bluuebunny/crossref_metadata_2025 huggingface dataset:
Matrix control of protein diffusion in biological membranes.
Authors: Koppel D E , Sheetz M P , Schindler M
Published: May 2006

Lateral diffusion coefficients of fluorescently labeled lipids and integral membrane proteins were determined in the membranes of normal and spectrin-deficient spherocytic mouse erythrocytes by the technique of fluorescence redistribution after photobleaching. The results were used to generate a mathematical description of a matrix-control model of membrane protein diffusion. In the spherocytic cells, which lack the principal components of the cytoskeletal matrix of normal cells, the diffusion coefficients of lipid (1.5 +/- 0.5 X 10(-8) cm2/s) and protein (2.5 +/- 0.6 X 10(-9) cm2/s) differ only by a factor of 6, close to the difference predicted on the basis of size by the two-dimensional bilayer continuum model of Saffman and Delbrück [Saffman, P. G. l& Delbrück, M. (1975) Proc. Natl. Acad. Sci. USA 72, 3111-3113]. In contrast, the membranes of normal cells show a lipid diffusion coefficient (1.4 +/- 0.5 X 10(-8) cm2/s) that is some 300-fold greater than that of the membrane proteins (4.5 +/- 0.8 X 10(-11) cm2/s). Analysis of these results, based on the hypothesis that protein diffusion in normal membranes is sterically hindered by a labile matrix, yields an effective matrix surface viscosity consistent with the viscoelastic mechanical properties of the membranes. Thus, a relationship is established between the deformation characteristics of the membrane and the lateral mobility of proteins suspended in the membrane.
Crystalline arrays of membrane-bound acetylcholine receptor.
Authors: Kistler J , Stroud R M
Published: May 2006

Electron micrographs of tubular structures with a crystalline arrangement of membrane-bound acetylcholine receptor oligomers have been analyzed by digital image reconstruction. The receptor molecules are oriented synaptic side out, and in projection they appear to be asymmetric and have a defined orientation. All four subunits are contained in the oligomers as demonstrated by immunoelectron microscopy; these structures therefore appear to be suitable for subunit localization in the oligomer.
Alterations in chromatin structure during early sea urchin embryogenesis.
Authors: Savić A , Richman P , Williamson P , Poccia D
Published: May 2006

Sea urchin sperm before fertilization possess the longest nucleosome repeat length yet determined for any chromatin. By the time the fertilized egg gives rise to a blastula or gastrula embryo, the chromatin has a considerably shorter repeat length and, in addition, a sequence of different histone variants of H1, H2A, and H2B has appeared. We have investigated the relationship between these variations in histone composition and concomitant alterations in chromatin structure during the earliest stages of embryogenesis in two species of sea urchin. In contrast to the long repeat distance in sperm, chromatin loaded with cleavage stage histones has a much smaller repeat. Later stages containing predominantly alpha histones display an intermediate spacing. More detailed analysis of the events in the first cell cycle was carried out with polyspermically fertilized eggs. During the first 30 min after fertilization, in which sperm-specific H1 is completely replaced by cleavage-stage H1, the male pronuclear repeat remains unchanged. The decrease toward the repeat length of cleavage stages begins at about the time of DNA synthesis. Higher degrees of polyspermy extend the length of the cell cycle, including the duration of S phase and the length of time to reach the first chromosome condensation. At these higher degrees of polyspermy, the decrease in repeat length is also slowed. We conclude that the adjustment of the arrangement of nucleosomes in embryonic chromatin from that found in sperm can occur within the first cell cycle and that its timing is cell-cycle dependent. The adjustment is separable from a corresponding change in H1 composition.
Evidence for a lipoprotein carrier in human plasma catalyzing sterol efflux from cultured fibroblasts and its relationship to lecithin:cholesterol acyltransferase.
Authors: Fielding C J , Fielding P E
Published: May 2006

Immunoaffinity chromatography has been used to study the determinants of sterol efflux and net transport from cultured fibroblasts to human plasma medium. Sterol efflux was highly (approximately 80%) dependent upon a minor lipoprotein fraction containing apolipoprotein A-I unassociated with other apolipoproteins. The remaining activity was associated with the lipoprotein-free fraction of plasma and could be replaced by apoprotein-free albumin. Efflux was independent of lecithin:cholesterol acyltransferase (EC 2.3.1.43) activity. Net transport (i.e., the excess of efflux over influx) was completely inhibited by inhibition of lecithin:cholesterol acyltransferase or its removal by affinity chromatography on immobilized antibodies to apolipoprotein A-I or D (components of the transfer complex in human plasma). In uninhibited plasma, efflux and net transport rates had similar kinetics, suggesting that these were linked functions and that net transport was initiated by a carrier-dependent efflux step that, in the absence of lecithin:cholesterol acyltransferase activity, was associated with an equivalent influx of free sterol to the cells and that, in the presence of lecithin:cholesterol acyltransferase, was associated with esterification and transfer protein activity. The cholesterol carrier lipoprotein function (approximately 5% of plasma apolipoprotein A-I) appears to be the first step of lecithin:cholesterol acyltransferase-linked sterol transport from cells.
Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells.
Authors: Matthew E , Laskin J D , Zimmerman E A , Weinstein I B , Hsu K C , Engelhardt D L
Published: May 2006

