bluuebunny/crossref_metadata_2025 huggingface dataset:
Regions of Single-Stranded DNA in the Growing Points of Replicating Bacteriophage T7 Chromosomes
Authors: Wolfson John , Dressler David
Published: May 2006

In partially replicated T7 chromosomes, the points where parental strands are separating and new DNA is being synthesized can be seen in the electron microscope to contain regions of single-stranded template DNA. The single-stranded regions are located on only one of the two daughter arms of the replicating chromosome. Inman and Schnös observed such single-stranded regions in 50% of the growing points of replicating lambda DNA, and, as reported in this paper, we find them in about 85% of the growing points of T7 DNA. Both studies support the conclusion that DNA synthesis involves the direct elongation of one daughter strand in the growing point. Evidently, this elongation is accompanied by the unwinding of the parental double helix to expose a region of single-stranded DNA which is then converted to the duplex state by a discontinuous mechanism involving the synthesis of DNA fragments.
The Interaction of Histone IV with Nucleoside Triphosphates
Authors: Wickett R. , Isenberg I.
Published: May 2006

The nucleoside triphosphates ATP, GTP, TTP, and CTP induce conformational changes in histone IV at concentrations that are of the same magnitude as the histone concentration. The purine nucleoside triphosphates are much more effective than the pyrimidine nucleoside triphosphates.
Initiation of DNA Synthesis: Synthesis of ϕX174 Replicative Form Requires RNA Synthesis Resistant to Rifampicin
Authors: Schekman Randy , Wickner William , Westergaard Ole , Brutlag Douglas , Geider Klaus , Bertsch Leroy L. , Kornberg Arthur
Published: May 2006

Conversion of single-stranded DNA of phage ϕX174 to the double-stranded replicative form in
Escherichia coli
uses enzymes essential for initiation and replication of the host chromosome. These enzymes can now be purified by the assay that this phage system provides. The ϕX174 conversion is distinct from that of M13. The reaction requires different host enzymes and is resistant to rifampicin and streptolydigin, inhibitors of RNA polymerase. However, RNA synthesis is essential for ϕX174 DNA synthesis: the reaction is inhibited by low concentrations of actinomycin D, all four ribonucleoside triphosphates are required, and an average of one phosphodiester bond links DNA to RNA in the isolated double-stranded circles. Thus, we presume that, as in the case of M13, synthesis of a short RNA chain primes the synthesis of a replicative form by DNA polymerase. Initiation of DNA synthesis by RNA priming is a mechanism of wide significance.
The Contribution of the α and β Chains to the Kinetics of Oxygen Binding to and Dissociation from Hemoglobin
Authors: Gibson Quentin H.
Published: May 2006

A new type of experiment in which hemoglobin is exposed briefly to oxygen has shown that the half-time of dissociation of oxygen from some partly oxygenated intermediates is about 1 msec at 20° and 10 msec at 2°. The rapid dissociation occurs selectively from one type of chain, provisionally identified as the β-chain. Chains that show the rapid rate of dissociation of oxygen also bind rapidly. It follows that the kinetic equivalent of the Adair equation and the Monod-Wyman-Changeux model are quite unsuited to represent the kinetics of the oxygen-hemoglobin reaction. The reaction of oxygen with hemoglobin closely resembles that of the alkyl isocyanides and differs radically from that of carbon monoxide.
Autonomy of Mitochondria in
Saccharomyces cerevisiae
in Their Production of Messenger RNA
Authors: Mahler Henry R. , Dawidowicz Karl
Published: May 2006

In
ts
-
136, a temperature-sensitive mutant of
Saccharomyces cerevisiae
, nuclear and mitochondrial RNA production can be inhibited selectively by exposure to 36° and ethidium bromide, respectively. Using the programming of mitochondrial polysomes, as measured by their ability to form nascent polypeptide chains, as an assay for functional messenger RNA, we have determined its response to temperature shifts and ethidium bromide. Only ethidium bromide produced a measurable effect; in contrast the cell-sap system responded exclusively to temperature shifts. We conclude that transcription of mitochondrial DNA is sufficient and that import of messenger RNA transcribed from nuclear chromosomes makes no measurable contribution to intramitochondrial protein synthesis.
Biosynthesis of the Photosystem I Chlorophyll-Protein Complex in Greening Leaves of Higher Plants
Authors: Alberte Randall S. , Thornber J. Philip , Naylor Aubrey W.
Published: May 2006

Biosynthesis of photosystem I chlorophyll-protein complex, derived from chloroplast lamellae solubilized with Triton X-100, was examined in rapidly greening primary leaves of the jack bean. It was found that the chlorophyll/P700 ratio of the complex was low (35/1) in the early stages of greening, and increased steadily to a stable level (75/1) during continued chloroplast development. It is suggested from these results that in the initial stages of greening, and probably throughout synthesis, the P700-chlorophyll-protein molecules are first inserted into the developing lamellae, and those photosystem I chlorophyll-protein molecules functioning solely in a light-harvesting capacity are added subsequently.
Photooxidation of Chlorophyll
b
by Quinones Studied by Chemically-Induced Dynamic Nuclear Polarization
Authors: Tomkiewicz Micha , Klein Melvin P.
Published: May 2006

