bluuebunny/crossref_metadata_2025 huggingface dataset:
Absolute rates of protein synthesis during meiotic maturation of mammalian oocytes in vitro.
Authors: Schultz R M , LaMarca M J , Wassarman P M
Published: May 2006

Measurements of the rates of incorporation of [35S]methionine into protein and the specific activities of endogenous free methionine pools have been used to calculate the absolute rates of protein synthesis in mouse oocytes during spontaneous meiotic maturation in vitro. Fluorodinitro[3H]benzene was used to determine the specific activity of the oocyte's free methionine pool. It was found that the absolute rate of protein synthesis decreased from 43 to 31 pg/hr per oocyte during meiotic progression from dictyate to metaphase II (meiotic maturation), while the size of the intracellular free methionine pool decreased from 61 to 35 fmol per oocyte during the same period. Comparable measurements made on ovulated mouse oocytes that had undergone meiotic maturation in vivo strongly suggest that the decrease in the absolute rate of protein synthesis observed during meiotic maturation in vitro is physiologically significant. An alternative method that depends upon differential expansion of the oocyte's endogenous methionine pool was also used to determine absolute rates of protein synthesis. The results of these experiments are in excellent agreement with those obtained by using fluorodinitro[3H]benzene, indicating that the oocyte's free methionine pool is not compartmentalized.
Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation.
Authors: Szoka F , Papahadjopoulos D
Published: May 2006

Large unilamellar and oligolamellar vesicles are formed when an aqueous buffer is introduced into a mixture of phospholipid and organic solvent and the organic solvent is subsequently removed by evaporation under reduced pressure. These vesicles can be made from various lipids or mixtures of lipids and have aqueous volume to lipid ratios that are 30 times higher than sonicated preparations and 4 times higher than multilamellar vesicles. Most importantly, a substantial fraction of the aqueous phase (up to 62% at low salt concentrations) is entrapped within the vesicles, encapsulating even large macromolecular assemblies with high efficiency. Thus, this relatively simple technique has unique advantages for encapsulating valuable water-soluble materials such as drugs, proteins, nucleic acids, and other biochemical reagents. The preparation and properties of the vesicles are described in detail.
Polycationic macromolecules inhibit cilia-mediated ovum transport in the rabbit oviduct.
Authors: Norwood J T , Hein C E , Halbert S A , Anderson R G
Published: May 2006

In both in vitro and in vivo experiments, polycationic macromolecules,such as poly(L-lysine), inhibited the transport of either surrogate or freshly ovulated cumulus masses across the oviduct epithelial surface without affecting the ciliary beat. Whereas transport across the fimbria in vivo was completely inhibited, transport down the ampulla was 3 to 7 times slower than normal. The effects of these polycations suggest that cilia-mediated ovum transport may involve the formation of transient adhesive bonds between the tip of the cilium and elements of the cumulus mass during each beat cycle of the cilium.
Energetics of sodium transport in toad urinary bladder.
Authors: Canessa M , Labarca P , DiBona D R , Leaf A
Published: May 2006

The ratio of the rate of transepithelial sodium transport, JNa, across the isolated toad urinary bladder to the simultaneously measured rate of transport-dependent metabolism, JsbCO2, has been measured as a function of the transepithelial electrical voltage, deltapsi. The ratio remains constant with a mean value of 18 to 20 over the range of imposed voltages of 0 to +70 mV. With increasing hyperpolarization of the bladder, JNa decreases and the calculated electromotive force or apparent "ENa" of the sodium pump increases. From thermodynamic and kinetic arguments it is shown that the apparent "ENa" approaches the maximal electrochemical potential gradient, ENa, against which sodium can be transported by this tissue only when JNa approximately 0. At this unique condition F ENa (in which F is the Faraday constant) is the maximal free energy of the chemical reaction driving sodium transport and thus equal to the maximal extramitochondrial phosphorylation potential and the maximal free energy of the mitochondrial respiratory chain within the transporting cells.
Ethylene biosynthesis: Identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene
Authors: Adams D. O. , Yang S. F.
Published: May 2006

L-[
U
-
14
C]Methionine fed to apple tissue was efficiently converted to ethylene when the tissue was incubated in air. In nitrogen, however, it was not metabolized to ethylene but was instead converted to 1-aminocyclopropane-1-carboxylic acid (ACC). When apple tissues were fed with L-[
methyl
-
14
C]methionine or L-[
35
S]methionine and incubated in nitrogen, radioactivity was found subsequently in methylthioribose. This suggests that methionine is first converted to
S
-adenosylmethionine which is in turn fragmented to ACC and methylthioadenosine. Methylthioadenosine is then hydrolyzed to methylthioribose. The conclusion that ACC is an intermediate in the conversion of methionine to ethylene is based on the following observations: Labeled ACC was efficiently converted to ethylene by apple tissue incubated in air; the conversion of labeled methionine to ethylene was greatly decreased in the presence of unlabeled ACC, but the conversion of labeled ACC to ethylene was little affected by the presence of unlabeled methionine; and 2-amino-4-(2′-aminoethoxy)
trans
-3-butenoic acid, a potent inhibitor of pyridoxal phosphate-mediated enzyme reactions, greatly inhibited the conversion of methionine to ethylene but did not inhibit conversion of ACC to ethylene. These data indicate the following sequence for the pathway of ethylene biosynthesis in apple tissue: methionine →
S
-adenosylmethionine → ACC → ethylene. A possible mechanism accounting for these reactions is presented.
Conservative segregation of parental histones during replication in the presence of cycloheximide
Authors: Riley Donald , Weintraub Harold
Published: May 2006