We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed that there are differences between the binding characteristics in intact cells and in membrane fractions prepared from these cells. Scatchard analysis of the binding data from membrane fractions gave a linear plot (Kd = 9.1 X 10(-8) M). With intact cells, a curvilinear Scatchard plot was obtained. This was resolved into two components defining binding sites with affinity constants of 1.7 X 10(-9) M and 4.6 X 10(-7) M. Thus, it appears that [3H]diazepam binding in intact cells is more complex than in isolated membranes. Several related benzodiazepines, including flunitrazepam, Ro-5-4864, nitrazepam, oxazepam, lorazepam, Ro-5-3072, chlordiazepoxide, and clonazepam also induced melanogenesis. When these compounds were tested for their ability to inhibit [3H]diazepam binding, flunitrazepam, diazepam, and Ro-5-4864 were found to be the most effective inhibitors. These three compounds were also the most potent in inducing melanogenesis. Our results suggest that the benzodiazepines modulate cell differentiation. The presence of high-affinity binding sites in this homogeneous, easily grown cell line may provide a useful model for studies on the mechanism of action of these compounds.
Histidine modification with diethyl pyrocarbonate shows heterogeneity of benzodiazepine receptors.
Authors: Burch T P , Ticku M K
Published: May 2006

The effect of diethyl pyrocarbonate modification of histidine on the specific binding of [3H]diazepam and its enhancement with muscimol and (+/-)-pentobarbital was investigated. Diethyl pyrocarbonate treatment produced a dose-related inhibition of specific [3H]diazepam binding to rat brain membranes with a maximal inhibition of approximately 40% at 1 mM. Scatchard analysis of the binding data showed that diethyl pyrocarbonate, while having no effect on the affinity (Kd), decreased the binding capacity (Bmax) of diazepam from a control value of 1543 +/- 116 fmol/mg of protein to 789 +/- 79 fmol/mg of protein (mean +/- SD; P less than 0.005; n = 4). Under conditions in which approximately 40% of the diazepam binding sites were modified by diethyl pyrocarbonate treatment, the ability of muscimol and pentobarbital to enhance diazepam binding was not altered. These results suggest that a histidine residue is critical for a part (approximately 40%) of the benzodiazepine binding sites and that there may exist a heterogeneity of benzodiazepine binding sites. Furthermore, these results indicate that perhaps only a portion of the benzodiazepine binding sites are functionally coupled to the gamma-aminobutyric acid receptor-ionophore complex.
Changes in protein synthesis underlying functional plasticity in immature monkey visual system.
Authors: Kennedy C , Suda S , Smith C B , Miyaoka M , Ito M , Sokoloff L
Published: May 2006

Local rates of cerebral protein synthesis were determined in newborn rhesus monkeys subjected to either acute or chronic monocular visual deprivation. Chronic monocular deprivation resulted in decreased rates of protein synthesis in the laminae of the lateral geniculate nuclei innervated by the deprived eye whereas rates of protein synthesis were normal in geniculate laminae innervated by the functioning eye. Acute monocular deprivation produced no differential changes in rates of protein synthesis in any of the geniculate laminae.
Molecular parameters and olfaction in the oriental fruit fly
Dacus dorsalis
Authors: Metcalf Robert L. , Metcalf Esther R. , Mitchell W. C.
Published: May 2006

The methyl eugenol receptor of the male oriental fruit fly (
Dacus dorsalis
) has been further characterized by evaluating the role of the linear free energy parameters ∏ and σ in the depolarization of the receptor by 37 substituted 3,4-dimethoxybenzenes. There was a strong positive correlation between the hydrophobic character of the primary substituent and intense odor and a positive correlation between the electron donating property of the primary substituent and intense odor. Maximum odor intensity was also associated with substituents of 3 atomic diameters and was improved by a center of unsaturation. Preference tests suggest that this simple and versatile odor receptor can serve as a model for investigation of molecular interactions between receptors and odorant molecules.
Administration of 3-methylcholanthrene to rats increases the specific hybridizable mRNA coding for cytochrome P-450c.
Authors: Bresnick E , Brosseau M , Levin W , Reik L , Ryan D E , Thomas P E
Published: May 2006

Poly(A)+-RNA obtained from the livers of 3-methylcholanthrene (3MC)-treated rats was translated into cytochrome P-450c in a cell-free reticulocyte system. In this translational system, no precursor cytochrome P-450c was observed. The mRNA responsible for the synthesis of this cytochrome was isolated by immunoprecipitation of liver polyribosomes obtained at 15 hr after 3MC treatment, and a cDNA was constructed by the reverse transcriptase reaction. The cDNA was further purified by hybridizing at a high R0t (product of RNA concentration and incubation time) to poly(A)+-RNA isolated from control rat liver, and the nonhybridized, single-stranded cDNA was isolated by hydroxylapatite chromatography. This cDNAp-450c was employed in hybridization reactions with poly(A)+-RNA isolated from the livers of rats treated with 3MC for various times. These studies indicated a maximal induction of mRNAp-450c at about 15 hr after 3MC injection, although levels of this mRNA were significantly increased by 7 hr. The mRNAp-450c concentration had diminished by 24 hr but remained higher than control levels for at least 48 hr. These studies establish an effect of 3MC upon the accumulation of mRNAp-450c in rat liver.
X-ray analysis (1. 4-Å resolution) of avian pancreatic polypeptide: Small globular protein hormone
Authors: Blundell T. L. , Pitts J. E. , Tickle I. J. , Wood S. P. , Wu C.-W.
Published: May 2006

