250--7560 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1--2 hr. This nonradiographic method of probe detection should be of general utility for genetic studies using Southern, RNA, or dot-blot hybridization protocols.
A quantitative genetic model of reciprocal altruism: a condition for kin or group selection to prevail.
Authors: Aoki K
Published: May 2006

A condition is derived for reciprocal altruism to evolve by kin or group selection. It is assumed that many additively acting genes of small effect and the environment determine the probability that an individual is a reciprocal altruist, as opposed to being unconditionally selfish. The particular form of reciprocal altruism considered is TIT FOR TAT, a strategy that involves being altruistic on the first encounter with another individual and doing whatever the other did on the previous encounter in subsequent encounters with the same individual. Encounters are restricted to individuals of the same generation belonging to the same kin or breeding group, but first encounters occur at random within that group. The number of individuals with which an individual interacts is assumed to be the same within any kin or breeding group. There are 1 + i expected encounters between two interacting individuals. On any encounter, it is assumed that an individual who behaves altruistically suffers a cost in personal fitness proportional to c while improving his partner's fitness by the same proportion of b. Then, the condition for kin or group selection to prevail is [Formula: see text] if group size is sufficiently large and the group mean and the within-group genotypic variance of the trait value (i.e., the probability of being a TIT-FOR-TAT strategist) are uncorrelated. Here, C, Vb, and Tb are the population mean, between-group variance, and between-group third central moment of the trait value and r is the correlation between the additive genotypic values of interacting kin or of individuals within the same breeding group. The right-hand side of the above inequality is monotone decreasing in C if we hold Tb/Vb constant, and kin and group selection become superfluous beyond a certain threshold value of C. The effect of finite group size is also considered in a kin-selection model.
Anti-acetylcholine receptor antibodies directed against the alpha-bungarotoxin binding site induce a unique form of experimental myasthenia.
Authors: Gomez C M , Richman D P
Published: May 2006

Of seven rat monoclonal antibodies directed against nicotinic acetylcholine receptor, the three whose binding was blocked by alpha-bungarotoxin produced acute paralysis in chicken hatchlings, whereas the four others had no effect. In the affected animals, weakness and decremental electromyographic responses appeared within 1 hr after intravenous injection and both abnormalities improved after anticholinesterase administration. No histological changes were seen in the muscle of injected animals. These data suggest that antibodies binding in relationship to the cholinergic binding site, and presumably producing pharmacologic blockade of acetylcholine receptor, may play an important role in the pathogenesis of the defective neuromuscular transmission in myasthenia gravis.
Sequential expression of new gene programs in inducer T-cell clones.
Authors: Freeman G J , Clayberger C , DeKruyff R , Rosenblum D S , Cantor H
Published: May 2006

We have prepared a cDNA probe that detects genes that are rapidly and abundantly expressed after exposure of inducer T-lymphocyte clones to antigen or mitogen. All inducer cells tested express a characteristic set of new mRNA, and these mRNAs are not expressed after activation of other lymphocytes. This initial burst of mRNA synthesis is paralleled by synthesis and secretion of a family of polypeptides that mediate inducer cell activity, including T- and B-cell growth factors, interferon, and molecules that bind to antigen. Expression of this initial genetic program precedes mitosis and is replaced within 74 hr by a different genetic program, which may control further cell division. The action of these sequential sets of genetic programs defines two stages of the cell's differentiation and accounts for altered expression of the cell's immunological functions.
Characterization of a specific phorbol ester aporeceptor in mouse brain cytosol.
Authors: Leach K L , James M L , Blumberg P M
Published: May 2006