Long stretches of protein-free, nonbeaded DNA were observed electron microscopically in nuclear spreads prepared from cells that had replicated their DNA in the absence of protein synthesis. The amount of this DNA increased with increasing time of replication in the presence of cycloheximide and was greatly decreased when replication was inhibited with 1-β-D-arabinofuranosylcytosine (cytosine arabinoside). This DNA is considered to be “free” DNA because it has the same diameter as marker PM2 DNA and it is preferentially sensitive to DNase I digestion. Reversal of the cycloheximide block resulted in a burst of histone synthesis and repair of the depleted chromatin within 5 min. In addition, 26 presumptive replication forks were observed with beaded chromatin on two arms and free DNA on the third. These results suggest that new histones are usually deposited onto new DNA, that the cellular histone pool is very small, that histone migration is minimal
in vivo
for at least 18 hr, that for most fibers nuclesome assembly and segregation is conservative for stretches of DNA as long as 100 kbases, and that some part of the octameric histone core may remain bound to DNA during the replication process. The regularity we have observed for the assembly and segregation of nucleosomes is likely to be important for our understanding of how chromosomal information is segregated during development.
Inverted repeats in chloroplast DNA from higher plants
Authors: Kolodner Richard , Tewari K. K.
Published: May 2006

The circular chloroplast DNAs from spinach, lettuce, and corn plants have been examined by electron microscopy and shown to contain a large sequence repeated one time in reverse polarity. The inverted sequence in spinach and lettuce chloroplast DNA has been found to be 24,400 base pairs long. The inverted sequence in the corn chloroplast DNA is 22,500 base pairs long. Denaturation mapping studies have shown that the structure of the inverted sequence is highly conserved in these three plants. Pea chloroplast DNA does not contain an inverted repeat. All of the circular dimers of pea chloroplast DNA are found to be in a head-to-tail confirmation. Circular dimers of spinach and lettuce were also found to have head-to-tail conformation. However, approximately 70-80% of the circular dimers in preparations of lettuce and spinach chloroplast DNA were found to be in a head-to-head conformation. We propose that the head-to-head circular dimers are formed by a recombination event between two circular monomers in the inverted sequence.
Rapid turnover of acetyl groups in the four core histones of simian virus 40 minichromosomes.
Authors: Chestier A , Yaniv M
Published: May 2006

The four core histones (H2a, H2b, H3, and H4) bound to simian virus 40 minichromosomes isolated from infected cells contain rapidly labeled acetyl groups in internal positions of the histone polypeptide chain. Upon chase, these acetyl residues decay with a half-life of less than 15 min. The acetyl groups are incorporated in histones bound to mature chromosomes and not in newly synthesized histones bound to replicating viral chromosomes. The rate of acetate incorporation is not related to the degree of steady state acetylation of the individual viral or cellular histones. This rate is 4-fold higher for the viral chromatin than for its cellular counterpart isolated from the same nuclei. The possible role for histone acetylation in viral genome expression is discussed.
Transplantation of cultured bovine corneal endothelial cells to rabbit cornea: clinical implications for human studies.
Authors: Gospodarowicz D , Greenburg G , Alvarado J
Published: May 2006

Rabbit corneas denuded of their endothelium were coated with bovine corneal endothelial cells (from steers) previously maintained in tissue culture for short (20 generations) or prolonged (200 generations) periods. When grafted back into female rabbits, the corneal buttons remained clear and showed no edema. In contrast, denuded corneas coated with bovine keratocytes and grafted into rabbits became opaque and edematous within 7 days and remained so thereafter. Bovine corneal endothelial cells of the grafted corneas, which had remained clear for over 100 days, proliferated actively when put back into tissue culture. The corneal endothelial cells of the graft were characteristic of the male (XY). The chromosome number of the endothelium of the recipient rabbit was 2n = 44 with sex chromosomes characteristic of the female (XX). Results of the karyotype analysis show that there was no invasion of the corneal button by the recipient endothelium and, conversely, no invasion of the recipient endothelium by the endothelium on the corneal button. These results demonstrate that cultured corneal endothelial cells remain functional in vitro and can replace a damaged or nonfunctional endothelium in vivo.
Absence of virus-induced lymphocyte suppression and interferon production in multiple sclerosis
Authors: Neighbour P. Andrew , Bloom Barry R.
Published: May 2006

Lymphocytes from normal adult donors exposed
in vitro
to inactivated measles virus were found to exert significant suppression (33.9%) of the concanavalin A responses of cryopreserved, autochthonous responder cells. In marked contrast, lymphocytes from multiple sclerosis patients exhibited significantly reduced suppression (1.5%), and in 80% of cases failed to suppress at all. The degree of suppression increased slightly with age of the patient but did not vary with the clinical stage of disease. There was no apparent genetic restriction of suppressor activity. Although specificity of this phenomenon for measles virus has not been established, no differences in the responses of lymphocytes from normal or multiple sclerosis patient donors were found with subacute sclerosing panencephalitis, Sendai, canine distemper, mumps, or influenza viruses.