The crystal structure of avian pancreatic polypeptide (aPP), a 36-residue polypeptide with some hormonal properties, has been determined by using single isomorphous replacement and anomalous scattering to 2.1-Å resolution. The phases were extended to 1.4-Å resolution by using a modified tangent formula. The molecule contains two regions of secondary structure—an extended polyproline-like helix (residues 1-8) and an α-helix (residues 14-31)—that run roughly antiparallel. The packing together of nonpolar groups from these regions gives the molecule a hydrophobic core in spite of its small size. The aPP molecules form a symmetrical dimer in the crystal stabilized principally by interlocking of nonpolar groups from the α-helices. The aPP dimers are crosslinked by coordination of Zn
2+
; three aPP molecules contribute ligands to each zinc. The coordination geometry is a distorted trigonal bipyramid. The properties of the aPP molecule in solution are consistent with expectations based on the crystal structure. The aPP molecule has several general features in common with the pancreatic hormones insulin and glucagon. All three hormones have complex mechanisms for self-association. Like insulin, aPP seems to have a stable monomeric structure but its biological activity seems to depend on the more flexible COOH-terminal region analogous to the flexible NH
2
-terminal region of glucagon.
Somatomedin receptor of human placenta: solubilization, photolabeling, partial purification, and comparison with insulin receptor.
Authors: Bhaumick B , Bala R M , Hollenberg M D
Published: May 2006

Using a recently isolated human basic somatomedin (basic SM) similar to insulin-like growth factor I (IGF-I), we studied both the photoaffinity-labeled and unlabeled basic-SM receptor solubilized from human placental cell membranes. Unlike the result with the insulin receptor, high yields of soluble basic-SM-binding activity are obtained with Triton X-100. The soluble basic-SM receptor retains high-affinity (Kd approximately 0.3 nM) peptide-specific binding of basic SM, similar to the binding present in particulate placenta membranes; the receptor exhibits a comparatively low affinity for insulin (Kd approximately 3 microM). On Sepharose 6B, like the crude soluble insulin receptor, the basic-SM receptor migrates as a species with an apparent Stokes radius of 7.2 nm; unlike the insulin receptor, the basic-SM receptor does not, under similar conditions, yield a smaller binding species (apparent Stokes radius 3.8 nm). Upon photoaffinity labeling with 125I-labeled basic SM, one principal specifically labeled constituent is detected. Upon gel electrophoresis in the presence of 2-mercaptoethanol, the photolabeled constituent, like the insulin receptor, migrates as a species with an apparent molecular weight of about 140,000; in the absence of reducing agent, a molecular weight greater than 240,000 is observed. Lectin-agarose affinity chromatography yields a 30-fold purification both of the basic-SM-binding activity and the photolabeled constituent. Anti-insulin receptor antibody does not appear to precipitate the basic-SM receptor. We conclude that the basic-SM receptor of human placenta is a glycoprotein, remarkably similar to (an isoreceptor) but distinct from the insulin receptor previously characterized in this tissue.
Distinct high-affinity binding sites for benzomorphan drugs and enkephalin in a neuroblastoma--brain hybrid cell line.
Authors: McLawhon R W , West R E , Miller R J , Dawson G
Published: May 2006

The high-affinity binding of benzomorphan drugs (ethylketocyclazocine and N-allylnorcyclazocine) and [DAla2,DLeu5] enkephalin was examined in a mouse neuroblastoma--Chinese hamster brain clonal hybrid cell line (NCB-20). Scatchard analysis of saturation binding isotherms indicated the presence of a single binding site for 3H-labeled [DAla2,DLeu5]enkephalin (Kd = 3 nM) and multiple binding sites for [3H]ethylketocyclazocine (Kd = 4 and 20 nM) and N-[3H]allylnorcyclazocine (Kd = 0.5 and 15 nM). Both ethylketocyclazocine and N-allylnorcyclazocine competed (Ki = 10 and 30 nM, respectively) with [3H][DAla2,DLeu5]enkephalin binding in NCB-20 cells but neither [DAla2,DLeu5]enkephalin nor morphine could completely inhibit the specific binding of [3H]ethylketocyclazocine (7 nM) or N-[3H]allylnorcyclazocine (3 nM). Furthermore, not all benzomorphan drugs (e.g., ethylketocyclazocine) were totally efficacious in displacing 3 nM N-[3H]allylnorcyclazocine binding in the presence or absence of high concentrations of [DAla2,DLeu5]enkephalin. The data presented suggest that benzomorphan drugs interact with three distinct high-affinity binding sites: (i) a site that binds enkephalin and morphine in addition to ethylketocyclazocine and N-allylnorcyclazocine; (ii) a site that binds both ethylketocyclazocine and N-allylnorcyclazocine but not enkephalin and morphine; and (iii) a site that binds N-allylnorcyclazocine but not enkephalin, morphine, or ethylketocyclazocine. The first of these sites was comparable to the delta opiate receptor expressed in NG108-15 and N4TG1 cell lines based on the potency series obtained for various opiates and benzomorphan drugs in competition studies with [3H][DAla2,DLeu5]-enkephalin. However, the specific high-affinity benzomorphan binding sites thus far are unique and may represent biochemical correlates of kappa and sigma opiate receptors which have been proposed to exist on the basis of physiological studies.
Cytoplasmic transfer of the mitogenic response to platelet-derived growth factor.
Authors: Smith J C , Stiles C D
Published: May 2006