In the presence of phosphatidylserine, [20-3H]-phorbol 12,13-dibutyrate [( 3H]PBt2) bound specifically to a single class of binding sites in mouse brain cytosol (supernatant at 100,000 X g). The dissociation constant for binding was 3.1 X 10(-9) M, and at saturation 23.2 pmol of [3H]PBt2 was bound per mg of cytosolic protein. Less than 1 pmol of [3H]PBt2 per mg bound in the absence of phospholipids. Phosphatidic acid, sphingomyelin, and phosphatidylinositol also were able to reconstitute binding activity, whereas phosphatidylcholine and phosphatidylethanolamine were relatively ineffective. [3H]PBt2 binding was inhibited by phorbol 12-myristate 13-acetate (Ki = 4.4 X 10(-11) M), phorbol 12,13-didecanoate (Ki = 7.7 X 10(-9) M), phorbol 12,13-diacetate (Ki = 4.4 X 10(-7) M) and 4-O-methylphorbol 12-myristate 13-acetate (Ki = 5.1 X 10(-7) M). The apparent Ki values of the phorbol-related diterpenes for inhibiting binding agreed reasonably closely with the values previously determined for mouse brain membrane binding. The biologically inactive derivatives phorbol (30 microM) and 4 alpha-phorbol 12,13-didecanoate (30 microM) did not inhibit binding. The aporeceptor was eluted in one peak during Ultrogel 44 column chromatography, corresponding to a molecular weight of approximately equal to 77,000. Calcium phospholipid-dependent protein kinase C activity was eluted with a profile similar to that of the cytosolic aporeceptor-binding activity.
Testing an alternative model for the ribosomal peptide elongation cycle.
Authors: Rheinberger H J , Nierhaus K H
Published: May 2006

A kinetic analysis of poly(U)-dependent poly(Phe) synthesis with [14C]tRNAPhe and [3H]phenylalanine demonstrated that, in the course of efficient poly(Phe) synthesis, two tRNAs are present per 70S ribosome at all times, although at least 70% of the poly(Phe)-tRNAPhe is found at the peptidyl-tRNA (P) site. Together with our recent observation of a third tRNA-binding site on Escherichia coli ribosomes, these findings suggest a model for the peptide elongation cycle in which two tRNA molecules are present on the ribosome at both the pre- and the post-translocational state. This model predicts that deacylated tRNA is not released from the P site but translocated to the exit (E) site before release occurs. A series of translocation experiments with deacylated [14C]tRNAPhe at the P site and oligo [( 3H]Phe)-tRNA at the aminoacyl-tRNA (A) site proved that efficient elongation factor G-dependent translocation is not accompanied by a corresponding [14C]tRNAPhe release. However, significant [14C]tRNAPhe release was observed after translocation when an aminoacyl-tRNA was bound to the A site. Thus, deacylated tRNA is not released from the P site but is translocated to the E site, which therefore must be located "upstream" adjacent to the P site. Furthermore, the trigger for the release of deacylated tRNA from the E site is the binding of aminoacyl-tRNA to the A site.
Structure and biological activity of v-raf, a unique oncogene transduced by a retrovirus.
Authors: Rapp U R , Goldsborough M D , Mark G E , Bonner T I , Groffen J , Reynolds F H , Stephenson J R
Published: May 2006

We have molecularly cloned a unique acutely transforming replication-defective mouse type C virus (3611-MSV) and characterized its acquired oncogene. The viral genome closely resembles Moloney (M) murine leukemia virus (MuLV), except for a substitution in M-MuLV in the middle of p30 and the middle of the polymerase gene (pol). Heteroduplex analysis revealed that 2.4 kilobases of M-MuLV DNA were replaced by 1.2 kilobases of cellular DNA. The junctions between viral and cellular sequences were determined by DNA sequence analysis to be 517 nucleotides into the p30 sequence and 1,920 nucleotides into the polymerase sequence. Comparison of the transforming gene from 3611-MSV, designated v-raf, with previously isolated retrovirus oncogenes either by direct hybridization or by comparison of restriction fragments of their cellular homologs shows it to be unique. Transfection of NIH 3T3 cells with cloned 3611-MSV proviral DNA leads to highly efficient transformation and the recovered virus elicits tumors in mice typical of the 3611-MSV virus. Transfected NIH 3T3 cells express two 3611-MSV-specific polyproteins (P75 and P90), both of which contain NH2-terminal gag gene-encoded components linked to the acquired sequence (v-raf) translational product. The cellular homolog, c-raf, is present in one or two copies per haploid genome in mouse and human DNA.
Cloning of human purine-nucleoside phosphorylase cDNA sequences by complementation in Escherichia coli.
Authors: Goddard J M , Caput D , Williams S R , Martin D W
Published: May 2006