Supernates of measles-treated lymphocytes from normal donors possessed both suppressive and antiviral activities. Both activities were resistant to pH 2 treatment and were neutralized by an anti-human leukocyte interferon antiserum, strongly suggesting that interferon (probably type I) was the mediator of suppression. Consistent with their inability to suppress concanavalin A responses, lymphocytes from multiple sclerosis patients failed to produce significant amounts of interferon in response to measles challenge
in vitro
. These results extend previous observations that multiple sclerosis patients are unable to respond appropriately to measles virus antigen
in vitro
.
Modification of the blood-brain barrier: increased concentration and fate of enzymes entering the brain.
Authors: Barranger J A , Rapoport S I , Fredericks W R , Pentchev P G , MacDermot K D , Steusing J K , Brady R O
Published: May 2006

The blood-brain barrier of rats was opened reversibly by infusing a hyperosmotic solution of arabinose into the external carotid artery. Permeability was increased maximally in the first 15 min and remained slightly elevated at 1 hr. Osmotic barrier opening significantly increased brain uptake of intravenously injected alpha-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.2.4) (derived from human placenta) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). By injection of 4 X 10(5) units of alpha-mannosidase into an animal, brain activity rose to about twice the normal control activity of the enzyme. After 30 min, activity of administered enzyme in the extracellular space of the brain was calculated to be 30% of the serum concentration. Biochemical and histological studies with horseradish peroxidase showed that exogenously administered enzyme entered the brain extracellular space immediately after barrier opening and was incorporated within neuronal lysosomal packets during the next 24 hr. Measurable peroxidase activity was found in brain as much as 72 hr after osmotic treatment. The results demonstrate that the blood-brain barrier can be reversibly opened to enzymes, that a glycoprotein enzyme is incorporated into neuronal lysosomes, and that the brain may now be considered a potential target for enzyme replacement therapy in heritable metabolic disorders.
Utilization of trimethylamine and other N-methyl compounds for growth and methane formation by Methanosarcina barkeri.
Authors: Hippe H , Caspari D , Fiebig K , Gottschalk G
Published: May 2006

A number of N-methyl compounds, including several methylamines, creatine, sarcosine, choline, and betaine, were readily fermented by enrichment cultures yielding methane as a major product. Methylamine, dimethylamine, trimethylamine, and ethyldimethylamine were fermented by pure cultures of Methanosarcina barkeri; except for ethyldimethylamine, these amines are considered important substrates of this methanogenic microorganism. Creatine, sarcosine, choline, and betaine were fermented to methane only by mixed cultures. During growth of M. barkeri on methyl-, dimethyl-, or trimethylamine, methanol was not excreted into the medium. The fermentation of trimethylamine gave rise to an intermediary accumulation of methyl- and dimethylamine in the medium. An accumulation of methylamine during the fermentation of dimethylamine was not observed. Methane and ammonia were produced from the three methylamines by M. barkeri in amounts expected on the basis of the appropriate fermentation equations. The growth yield was 5.8 mg of cells (dry weight) per mmol of methane and was not dependent on the kind of methyl compound used as substrate.
Growth of a rat neuroblastoma cell line in serum-free supplemented medium.
Authors: Bottenstein J E , Sato G H
Published: May 2006

The rat neuroblastoma B104 cell line, which originated in the central nervous system, was able to proliferate in the absence of serum in synthetic medium supplemented with insulin, transferrin, progesterone, selenium, and putrescine. When added individually, each supplement had little or no effect; however, in combination there was a marked synergistic effect on cell number. The cells attained the same saturation density in this medium as in medium with 10% fetal calf serum added. More extensive process formation was observed in the supplemented medium, and other differentiated properties were retained as well.
Cholecystokinin octapeptide-like immunoreactivity: histochemical localization in rat brain.
Authors: Innis R B , Corrêa F M , Uhl G R , Schneider B , Snyder S H
Published: May 2006

Cholecystokinin octapeptide-like (CCK-OP-like) immunoreactivity was localized in the rat brain by using the indirect immunofluorescence method. Specificity in immunohistochemical studies was demonstrated by the virtual elimination of staining with either preimmune sera or sera preadsorbed with CCK-OP and by the achievement of similar fluorescent patterns with two different primary anti-CCK-OP sera. CCK-OP-like fluorescence was localized in neuronal cell bodies, fibers, and varicose terminals. The most dense collections of CCK-OP cells occurred in the periaqueductal gray and in the dorsomedial hypothalamus. Substantial numbers of cells and fibers also were present in the medial/dorsal and perirhinal cortex; more limited groups of cells were found in the pyramidal layer of the hippocampus and in the dorsal raphe.
Mouse mammary epithelial cells on floating collagen gels: Transepithelial ion transport and effects of prolactin
Authors: Bisbee Chester A. , Machen Terry E. , Bern Howard A.
Published: May 2006