BALB/c 3T3 mouse cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA and divide. When cells are treated with PDGF and then fused to untreated cells, the resulting heterokaryons become competent to replicate their DNA. Cytoplasts derived from PDGF-treated cells are also able to transfer the growth response to untreated cells. After cytoplasmic transfer to another cell, the strength of the PDGF-induced mitogenic signal is attenuated by a factor roughly proportional to the increase in total cytoplasmic volume. When RNA synthesis is blocked during PDGF treatment, cells do not acquire the capacity to transfer the PDGF growth signal to untreated cells. By contrast, exposure to cycloheximide during PDGF treatment has no effect. These observations suggest that cytoplasmic transfer of the growth response to PDGF (competence) is mediated by a PDGF-induced stable RNA rather than by PDGF itself or a PDGF--receptor complex. The onset of DNA synthesis in PDGF--control heterokaryons occurs a minimum of 11 hr after cell fusion. Thus the substance that is transferred in these cell fusions is not directly involved in DNA synthesis; rather, it seems to trigger a sequence of events culminating in DNA synthesis.
Change in state of phosphorylation of acetylcholine receptor during maturation of the electromotor synapse in Torpedo marmorata electric organ.
Authors: Saitoh T , Changeux J P
Published: May 2006

Two populations of membrane fragments, both rich in acetylcholine receptor (AcChoR), appeared during subcellular fractionation by ultracentrifugation of neonatal Torpedo marmorata electric organs. One of these equilibrated at 38.5% (wt/wt) sucrose, as did AcChoR-rich membranes from adult fish; the other equilibrated at 36.8% sucrose. AcChoR purified from these light membrane fractions gave the same subunit profile as adult AcChoR (after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) but was more susceptible to heat inactivation and focused at an isoelectric point more alkaline by 0.1 pH unit. Treatment of adult AcChoR with Escherichia coli alkaline phosphatase decreased its thermal stability and shifted its isoelectric point towards alkaline pH. However, identical treatment did not affect AcChoR purified from neonatal light membrane fractions. The gamma and delta chains of AcChoR can be phosphorylated in vitro by endogenous protein kinases, which copurify with AcChoR-rich membranes. Treatment of AcChoR from neonatal light membranes by E. coli alkaline phosphatase enhanced the phosphorylation of the gamma and delta chains but did so to a smaller extent than in the case of adult AcChoR. In conclusion, adult AcChoR appears to be more phosphorylated than AcChoR from neonatal light membranes, indicating that its state of phosphorylation changes during development.
Immune response potential to poly(Tyr,Glu)-poly(DLAla)--poly(Lys) of human T cells of different donors.
Authors: Katz D , Bentwich Z , Eshhar N , Löwy I , Mozes E
Published: May 2006

Human peripheral blood T cells of normal donors were activated in vitro with autologous adherent cells pulsed with poly(LTyr,LGlu)-poly(DLAla)--poly(LLys) [abbreviated (T,G)-A--L]. The "educated" T cells were tested: (i) for their ability to produce a (T,G)-A--L-specific T cell-replacing factor in the cooperation with B cells for antibody responses in vivo or in vitro and (ii) for their ability to proliferate in the presence of a second stimulus of (T,G)-A--L. Results of screening of 66 donors demonstrated that educated T cells of about 50% of the donors produced an active (T,G)-A--L-specific factor, whereas activated cells of only half of the factor producers were capable of proliferating in the presence of the antigen. Thus, as reported for all other species studied, human individuals differ in their response potential to (T,G)-A--L.
Cryoprecipitogogue from normal serum: mechanism for cryoprecipitation of immune complexes.
Authors: Hardin J A
Published: May 2006

We designed this study to examine the relationship of cryoglobulins to immune complexes in sera of patients with rheumatic or infectious diseases. Polyethylene glycol was used to precipitate large proteins from normal serum and then was dialyzed away. The precipitated proteins were soluble in warm phosphate-buffered saline but at 4 degrees C they reversibly reprecipitated. As they reprecipitated, they selectively coprecipitated cold-soluble immune complexes. By NaDodSO4/polyacrylamide gel electrophoresis, these normal, nonimmunoglobulin cryoproteins were similar to the nonimmunoglobulin constituents of washed cryoimmunoglobulins from patients with rheumatic or infectious diseases. These findings suggest that cryoprecipitability is a property not of immune complexes themselves but of a group of large normal serum proteins. In inflammatory diseases, the concentrations of some of these proteins, responding as acute-phase reactants, may increase to the point where intermolecular attractive forces become prominent, particularly in the cold. Cold-augmented molecular aggregation between these nonimmunoglobulin proteins and immune complexes could then act to decrease their collective solubility and result in the cryoprecipitation of otherwise cold-soluble immune complexes.
Monoclonal antibodies against beta nerve growth factor and their effects on receptor binding and biological activity.
Authors: Zimmermann A , Sutter A , Shooter E M
Published: May 2006