We have obtained cDNA clones that contain the entire coding region of the human purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) mRNA. The cDNA sequences were generated by reverse transcription of PNP-enriched mRNA obtained by immunoadsorption of HeLa cell polyribosomes with monospecific antibody to human PNP. cDNA molecules that were close in length to PNP mRNA were separated by agarose gel electrophoresis and inserted into the Pst I site of the plasmid pBR322. Plasmid DNA from the pooled clones was used to transform PNP-deficient Escherichia coli cells, and those transformants that phenotypically expressed PNP were isolated on selective media. The presence of human PNP in the selected bacterial cells was detected by immunoprecipitation with human PNP antibody.
Birds, behavior, and anatomical evolution.
Authors: Wyles J S , Kunkel J G , Wilson A C
Published: May 2006

Study of more than 200 species suggests that the anatomical differences among birds are as big as those among other vertebrates of comparable taxonomic rank. The result is notable because, for more than 100 years, many biologists have believed that birds are more uniform anatomically than other classes of vertebrates. Furthermore, assessment of biochemical and geological evidence suggests that the time scale for bird evolution could be quite short. Hence, birds may share with placental mammals the distinction of having had a high rate of anatomical evolution, compared to that in lower vertebrates. The rate appears to have been very high in songbirds and higher primates and extremely high in the genus Homo. In an attempt to explain such contrasts in rates of anatomical evolution, we advance the hypothesis that in higher vertebrates, behavior, rather than environmental change, is the major driving force for evolution at the organismal level. This hypothesis predicts accelerated anatomical evolution in species composed of numerous mobile individuals with the dual capacity for behavioral innovation and social propagation of new habits. Consistent with this hypothesis, we demonstrate a correlation between relative brain size and rate of anatomical evolution in land vertebrates.
Molecular cloning and characterization of the gene coding for human complement protein factor B.
Authors: Campbell R D , Porter R R
Published: May 2006

Four cosmid clones, each with an average insert size of 40 kilobase pairs and containing the factor B gene, were isolated from a human genomic DNA library. The clones were identified by hybridization with a 515-base-pair cDNA probe isolated by using a unique 17-base synthetic oligonucleotide probe from a human liver cDNA library. The cosmid clones were characterized by restriction endonuclease digestion and Southern blotting, and a partial restriction map of the DNA represented in the cosmids was constructed. The Bb portion of the factor B gene is about 4 kb in length. DNA sequence analysis has resulted in the determination of 3.3 kb of sequence at the 3' end of the gene. This region codes for amino acids 87-505 of Bb and includes the whole of the serine proteinase domain of the protein. The three active site residues of histidine, aspartic acid, and serine found at positions 267, 317, and 440 of the Bb sequence, respectively, lie on separate exons. Other functional regions within the serine proteinase domain are separated also by intervening sequences.
(2'-5')Oligoadenylate in rat liver: modulation after partial hepatectomy.
Authors: Etienne-Smekens M , Vandenbussche P , Content J , Dumont J E
Published: May 2006