Epithelial cells dissociated from midpregnant BALB/c mouse mammary glands were cultured for as long as 20 days as confluent monolayers on floating collagen gels. Detached gels bearing monolayers were placed in lucite Ussing chàmbers for measurement of transepithelial potential difference (PD), short-circuit current (I
sc
), resistance (R), and unidirectional fluxes of Na
+
and Cl
-
during short-circuit current conditions (PD = 0). With Hanks' solution bathing both sides of cultures maintained with insulin and cortisol, PD = -12.8 mV (serosal side ground), I
sc
= 24.6 μA/cm
2
, and R = 507 ω·cm
2
. Net absorption of Na
+
equaled I
sc
, and there was no net Cl
-
transport. PD and I
sc
were reduced 50% by mucosal addition of 10 μM amiloride and to zero by metabolic inhibition with nitrogen gas or by serosal addition of 0.1 mM ouabain. In similar cultures supplemented with prolactin, PD and I
sc
increased to -15.8 mV and 48.0 μA/cm
2
, respectively, and R decreased to 374 ω·cm
2
. Inhibitor effects were similar to those seen in prolactin-free cultures. Prolactin exposure resulted in a 3-fold increase in net absorption of Na
+
. Na
+
absorption was not equivalent to I
sc
, and there was little Cl
-
absorption; therefore, prolactin induced active transport of other, as yet unidentified, ions. These effects of prolactin require at least 3 days to occur and cannot be attributed to the known contamination with neurohypophysial hormones. The prolactin-induced increase in Na
+
absorption parallels its Na
+
-retaining ability in lower vertebrates and could be part of the mechanism that keeps milk Na
+
concentration low in intact glands.
Theory of protein-lipid and protein-protein interactions in bilayer membranes.
Authors: Owicki J C , McConnell H M
Published: May 2006

A model for protein-lipid interactions in bilayer membranes where the proteins are very dilute is extended to higher protein concentration, where appreciable lipid-mediated protein-protein interactions occur. It is found that proteins may change the lipid phase transition temperature and that they weaken the phase transition. There exists a critical protein concentration above which the sharp lipid phase transition is abolished. The model also qualitatively reproduces several experimental observations on the physical behavior of bilayers formed from mixtures of cholesterol and phosphatidylcholines.
Effect of tetanus toxin on the accumulation of the permeant lipophilic cation tetraphenylphosphonium by guinea pig brain synaptosomes
Authors: Ramos Sofia , Grollman Evelyn F. , Lazo Pedro S. , Dyer Sherry A. , Habig William H. , Hardegree M. Carolyn , Kaback H. Ronald , Kohn Leonard D.
Published: May 2006

Accumulation of the permeant lipophilic cation [
3
H]tetraphenylphosphonium (TPP
+
) by synaptosome preparations from guinea pig brain cerebral cortex is inhibited 1:10 by medium containing 193 mM K
+
and by veratridine. A further 1:10 to 1:15 decrease in TPP
+
uptake occurs under nitrogen and in the presence of mitochondrial inhibitors such as oligomycin, whereas starvation and succinate supplementation have no effect. These data indicate that, in analogy to intact neurons, there is an electrical potential (ΔΨ, interior negative) of -60 to -80 mV across the synaptosomal membrane that is due primarily to a K
+
diffusion gradient (K
+
in
→K
+
out
). The data also indicate that mitochondria entrapped within the synaptosome but not free mitochondria make a large contribution to the TPP
+
concentration gradients observed.

Conditions are defined in which tetanus toxin binds specifically and immediately to synaptosomes in media used to measure TPP
+
uptake. Under these conditions tetanus toxin induces dose-dependent changes in TPP
+
uptake that are blocked by antitoxin and not mimicked by biologically inactivated toxin preparations. The effect of tetanus toxin on TPP
+
uptake is not evident in the presence of 193 mM K
+
or veratridine but remains under conditions known to abolish the mitochondrial ΔΨ. Moreover, tetanus toxin has no effect on TPP
+
uptake by isolated synaptosomal mitochondria. The results thus define an
in vitro
action of tetanus toxin on the synaptosomal membrane that can be correlated with biological potency
in vivo
and is consistent with the
in vivo
effects of tetanus toxin on neuronal transmission.
Integration of bovine leukemia virus DNA in the bovine genome.
Authors: Kettmann R , Meunier-Rotival M , Cortadas J , Cuny G , Ghysdael J , Mammerickx M , Burny A , Bernardi G
Published: May 2006

DNA preparations from circulating leukocytes, lymph node tumors, and spleens of three bovine leukemia virus-infected cattle were fractionated by Cs2SO4/3,6-bis(acetatomercurimethyl)dioxane density gradient centrifugation. Bovine leukemia virus proviral sequences were found in large GC-rich fragments having a buoyant density in CsCl close to 1.708 g/cm3. Provirus integration, therefore, does not take place at random locations in the host genome, but in a specific class of DNA segments. Hybridization of cDNA synthesized on viral RNA to EcoRI and Xba I restriction fragments of the DNA from infected cells showed that: (i) only one copy of proviral DNA is integrated per haploid genome; (ii) different restriction patterns were found in the proviral DNAs present in the genomes of different animals, providing evidence for the existence of several strains or mutants; and (iii) different integration sites for the proviral DNA were found in the genome of different animals and of different infected cells in the same animal. The latter finding strongly suggests a polyclonal origin of bovine leukemia virus-infected cells.
Escherichia coli heat-labile enterotoxin: DNA-directed in vitro synthesis and structure.
Authors: Dorner F , Hughes C , Nahler G , Högenauer G
Published: May 2006