Two hybrid cell lines, MC beta-1 and MC beta-2, secreting monoclonal antibodies against mouse submaxillary gland beta nerve growth factor (beta NGF), were produced by interspecies hybridization of spleen cells from rats immunized with beta NGF and mouse myeloma cells. The antibodies secreted by the two hybridomas are of the IgG1 subclass and bind staphylococcal protein A. The equilibrium dissociation constant of the beta NGF--antibody complex was determined for the MC beta-1 antibodies in solid phase and in solution. On protein A-coated surfaces the Kd is 3 X 10(-10) M, 2 orders of magnitude lower than the Kd 2 X 10(-8) M obtained in solution. The antigenic site recognized by MC beta-1 antibodies is present on each protomer of the beta NGF dimer, and the binding affinity of the second antibody molecule is similar to that of the first. The MC beta-1 antibodies inhibit neurite outgrowth from sensory neurons. Because this inhibition directly correlates with the inhibition of binding to the higher affinity beta NGF receptors, it suggests that beta NGF complexed with two antibody molecules does not bind to the receptor and is biologically inactive.
Phanerozoic cycles of sedimentary carbon and sulfur
Authors: Garrels Robert M. , Lerman Abraham
Published: May 2006

A reservoir model of a Recent steady-state sedimentary system in which the reduced sulfur and oxidized sulfur reservoirs were coupled with the oxidized carbon and reduced carbon reservoirs was constructed. The time curve of the sulfur isotope ratios of the sedimentary sulfate reservoir was used to drive the model back to the beginning of Cambrian time (600 million years ago), producing the reservoir sizes and isotope values and material fluxes of the carbon-sulfur system. The predicted values of carbon isotope ratios of the carbonate reservoir agree well with observed values, showing that the model is basically sound. Some general conclusions from this success are (
i
) material flux rates in the carbon-oxygen-sulfur system of the geologic past (averaged over tens of millions of years) lie within about a factor of 2 of Recent rates. (
ii
) The oxidation-reduction balances of Phanerozoic time were dominated by reciprocal relationships between carbon and sulfur compounds. (
iii
) The rate of production of atmospheric oxygen by storage in sediments of organic carbon of photosynthetic origin increased from the Cambrian Period to the Permian Period and declined somewhat from the Permian Period to the Present. (
iv
) The storage of oxygen in oxidized sulfur compounds kept pace (within the limits of the data) with oxygen production. (
v
) Transfer of oxygen from CO
2
to SO
4
from the Cambrian to the Permian Period was several times the Recent free oxygen content of the atmosphere.
cDNA sequence of a new chicken embryonic rho-globin.
Authors: Roninson I B , Ingram V M
Published: May 2006

In order to use specific DNA probes for the study of developmentally regulated gene expression, we have prepared cDNA clones corresponding to chicken embryonic globins by inserting cDNA.mRNA hybrids into the Pst I site of the plasmid pBR322 by using poly(dG) and poly(dC) linkers. The nucleotide sequence of the insert of one clone, representing a nearly full-length copy of an embryonic beta-like globin cDNA, has been determined. The amino acid sequence of the globin encoded by this insert is identical to the sequence of embryonic rho-globin, except for four amino acid residues near the carboxy terminus. Comparison of mRNA sequences of the embryonic and adult chicken beta-globins indicates the presence of extensive deletions in the 3' untranslated region of the embryonic gene.
Primary structure and evolution of rat growth hormone gene.
Authors: Barta A , Richards R I , Baxter J D , Shine J
Published: May 2006

The rat growth hormone gene was isolated on a cloned 11.4-kilobase EcoRI-generated DNA fragment from a bacteriophage "library" of chromosomal DNA. The structural gene sequence, approximately 2.1 kilobases long, was identified by hybridization to the corresponding cloned rat growth hormone cDNA and shown to contain four intervening sequences. The complete primary structure of the gene and the 5' and 3'-flanking regions was determined. The mosaic structure of exons and introns can be related to the different biological activities of growth hormone and to the evolution from ancestral sequences of a gene that was the precursor to the growth hormone and the related prolactin and placental lactogen (chorionic somatomammotropin) genes. The largest intron was found to contain a dispersed repetitive DNA sequence flanked by perfect 18-base pair direct repeats. The mobility of sequences of this kind could play a role in the observed variation of intron sizes and in rearrangements of mammalian genes.
Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome.
Authors: Racaniello V R , Baltimore D
Published: May 2006

The complete 7410 nucleotide sequence of poliovirus type I genome was obtained from cloned cDNA. Double-stranded poliovirus cDNA was synthesized and inserted into the Pst I site of plasmid pBR322, and three clones were derived that together provided DNA copies of the entire poliovirus genome. Two of the clones contained inserts of 2.5 and 6.5 kilobases and represented all but the 5' 115 bases of poliovirus RNA. A third clone was generated from primer-extended DNA and contained sequences from the 5' end of the viral RNA. An open reading frame that was identified in the nucleotide sequence starting 743 bases from the 5' end of the RNA and extending to a termination codon 71 bases from the 3' end contained known poliovirus polypeptide sequence.
Replication of plasmid pT181 DNA in vitro: requirement for a plasmid-encoded product.
Authors: Khan S A , Carleton S M , Novick R P
Published: May 2006