(2'-5')Oligoadenylate synthetase [(2'-5')A synthetase], which synthesizes a series of oligoadenylates ppp-(A2'p)n5'A [collectively referred to as (2'-5')A], has been described previously in rat liver cells, where its concentration varied with the growth status of this organ--i.e., it decreased during the early phase of rat liver regeneration after partial hepatectomy. Because double-stranded RNA, the only known activator of this enzyme, has been detected in rat liver nuclei, (2'-5')A synthesis could occur in this tissue in vivo. Analysis of rat liver cell extract after HPLC by the endonuclease-based radiobinding assay revealed several components with retention times similar to (2'-5')A trimer- and tetramer-like material. A further characterization of these compounds by their susceptibility to alkaline phosphatase and snake venom phosphodiesterase, their resistance to micrococcal nuclease, and their ability to activate an endonuclease indicated the natural occurrence of oligonucleotides indistinguishable from authentic (2'-5')A in rat liver cells. Using the combination of the radiobinding assay and a simplified (2'-5')A extraction procedure that does not involve HPLC, we further show that the early drop of (2'-5')A synthetase activity during rat liver regeneration was accompanied by a similar decrease in intracellular (2'-5')A concentration. The three characteristic phases of the (2'-5')A synthetase kinetics during the first 40 hr of liver regeneration were mimicked by the kinetics of the synthesis of the (2'-5')A oligonucleotides themselves: between 6 and 20 hr after hepatectomy, there was a sharp decrease in (2'-5')A concentration; between 20 and 24 hr, the concentration of (2'-5')A reached a minimum; at 36 hr or after the first wave of DNA synthesis (the major event of liver regeneration), the (2'-5')A concentration returned to normal. In this characterization of the (2'-5')A oligonucleotide family in a functional tissue of an animal that had not been previously treated with interferon or infected with virus, the data are compatible with a physiological role of the (2'-5')A system acting as an intracellular component of the regulatory mechanisms leading to cell proliferation or differentiation.
Spontaneous activation of a human proto-oncogene.
Authors: Santos E , Reddy E P , Pulciani S , Feldmann R J , Barbacid M
Published: May 2006

It has been recently shown that malignant activation of the c-has/bas proto-oncogene in T24 human bladder carcinoma cells was mediated by a single point mutation. A deoxyguanosine located at position 35 of the first exon of this proto-oncogene was substituted by thymidine. These findings predicted that the resulting oncogene would code for a structurally altered p21 protein containing valine instead of glycine as its 12th amino acid residue. We now report the spontaneous activation of the human c-has/bas proto-oncogene during transfection of NIH/3T3 cells. As in T24 cells, this in vitro activated oncogene also acquired malignant properties by a single point mutation. In this case we have detected a G leads to A transition, which occurred at the same position as the mutation responsible for the activation of the T24 oncogene. These results predict that the p21 protein coded for by the spontaneously activated c-has/bas gene will incorporate aspartic acid as its 12th amino acid residue. Computer analysis of the secondary structure of c-has/bas encoded p21 proteins indicates that substitution of the glycine residue located at position 12, not only by aspartic acid or valine but also by any other amino acid, would result in the same structural alteration. These findings indicate that a specific conformational change is sufficient to confer transforming properties to this p21 protein. Moreover, they predict that any mutation affecting the coding properties of the 12th codon of the c-has/bas proto-oncogene will lead to its malignant activation.
RETRACTED: A precise termination site in the mouse beta major-globin transcription unit.
Authors: Salditt-Georgieff M , Darnell J E
Published: May 2006

Nascent labeled RNA from induced, globin-producing mouse erythroleukemia cells was hybridized to cloned regions of the beta major-globin gene. Transcription ceases about 1,000 bases downstream from the poly(A) site as indicated by protection from nuclease digestion of a discrete-sized RNA fragment that it shorter than the protecting cloned DNA fragment. This defines an apparently unique termination site for a protein-coding gene that is transcribed by RNA polymerase II.
A DNA repair gene required for the incision of damaged DNA is essential for viability in Saccharomyces cerevisiae.
Authors: Naumovski L , Friedberg E C
Published: May 2006

A diploid strain (RAD3/RAD3) of Saccharomyces cerevisiae was transformed with an integration plasmid containing an internal fragment of the cloned yeast RAD3 gene. Integration by homologous recombination inactivated one of the diploid RAD3 genes, creating a recessive mutation. This mutation is inferred to be lethal in haploid cells since sporulation of diploid transformants segregated two viable and two inviable spores per tetrad, while integration of plasmids containing one or the other end of the RAD3 gene resulted in diploid transformants that segregated normally--i.e., four viable spores in each tetrad. Evidence that integration of the internal fragment occurred specifically at one of the RAD3 genes in the diploid is provided by DNA . DNA hybridizations. In addition, transformation of a diploid strain heterozygous for the RAD3 gene (RAD3/rad3-2) (carrying a rad3 mutation that does not affect the viability of haploid cells) results in the rad- phenotype in half of the transformants, indicating that the RAD3 gene was inactivated in these cells.
Hepatitis B virus DNA in Kaposi sarcoma.
Authors: Siddiqui A
Published: May 2006