Escherichia coli heat-labile enterotoxin was synthesized in a cell-free system directed by DNA of the plasmid P307. Synthesis of the toxin, assayed by the elongation induced in Chinese hamster ovary cells, was strongly stimulated by cyclic AMP and occurred at physiological levels of Mg2+ only when the polyamine spermidine was present. Activity was abolished by heat and antisera prepared against the enterotoxins of both E. coli P263 and Vibrio cholera. Tritium-labeled enterotoxin was purified by immunoprecipitation and examined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. When gel slices were assayed for the ability to stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in erythrocyte ghosts, two peaks were found, one at Mr 26,000 and frequently, but not always, another at Mr 23,000. Detection of radiolabeled protein by fluorography and scintillation counting of gel slices revealed three prominent polypeptides, two corresponding to the peaks having adenylate cyclase-stimulating activity and a further one of Mr 11,500, identical to that of the cholera subunit B. The data suggest that the E. coli heat-labile enterotoxin synthesized in the cell-free system has a subunit structure.
Isolation and identification of bacteriophage phi X174 prohead.
Authors: Mukai R , Hamatake R K , Hayashi M
Published: May 2006

The morphogenesis of bacteriophage phi X174 has been investigated by using an in vitro DNA synthesizing system. An extract of a B-mutant-infected cell is capable of synthesizing infectious phage in vitro when the B gene function is provided by the addition of an ammonium sulfate fraction of a C-mutant-infected-cell extract. This fraction contains the omega complex, a complex of phage-coded proteins with S = 108; the B-mutant extract does not. The purified omega complex, isolated from the C-mutant extract, caused the synthesis and encapsidation of viral DNA when added to B-mutant extract. The omega complex contains the B protein but it is the intact omega complex that functioned in the in vitro complementation of the B-mutant extract because other fractions containing B protein but no omega complex had little or no complementing activity. The results indicate that the omega complex is the phi X174 prohead. The B protein is not found in either the 132S or 114S phage particles but is an essential component of the prohead. This suggests that it may have a scaffolding function.
Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation.
Authors: Ricciardi R P , Miller J S , Roberts B E
Published: May 2006

We describe a simple procedure for isolating specific mRNAs and for mapping them to the regions of the DNA from which they originate. The method involves hybridization of total cytoplasmic RNA to restriction fragments of DNA that have been fractionated in agarose gels and immobilized on nitrocellulose filters. The hybridization-selected RNAs are eluted and translated in a cell-free system in order to identify their encoded polypeptides. Optimal hybridization and filter washing conditions are given for selection of mRNAs from adenovirus 2-infected cells and transformed cells.
Highly active template-specific RNA-dependent RNA polymerase from barley leaves infected with brome mosaic virus.
Authors: Hardy S F , German T L , Loesch-Fries L S , Hall T C
Published: May 2006

The extraction of a template-dependent and template-specific RNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from a eukaryotic source is described. The enzyme, extracted from barley leaves infected with brome mosaic virus (BMV), is capable of incorporating high levels of radioactivity into trichloroacetic acid-insoluble products. The purification procedure included solubilization with nonionic detergent and precipitation with polyethylene glycol. The enzyme was more than 50 times more active than was a comparable preparation from mock-inoculated leaves and was stimulated more than 15-fold by the addition of BMV RNA to the reaction. Other viral RNA templates were less than 25% as efficient as was BMV RNA in stimulating UMP incorporation; poly(A), tRNA, and mRNA gave little stimulation and rRNA was inactive. Autoradiographic analysis after electrophoretic separation of the radioactive products from reaction mixtures containing BMV RNA template revealed prominent bands that coelectrophoresed with replicative forms of BMV RNAs. When BMV RNA template was enriched in RNA3 or RNA4, larger proportions of the products were replicative forms of RNA3 or RNA4, respectively.
Termination of transcription by bacteriophage T3 RNA polymerase: homogeneous 3'-terminal oligonucleotide sequence of in vitro T3 RNA polymerase transcripts.
Authors: Majumder H K , Maitra U , Rosenberg M
Published: May 2006

RNA was synthesized in vitro from a T3 DNA template by T3 RNA polymerase and subsequently separated into seven discrete size classes (molecular weights ranging between 0.21 x 10(6) and 6.2 x 10(6)) by electrophoresis in polyacrylamide slab gels. RNase T1-generated 3'-terminal oligonucleotide fragments were then selectively isolated from either the unfractionated total RNA or the gel-purified specific transcripts by chromatography on columns of dihydroxyboryl-cellulose. Sequence analysis of these oligonucleotide products indicated that the unfractionated transcripts as well as all the individual major RNA species examined had a unique sequence, (Gp)UpUpUpUpUpGOH, at their 3' termini. The specificity of this sequence, as well as the total lack of any sequence heterogeneity at the ends of these transcripts, indicates a high degree of specificity of termination during transcription in this system.
Beta-turns in nascent procollagen are sites of posttranslational enzymatic hydroxylation of proline.
Authors: Brahmachari S K , Ananthanarayanan V S
Published: May 2006