PT181 is a naturally occurring 4.5-kilobase Staphylococcus aureus plasmid encoding resistance to tetracycline. The plasmid has a copy number of about 20 per cell; a mutant, cop-608, that has a copy number of 800-1000 has been isolated. A cell-free extract has been developed that carries out complete replication of this plasmid. Extracts made from a strain containing the mutant have much greater replication activity than do extracts of strains containing pT181. In an extract from which endogenous DNA has been removed, DNA synthesis is dependent upon the addition of exogenous plasmid DNA. The replication system is specific for pT181 and related plasmids but it is inactive with other S. aureus plasmids. Furthermore, pT181 DNA does not replicate in extracts made from plasmid-negative strains or strains containing other plasmids. The results suggest that a specific plasmid-encoded substance is required for the replication of pT181 DNA.
In vitro reconstruction of the mitochondrial translation system of yeast.
Authors: Pfisterer J , Buetow D E
Published: May 2006

We have isolated the translation system from yeast mitochondria and have reconstructed it in vitro. This submitochondrial system, composed of mitochondrial ribosomes, tRNA, pH 5 fraction and mRNA, is maximally active at 10 mM Mg2+ and 100 mM KCl or NH4Cl. NH4+ is more stimulatory than K+. Added Escherichia coli tRNA gives less than half the activity obtained with added mitochondrial tRNA. Activity is enhanced with protease inhibitors but not with Ca2+, spermine, or spermidine. In contrast to heterologous translation systems, the present system produces products with molecular weights similar to those of products synthesized by yeast mitochondria in vivo and by intact yeast mitochondria in vitro. The results support the idea that the unique coding features of the mitochondrial genome require a unique translation system for accurate translation of mitochondrial mRNAs.
The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La.
Authors: Chung C H , Goldberg A L
Published: May 2006

In Escherichia coli, degradation of abnormal proteins is an energy-requiring process; it is decreased in mutants in the lon (capR or deg) gene. We find that the protein encoded by the lon gene is an ATP-dependent protease and is identical to protease La, recently described in E. coli. Both proteins are serine proteases that hydrolyze casein and globin, but not insulin, in the presence of ATP and Mg2+. Both respond to ATP, less well to other nucleoside triphosphates, and not to nonhydrolyzable ATP analogs. The purified lon protein has an apparent Mr of 450,000 and appears to be composed of four identical subunits. Its size, chromatographic behavior, and sensitivity to various inhibitors and heat are indistinguishable from those of protease La. Moreover, in a strain that carries additional copies of the lon+ allele on a plasmid, the content of protease La, but not of other proteases, is 2- to 10-fold greater than in the lon+ parent strain. Strains carrying the nonsense mutations capR9 and capR- also contain this ATP-dependent proteolytic activity, but it is present in substantially lower amounts and is inactivated by phosphocellulose chromatography, unlike the wild-type enzyme. Degradation of abnormal proteins in these lon- strains, which is slower than in the wild type, still requires ATP. Alterations in the ATP-dependent protease in the lon- mutants can account for the defect in intracellular proteolysis and perhaps also for the other phenotypic effects of this pleiotropic gene.
Cellular differentiation and neoplasia: characterization of subpopulations of cells that have neoplasia-related growth properties in Syrian hamster embryo cell cultures.
Authors: Nakano S , Ts'o P O
Published: May 2006

Cellular subpopulations having two of the growth properties of neoplastically transformed cells--lack of postconfluence inhibition of cell division (CI-) and anchorage independence of growth (AD-)--were found in cell cultures established from 10- to 13-day-old Syrian hamster embryos. The subpopulations having these properties decrease with increasing gestation period of the embryo as well as with continuing passage in vitro. The decrease in these subpopulations was also observed when they were cultured on a lethally irradiated confluent monolayer of contact-inhibited cells (cell mat), a selection condition for CI- cells. Therefore, negative selection cannot be the explanation for the loss of CI- cells in the population, leaving two other possibilities: either the loss of proliferative capacity of the CI- cells or the acquisition of sensitivity to postconfluence inhibition of cell division (CI+) of this subpopulation on in vitro culture or in vivo growth. The CI- subpopulations were isolated clonally from cell mats and were cultured continuously on both cell mats or plastic dishes. The results indicate that these cells did not lose proliferative capacity but acquired the contact-inhibited phenotype. This result, together with the fact that embryonic development in vivo also decreases CI/AD-subpopulations, suggests that the disappearance of these subpopulations is due to cellular differentiation of the CI-/AD- cells to become CI+/AD+ cells.
Characterization of immunologically significant unique B16 melanoma proteins produced
in vivo
and
in vitro
Authors: Gersten Douglas M. , Hearing Vincent J. , Marchalonis John J.
Published: May 2006

Proteins unique to B16 melanoma cells grown in culture and as solid tumors have been independently described recently. As both proteins have apparent
M
r
of 65,000-70,000, we have more fully compared their biochemical characteristics. The
M
r
65,000 protein, which is isolated from B16 cells in culture, has an aggregate molecular weight of >200,000 under nonreducing conditions. This
M
r
65,000 form is shed into the culture medium exclusively and is the predominant molecular species of serum-free culture supernatants. It was found that B700, a major membrane protein of B16 cells in solid tumors, and the
M
r
65,000 preparation from cultured cells were electrophoretically identical when compared under four separate sets of conditions. These two proteins also share immunologic determinants. The results suggest the identity of these two proteins isolated by completely different approaches.
Identification, characterization, and distribution of secretin immunoreactivity in rat and pig brain.
Authors: O'Donohue T L , Charlton C G , Miller R L , Boden G , Jacobowitz D M
Published: May 2006