DNA extracted from Kaposi sarcoma biopsies was analyzed for the presence of hepatitis B viral (HBV) DNA sequences by Southern blot hybridization. The probes used were (i) molecularly cloned subgenomic fragments of HBV DNA and (ii) HBV DNA extracted from Dane particles that included DNA sequences that encode hepatitis B surface antigen and core antigen. The limited study reported here demonstrates that HBV DNA exists mainly in an episomal state in the Kaposi sarcoma tissue and that these DNA forms are different from those found in hepatitis B virion. The presence of various DNA forms suggests that HBV DNA is engaged in DNA replication, which implies that the tissue is infected with HBV.
Resolution of the diadenosine 5',5"'-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase alpha.
Authors: Baril E , Bonin P , Burstein D , Mara K , Zamecnik P
Published: May 2006

A diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase alpha [deoxynucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells [DNA polymerase alpha 2 of Lamothe, P., Baril, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl. Acad. Sci. USA 78, 4723-4727]. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatography on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A.
Induction of ppp(A2'p)nA-dependent RNase in murine JLS-V9R cells during growth inhibition.
Authors: Jacobsen H , Krause D , Friedman R M , Silverman R H
Published: May 2006

We recently reported that interferon induces the synthesis of ppp(A2'p)nA(n = 2 to greater than or equal to 4) (2-5A)-dependent RNase in the murine cell line JLS-V9R. These cells normally contain very low levels of the nuclease; after interferon treatment, however, they develop levels approaching those found in murine L or Ehrlich ascites tumor cells. Here, we report a similar increase in the nuclease levels in JLS-V9R cells during the transition from the subconfluent actively growing state to the confluent stationary phase. Levels of 2-5A synthetase increased in parallel with the nuclease. The induced levels of both the nuclease and synthetase returned to low basal amounts after trypsinization, dilution, and culturing of the cells at subconfluent densities. The addition of anti-murine interferon (alpha + beta) antibodies to the medium did not affect the induction of the nuclease nor could any interferon be detected in the culture supernatants as determined by the lack of antiviral activity. The increase in the enzymes was not, therefore, due to the spontaneous production of interferon. The induction of the nuclease during confluency preceded an inhibition of [3H]-thymidine incorporation by the cells into DNA. The regulation of the 2-5A-dependent RNase in JLS-V9R cells may, therefore, be related to the control of cell growth.
Unequal crossing-over associated with asymmetrical synapsis between nomadic elements in the
Drosophila melanogaster
genome
Authors: Goldberg Michael L. , Sheen Jenq-Yunn , Gehring Walter J. , Green M. M.
Published: May 2006

The molecular structure of reciprocal duplications and deficiencies produced by unequal crossing-over at the white (
w
) locus of
Drosophila melanogaster
females heterozygous for the alleles

w
a

and

w
a4

has been examined. A transposable,
copia
-like element is found at the rearrangement breakpoints. Further characterization indicates that asymmetrical pairing between two copies of this element, which are at least 60 kilobases apart in the parental chromosomes, followed by a crossover within the paired elements, is responsible for the duplication and deficiencies observed. The frequency of these events is high compared with normal homologous exchange, implying that synaptic pairing during meiosis must be sufficiently flexible as to allow efficient recognition of sequences located in nonidentical positions on homologous chromosomes. These results suggest a possible mechanism for the generation of tandem duplications in eukaryotic organisms.
Mutant analysis shows that the Ca2+-induced K+ current shuts off one type of excitation in Paramecium.
Authors: Saimi Y , Hinrichsen R D , Forte M , Kung C
Published: May 2006

Two mutants of Paramecium tetraurelia, called "pantophobiacs," were found to lack most of the slow Ca2+-induced K+ outward current. Passive properties, the transient Ca2+ inward current, and the fast depolarization-induced K+ outward current remain normal. The mutant defect reduces the ability to shut off a normal, excited state of the membrane and results in repeated, long backward swimming instead of the wild-type jerks in response to a variety of ions, to heat, and to touch.