The selective hydroxylation of proline residues in nascent procollagen chains by prolyl hydroxylase (EC 1.14.11.2) can be understood in terms of the conformational feature of the -Pro-Gly-segments in linear peptides and globular proteins. The folded beta-turn conformation in such segments appears to be the conformational requirement for proline hydroxylation. The available data on the hydroxylation of native and synthetic substrates of prolyl hydroxylase are explained on the basis of the extent of beta-turn formation in them. Taken in conjunction with the conformational features of the hydroxyproline residue, our results bring out the conformational reason for the posttranslational proline hydroxylation which, it is proposed, leads to the "straightening" of the beta-turn segments into the linear triple-helical conformation.
Nucleotide sequences of trpA of Salmonella typhimurium and Escherichia coli: an evolutionary comparison.
Authors: Nichols B P , Yanofsky C
Published: May 2006

The complete nucleotide sequences of trpA of Salmonella typhimurium and Escherichia coli were determined. The nucleotide sequences are 24.8% divergent, compared with amino acid sequence divergence of 14.9%. Over half of the codons of each gene contain synonymous nucleotide changes. The pattern of synonymous nucleotide changes is consistent with the interpretation that such changes result from random mutational events. We do not find any evidence indicating that codon selection or RNA structure is of major selective value. We conclude that polypeptide function is the primary basis of selection in trpA and that most synonymous codon changes are selectively neutral.
Control of yeast cell types by mobile genes: A test
Authors: Kushner Peter J. , Blair Lindley C. , Herskowitz Ira
Published: May 2006

The yeast
Saccharomyces cerevisiae
changes cell types by switching the alleles of the mating type locus (
MAT
) from a to α and vice versa. In the cassette model, these switches—e.g., from a to α—occur when a replica of silent α information (an α “cassette”) replaces the resident a cassette at the mating type locus and is thereby expressed. We have identified a mutation in the locus postulated to be the silent α information (
HML
α) and find that a mutation is introduced into the mating type locus as a result of interconversion:
HML
α
-
MAT
α cells switch to
MAT
a and then to
MAT
α
-
. The
MAT
α
-
mutation leads to defective mating and behaves like some previously identified
MAT
α
-
mutations. These observations satisfy the prediction of the cassette and controlling element models that genetic information is transmitted from
HML
α to the mating type locus.
Manganese and zinc blockade of enzyme induction: Studies with microsomal heme oxygenase
Authors: Drummond George S. , Kappas Attallah
Published: May 2006

Heme oxygenase (decyclizing) [heme,hydrogen-donor:oxygen oxidoreductase (α-methene-oxidizing, hydroxylating), EC 1.14.99.3] is greatly induced in the kidney by the administration of nickel or tin. Manganese, when administered simultaneously with nickel or tin in an equimolar amount, substantially inhibited the induction of heme oxygenase. The extent of inhibition was 80% and 98%, respectively. In rats pretreated up to 8 hr with manganese, the level of induction of heme oxygenase by nickel or tin was markedly reduced in a time-dependent fashion. Manganese treatment after the inducing metal was relatively ineffective in preventing the induction of heme oxygenase. Manganese
in vitro
did not inhibit heme oxygenase in the microsomes isolated from either control or tin-induced rats and
in vivo
did not increase the rate of catabolism of the induced enzyme. Magnesium was unable to block nickel or tin induction of heme oxygenase. Zinc in equimolar amounts could also substantially reduce the extent of induction of renal heme oxygenase when administered simultaneously with nickel or tin. In addition, simultaneous zinc administration blocked to a considerable extent the induction of hepatic heme oxygenase by nickel, cobalt, or cadmium. These findings indicate the existence of metal—metal interactions that can greatly influence the regulatory mechanism for the induced synthesis of heme oxygenase, the rate-limiting enzyme in heme degradation.
Alterations in nociception and body temperature after intracisternal administration of neurotensin, beta-endorphin, other endogenous peptides, and morphine.
Authors: Nemeroff C B , Osbahr A J , Manberg P J , Ervin G N , Prange A J
Published: May 2006

The antinociceptive and hypothermic effects of intracisternal administration of 11 endogenous neuropeptides and morphine were evaluated in mice. Of the substances tested, only neurotensin (NT) and beta-endorphin exerted significant antinociceptive and hypothermic effects; NT was the most potent in inducing hypothermia whereas beta-endorphin was the most potent antinociceptive agent via this route of administration. Both NT, and beta-endorphin were, on a molar basis, considerably more potent antinociceptive agents than morphine, [Met]enkephalin, or [Leu]enkephalin. NT-induced analgesia and hypothermia both were significantly dose-dependent. Substance P was found to produce significant hyperalgesia and hyperthermia. Bombesin produced a significant hypothermic effect, whereas somatostatin and luteinizing hormone-releasing hormone (luliberin) produced hyperthermia. None of the other peptides studies [bradykinin, thyrotropin-releasing factor (thyroliberin), melanocyte-stimulating hormone release-inhibiting factor (melanostatin), somatostatin, [Met]enkephalin, and [Leu]enkephalin] produced any significant alterations in colonic temperature or response to a noxious stimulus with the doses tested. These data demonstrate that NT and beta-endorphin, two endogenous brain peptides, are potent in inducing hypothermia and in producing an antinociceptive state.
Photoreactive labeling of M13 coat protein in model membranes by use of a glycolipid probe.
Authors: Hu V W , Wisnieski B J
Published: May 2006