Secretin immunoreactivity was detected in the central nervous system of the rat and pig with a highly specific radioimmunoassay. The secretin immunoreactivity in the rat and pig brain and duodenum extracts was fractionated by using a reverse-phase high-pressure liquid chromatographic system. The immunoreactive secretin from pig brain and duodenum coeluted precisely with synthetic porcine secretin. However, immunoreactive secretin extracted from rat brain and duodenum eluted slightly before porcine secretin. These data suggest a slight difference in the structure of rat and pig secretin. The detection of secretin in the brain lays the groundwork for studies to determine the role of the peptide in central nervous system function.
Transformation of catecholaminergic precursors into glucagon (A) cells in mouse embryonic pancreas.
Authors: Teitelman G , Joh T H , Reis D J
Published: May 2006

In embryonic mice, the catecholamine biosynthetic enzyme tyrosine hydroxylase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] can be visualized immunocytochemically in a population of cells in epithelial cords of the developing pancreas. These embryonic catecholamine cells, first seen by day 11, are large and vacuolated and have a folded nuclear membrane. One day later, at day 12, glucagon is first detected immunocytochemically in pancreatic cells similar in location and morphology to the embryonic catecholamine cells. By use of a method for detecting both antigens in the same cell, both the hydroxylase and glucagon can be visualized between day 12 and day 14 in 10-40% of stained cells. From day 14, the number of cells stained for hydroxylase decreases; they cannot be detected after day 18. In contrast, the cells containing glucagon increase during development and persist throughout life. Endocrine cells of the embryonic pancreas also contain dopa decarboxylase but not dopamine-beta-hydroxylase or phenylethanolamine-N-methyl transferase. In adult mice, small cells containing tyrosine hydroxylase but differing in location and morphology from the embryonic catecholaminergic cells are seen in pancreatic islets. The adult catecholaminergic cells never store glucagon. We suggest that adult glucagon (A)-containing cells arise from transformation in situ of cells that transiently express a catecholaminergic (probably dopaminergic) phenotype. These results suggest that one class of peptidergic cells may arise from transformation of an aminergic precursor.
Differentiation of the fast Na+ channel in embryonic heart cells: interaction of the channel with neurotoxins.
Authors: Renaud J F , Romey G , Lombet A , Lazdunski M
Published: May 2006

The sensitivity of embryonic cardiac cells to tetrodotoxin (TTX) increases with age. At the early embryonic stage, the maximum upstroke velocity is not affected by the presence of TTX. In the course of both in ovo and in vitro development, this velocity reaches an adult-like value of 90-120 V/sec, which is decreased in the presence of TTX to 5-10 V/sec. The differentiation of the Na+ channel has been followed by using three types of specific toxins: (i) TTX or a tritiated derivative of it, (ii) a polypeptide toxin extracted from sea anemone, and (iii) the alkaloidic toxins veratridine and batrachotoxin. Electrophysiological, including voltage-clamp experiments, and biochemical studies have shown (i) that the TTX receptor and the fast Na+ channel machinery exist even when action potentials are insensitive to TTX--the channel is then in a nonfunctional or silent form that is revealed (or chemically activated) by both the alkaloids and the polypeptide toxin--and (ii) that the total number of Na+ channels increases during development by a factor of 4 or 5. In monolayers of cardiac cells insensitive to TTX in which all Na+ channels are in a nonfunctional form, the rate of degradation of the TTX receptor follows first-order kinetics with a half-time of 9 hr. In aggregates fully sensitive to TTX, the number of TTX receptors remains perfectly stable 24 hr after blockade of protein synthesis.
Liver microsomal cytochrome P-450 and the oxidative metabolism of arachidonic acid.
Authors: Capdevila J , Chacos N , Werringloer J , Prough R A , Estabrook R W
Published: May 2006

Arachidonic acid is oxidatively metabolized by rat liver microsomes at a rate of approximately 5 nmol per min per mg of protein at 25 degrees C. This reaction is dependent on the presence of NADPH and oxygen. Studies with various inhibitors indicate a role for membrane-bound cytochrome P-450 in the transformation of arachidonic acid to a mixture of hydroxy acid derivatives. The stoichiometry of the reaction conforms to that of a monooxygenase reaction--i.e., one mole of NADPH is oxidized per mole of oxygen utilized--suggesting a reaction mechanism different from that proposed for lipid peroxidation reactions. No evidence for the formation of prostaglandin-like metabolites was obtained. The diene character of some of the metabolites formed suggests another role for cytochrome P-450--i.e., participation in hydrogen abstraction reactions for the activation of various substrates.
Identification of a RNA polymerase II initiation site in the long terminal repeat of Moloney murine leukemia viral DNA.
Authors: Fuhrman S A , Van Beveren C , Verma I M
Published: May 2006

We have used a soluble in vitro RNA polymerase II transcription system to define the site of initiation of Moloney murine leukemia viral RNA synthesis. Molecularly cloned integrated and unintegrated Moloney murine leukemia virus DNAs were used as templates. The 5' ends of in vitro transcripts and virion RNA of Moloney murine leukemia virus were compared by nuclease S1 protection experiments. Our results indicate that viral sequences upstream of the in vivo cap site are implicated in the transcription of viral RNA and that the 5' end of an in vitro transcript derived from an integrated Moloney murine leukemia virus clone corresponds to the 5' end of viral genomic RNA.
Molecular regulation of receptors: interaction of beta-estradiol and progesterone with the muscarinic system.
Authors: Sokolovsky M , Egozi Y , Avissar S
Published: May 2006

The effects of various substrates on the binding of agonists to muscarinic receptors were studied in the rat hypothalamus and adenohypophysis by competition experiments using the highly specific tritiated muscarinic antagonist N-methyl-4-piperidyl benzilate. It was found that agonist binding properties were affected only by the steroid sex hormones (beta-estradiol and progesterone), both of which resulted in a decrease in the proportion of high-affinity binding sites and a decrease in the dissociation constant. This suggests a link between the muscarinic system and the mechanism by which these steroids exert their gonadotropin-releasing effect on the adenohypophysis. We propose a model to depict the putative relationship between the muscarinic system and other receptor systems, including that which controls the steroid sex hormones.
Growth control of prostatic carcinoma cells in serum-free media: interrelationship of hormone response, cell density, and nutrient media.
Authors: Kaighn M E , Kirk D , Szalay M , Lechner J F
Published: May 2006

Two established prostatic carcinoma cell lines have been grown in long-term culture in a defined medium (PFMR-4) free of serum, hormones, or growth factors. Growth of both lines in serum-free medium was population dependent. This cell-density requirement could be replaced by mitomycin C-inactivated feeder cells, homologous conditioned medium, or fetal bovine serum, but not by hormones or growth factors. The cells responded to these factors only at high density. The nature of this hormonal response was dependent on the kind of basal nutrient medium used. Growth in PFMR-4 with added insulin was more rapid than that in DME/F12 medium with any combination of hormones or growth factors and was substantially greater than growth in DME/F12 medium with insulin alone. The results demonstrate that whereas these two prostatic carcinoma lines (PC-3 and DU 145) do not require hormones for survival or growth, they do respond to certain hormones under appropriate conditions. These conditions include both the type of basal nutrient medium used and the population density.
Extraordinarily high evolutionary rate of pseudogenes: evidence for the presence of selective pressure against changes between synonymous codons.
Authors: Miyata T , Hayashida H
Published: May 2006

Comparisons of nucleotide sequences of several pseudogenes described to date, including alpha- and beta-globin and immunoglobulin kappa-type variable domain pseudogenes, with those of functional counterparts revealed that pseudogenes accumulate mutations at an extremely high rate uniformly over their entirety. It is remarkable that the evolutionary rate exceeds the rate of changes between synonymous codons, the highest known rate, in functional genes. Because no pseudogenes appear to function, this result strongly supports the neutral theory. In addition this result apparently indicates the presence of selective pressure against changes between synonymous codons in functional genes. Close examinations of codon utilization patterns in pseudogenes and functional genes revealed a significant correlation between the rate of changes at synonymous codon sites and the strength of bias in code word usage. This implies that even synonymous codon changes are not completely free from selective pressure but are constrained in part, although presumably weakly, depending on the degree of bias in code word usage. We also reexamined alignment between mouse beta h3 (pseudogene) and beta maj sequences and found a unique structure of the beta h3 that is homologous in sequence to the beta maj gene overall but contains a long deletion (about 150 base pairs) in the middle of the gene.
Genetic organization and transcriptional regulation in the qa gene cluster of Neurospora crassa.
Authors: Patel V B , Schweizer M , Dykstra C C , Kushner S R , Giles N H
Published: May 2006

A transcription map of the qa gene cluster of Neurospora crassa has been constructed by using cloned DNA fragments as hybridization probes. The mRNAs encoded in the previously identified qa-2 (3-dehydroquinate hydro-lyase, EC 4.2.1.10), qa-4 (dehydroshikimate dehydratase), qa-3 (quinate:NAD+ 3-oxidoreductase, EC 1.1.1.24), and qa-1 (regulatory protein) genes have been characterized. In addition, mRNAs encoded in two new genes in this cluster (qa-x, qa-y) have been identified. Regulation of this system occurs at the level of transcription and is under the combined control of quinic acid and the qa-1 protein. The qa cluster represents a group of adjacent coding sequences which occupy approximately 18 kilobases in linkage group VII. The expression of the qa-1 gene appears to be constitutive but also autoregulated. mRNAs encoded in genes flanking the qa gene cluster have also been identified.
Isolation and partial characterization of an antigen-specific T-cell factor associated with the suppression of delayed type hypersensitivity.
Authors: Rosenstein R W , Murray J H , Cone R E , Ptak W , Iverson G M , Gershon R K
Published: May 2006

Antigen-specific factors associated with immunosuppressive activity, released by cultured T cells from mice tolerant to the haptens trinitrophenyl, dinitrophenyl and oxazolone, were purified by hapten affinity chromatography. Their binding specificity for antigens paralleled their immunoregulatory activity. Like some immunoglobulin molecules, these factors had blocked NH2 termini and could be bound to Fc-like receptors on macrophages. However, neither immunoglobulin constant region determinants (isotypes) nor antigens encoded by the major histocompatibility complex were detected on the suppressive factors. The purified factors occurred as 68,000-dalton proteins and non-covalently linked dimers. No associated immunoglobulin light chain molecules were detected. The factors showed a marked propensity toward degradation with major breakdown products of 45,000-50,000 and 25,000-30,000 daltons. These results suggest that these molecules are the T-cell products analogous to B-cell immunoglobulin (equivalent to heavy chains) and that they may be the antigen-specific components which act in conjunction with major histocompatibility-controlled gene products to perform antigen-specific suppression.