Coliphage M13 coat protein in synthetic bilayer membranes was labeled by use of 12-(4-azide-2-nitrophenoxy)stearoyl[1-14C]glucosamine, a photoreactive glycolipid probe that spontaneously inserts into membranes. In this model system, the probe preferentially labeled the proteins over the lipids. Experiments designed to test the probe's restriction to integral membrane proteins revealed that extrinsic proteins as well as external peptide fragments of integral membrane proteins were not accessible to the photogenerated nitrene on the fatty acid chain. Only integral membrane peptides were labeled by the membrane-bound probe. These results indicate that protein labeling can be effected specifically from within the hydrocarbon milieu of a model membrane system.
Purification of chicken intestinal receptor for 1,25-dihydroxyvitamin D.
Authors: Pike J W , Haussler M R
Published: May 2006

The 3.3S chicken intestinal receptor for 1,25-dihydroxyvitamin D [1,25-(OH)2D] has been purified approximately 86,000-fold from the cytosolic fraction. The receptor was selectively precipitated with Polymin P from high-speed supernatants derived from 800 g of intestinal mucosa and then sequentially chromatographed on DNA-cellulose. Sephacryl, blue dextran-Sepharose, DNA-cellulose, and heparin-Sepharose. Polyacrylamide gel electrophoresis of 8-10 microgram of the purified receptor in sodium dodecyl sulfate indicated the presence of one major and three minor protein bands of molecular weights 50,000-65,000. Sucrose gradient analysis of the purified material in 0.3 M KCl suggested that a fraction of the receptor remained complexed to the 1,25-(OH)2D and that its sedimentation property of 3.3S remained unchanged. These results represent a major purification of the chick intestinal receptor for 1,25-(OH)2D, an extremely rare and labile protein whose isolation is estimated to require a 200,000-fold purification. Of primary importance is the observation tht affinity ligands such as DNA and blue dextran can effect major purification of this protein, lending credence to the hypothesis that the 1,25-(OH)2D receptor functions within the cell nucleus by altering the expression of specific genes.
Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.
Authors: Livneh Z , Elad D , Sperling J
Published: May 2006

Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease.
Hybrid mammalian cells assemble hybrid ribosomes.
Authors: Wejksnora P J , Warner J R
Published: May 2006

Hybrid cell lines formed by fusion of mouse 3T3 cells and Chinese hamster ovary (CHO) cells resistant to emetine, which have an altered 40S ribosomal protein, are generally sensitive to emetine. From most hybrid lines it was possible to select sublines resistant to emetine. The ribosomal components of three lines were studied: A34, an emetine-sensitive hybrid; A34/R3, an emetine-resistant derivative of A34; and A72, an emetine-sensitive hybrid that did not give rise to emetine-resistant sublines. Genetic and biochemical evidence suggests that in A34 both the mouse emetine sensitivity gene and the hamster emetine resistance gene are active, whereas in A34/R3 only the hamster emetine resistance gene is active and in A72 only the mouse emetine sensitivity gene is active. The ribosomes of all three sublines contained both mouse and hamster RNA, predominantly mouse. However, the 60S subunits had roughly equal amounts of the three mouse and hamster proteins that could be distinguished by two-dimensional electrophoresis, suggesting the association of mouse RNA with hamster ribosomal proteins. The emetine-resistant and emetine-sensitive 40S subunits could be separated by sedimentation in 0.5 M KCl. Resistant subunits contained predominantly mouse RNA, presumably associated with the hamster protein conferring emetine resistance. We conclude that hybrid cells can form hybrid ribosomes and that the amounts of ribosomal RNA and ribosomal protein of each species are not closely coupled.
Biotinyl 5'-adenylate: corepressor role in the regulation of the biotin genes of Escherichia coli K-12.
Authors: Prakash O , Eisenberg M A
Published: May 2006

A DNA filter-binding technique was used to study the interaction of the biotin repressor and operator site. From a biotin saturation curve, the concentration for half-maximal binding (K0.5) was calculated to be 1 microM. However, in a similar study with the in vitro coupled transcription-translation system in which biotin served as the corepressor, the K0.5 for repression was 7.1 nM. This marked difference of over 2 orders of magnitude was attributed to the activation of biotin by the partially purified repressor preparation in the in vitro system. The activated product formed from biotin, ATP, and repressor preparation was identified as biotinyl 5'-adenylate by paper chromatography and hydroxamic acid formation. Synthetic biotinyl 5'-adenylate was as effective as biotin in the in vitro system (K0.5, 10 nM) and much more effective than biotin in the DNA-binding assay (K0.5 1.1 nM versus 1 microM). These studies indicate that biotinyl 5'-adenylate has a more direct role in the regulation of the biotin genes than does biotin per se.
Glutathione: interorgan translocation, turnover, and metabolism.
Authors: Griffith O W , Meister A
Published: May 2006

Glutathione is translocated out of cells; cells that have membrane-bound gamma-glutamyl transpeptidase can utilize translocated glutathione, whereas glutathione exported from cells that do not have appreciable transpeptidase enters the blood plasma. Glutathione is removed from the plasma by the kidney and other organs that have transpeptidase. Studies in which mice and rats were treated with buthionine sulfoximine, a selective and potent inhibitor of gamma-glutamylcysteine synthetase and therefore of glutathione synthesis, show that glutathione turns over at a significant rate in many tissues, especially kidney, liver, and pancreas; the rate of turnover in mouse skeletal muscle is about 60% of that in the kidney. Experiments on rats surgically deprived of one or both kidneys and treated with the gamma-glutamyl transpeptidase inhibitor D-gamma-glutamyl-(o-carboxy)phenylhydrazide establish that extrarenal gamma-glutamyl transpeptidase activity accounts for the utilization of about one-third of the total blood plasma glutathione. Normal animals treated with the transpeptidase inhibitor excrete large amounts of glutathione in their urine. They also excrete gamma-glutamylcysteine, suggesting that cleavage of glutathione at the cysteinylglycine bond may be of metabolic significance. The present and earlier findings lead to a tentative scheme (presented here) for the metabolism and translocation of glutathione, gamma-glutamyl amino acids, and related compounds.
Lateral mobility of an amphipathic apolipoprotein, ApoC-III, bound to phosphatidylcholine bilayers with and without cholesterol
Authors: Vaz Winchil L. C. , Jacobson Kenneth , Wu En-Shinn , Derzko Zenon
Published: May 2006

The technique of fluorescence recovery after photobleaching was used to investigate the lateral mobility of a fluorescein-labeled amphipathic apolipoprotein, ApoC-III, bound to multibilayers prepared from dipalmitoyl phosphatidylcholine, egg phosphatidylcholine, and a 1:1 (molar ratio) mixture of egg phosphatidylcholine and cholesterol. In dipalmitoyl phosphatidylcholine bilayers the lateral diffusion coefficient (
D
) for the protein is about 2 × 10
-9
cm
2
sec
-1
at 20°C and about 9 × 10
-8
cm
2
sec
-1
at 45°C. Plots of
D
versus temperature in this system show a transition between about 30 and 35°C. Arrhenius activation energies for the diffusion in this case between 15 and 30°C and between 35 and 45°C are 28.5 and 7.0 kcal mol
-1
, respectively (1 calorie = 4.18 joules). In egg phosphatidylcholine bilayers,
D
is about 3 × 10
-8
cm
2
sec
-1
at 20°C and the Arrhenius activation energy for diffusion is 8.1 kcal mol
-1
between 15 and 35°C in this system. In bilayers prepared from an equimolar mixture of egg phosphatidylcholine and cholesterol
D
at 20°C is about 1.4 × 10
-9
cm
2
sec
-1
and the Arrhenius activation energy for the diffusion of the protein in this system between 15 and 35°C is 15.1 kcal mol
-1
. Light-scattering and fluorescence-polarization results indicate that binding of this protein does not affect the gel-to-liquid crystalline phase transition of bilayer membranes but does mediate a major, reversible aggregation of the vesicles at about 33°C. These results lend support to the view that ApoC-III resides in the head-group region of the bilayer and suggest that its lateral diffusion coefficient represents an upper bound for integral membrane proteins.
Globin synthesis and erythroid differentiation in a Friend cell variant deficient in heme synthesis.
Authors: Rutherford T R , Harrison P R
Published: May 2006

Friend erythroleukemia cells of line Fw are noninducible for hemoglobin synthesis by dimethyl sulfoxide, butyric acid, and other agents. However, these agents were found to induce hemoglobin synthesis if the cells were also treated with exogenous hemin. Butyric acid (or, to a lesser extent, dimethyl sulfoxide) by itself induced accumulation of the erythroid-specific membrane protein spectrin. The basis of the control of globin gene expression by hemin was investigated. Hemin does not control globin and total protein synthesis via the action of the hemin-controlled repressor. Rather, hemin, alone or in combination with dimethyl sulfoxide, induces accumulation of globin mRNA and nuclear globin RNA sequences. Direct measurements of heme synthesis indicate that Fw cells may be significantly deficient in heme metabolism.
Amplified dihydrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes.
Authors: Kaufman R J , Brown P C , Schimke R T
Published: May 2006

Selection of mammalian cells in progressively increasing concentrations of methotrexate results in selective amplification of DNA sequences coding for dihydrofolate reductase (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). In some cell variants the amplified genes are stable with growth in the absence of methotrexate, whereas in other variants the amplified genes are lost from the population. We have previously reported that in a stably amplified variant of Chinese hamster ovary cells, the genes are localized to a single chromosome. Herein we report that in mouse S-180 and L5178Y cell lines unstably amplified dihydrofolate reductase DNA sequences are associated with small, paired chromosomal elements denoted "double minute chromosomes," whereas in stably amplified cells of the same origin, the genes are associated with large chromosomes.