The photooxidation of chlorophyll
b
by 1,4-benzoquinone and 2,6-dimethyl-1,4-benzoquinone was investigated by the technique of chemically-induced dynamic nuclear polarization. Polarization of the proton magnetic resonance lines of the quinone was detected. A mechanism for the photooxidation was postulated that invokes the reaction of the quinone with the excited singlet state of the chlorophyll to form a radical pair. This mechanism, together with a theoretical model and parameters taken from the literature, yields a theoretical proton magnetic resonance spectrum for the polarized quinone that agrees well with that observed experimentally.
Inactivation System of the Mammalian X Chromosome
Authors: Brown Spencer W. , Chandra H. Sharat
Published: May 2006

In female mammals, one of the two X chromosomes present is inactivated during early development. In marsupials, the paternal X is inactivated; in eutherians, one of the two X chromosomes is inactivated at random. A mechanism is proposed to explain the cytogenetic data on inactivation and the derivation of the eutherian system from the marsupial system. In the marsupial system, a site on the X chromosome is sensitive to paternal origin: when the X chromosome is of maternal origin, this sensitive site is responsible for influencing an adjacent site, the receptor, to maintain the X in an active state; the paternal X becomes inactive. Transposition of the sensitive site to an autosome in eutherians would have two consequences. Since the receptor site of the X chromosome is no longer adjacent, the autosomal sensitive site of maternal origin would activate an X at random. The number of active X chromosomes would conform to the number of maternal sensitive sites and thus, generally, to the number of maternal sets of autosomes. The response of the sensitive site to its passage through the male may be designated as imprinting, a term used by Crouse to indicate that the behavior of
Sciara
chromosomes is determined by parental origin.
An Alteration in Glucose Metabolism Associated with a Defect in Ketone Body Metabolism
Authors: Tildon J. Tyson
Published: May 2006

Skin fibroblasts (CC-69) cultured from a patient with a unique syndrome of ketoacidosis associated with coenzyme A transferase (EC 2.8.3.5) deficiency showed an altered pattern of carbohydrate metabolism. These cells used glucose at a rate significantly less than controls (125 against 680 nmol/mg per hr). The oxidation of [
6
-
14
C]glucose to
14
CO
2
by these cells was also significantly diminished (12 against 350 pmol/mg per hr), but [
2
-
14
C]pyruvate and [
1,4
-
14
C]succinate oxidation by these cells did not differ from that by control cells. Measurements of glycolytic intermediates showed a reduction of several intermediates in the CC-69 cells that confirmed an inhibition of glycolysis between fructose-1,6-bisphosphate and pyruvate. The apparent inhibition in these cells could be reversed by an extended incubation of the cells in a buffered glucose solution. After 18 hr of incubation in 2.5 mM glucose, glucose uptake by the CC-69 cells increased 20-fold to 2560 nmol/mg per hr, whereas the rate for control cells remained constant at 640 ± 90. Concomitant with this increase, [
6
-
14
C]glucose oxidation rose from 8 to 2261 pmol/mg per hr while controls remained constant at 428 ± 175. This change was not due to new enzyme formation because incubation with puromycin had no effect on the increased use of glucose. Mixing experiments demonstrated no transfer of a permeable inhibitor or activating substances. In view of the deficiency of coenzyme A transferase in these cells, the data suggest an indirect regulatory role for this enzyme in peripheral tissue glycolysis.
Electron Spin Resonance Studies of Spin-Labeled Mammalian Cells by Detection of Surface-Membrane Signals
Authors: Kaplan Joseph , Canonico Peter G. , Caspary William J.
Published: May 2006

Lipid-soluble spin labels were incorporated into human lymphocytes and mouse L-cells and the resulting electron spin resonance spectra were compared with spectra obtained from similarly labeled human erythrocytes. Spin labels were found in all subcellular fractions of the nucleated cells that contained membranes. Spinlabeled cells remained viable and capable of replicating
in vitro
. Electron spin resonance signals from spin-labeled nucleated cells underwent a time- and temperature-dependent decay that was reversed by bathing the cells in K
3
Fe(CN)
6
. The demonstration of a relative cell impermeability to ferricyanide, as measured by both colorimetric and radioisotopic label methods, indicated that only spin-labeled molecules in the surface membrane were reactivated when ferricyanide was added to spin-labeled cells after the electron spin resonance signal had decayed.
Dansyl-Galactoside, a Fluorescent Probe of Active Transport in Bacterial Membrane Vesicles
Authors: Reeves John P. , Shechter Emanuel , Weil Rudolf , Kaback H. R.
Published: May 2006

A fluorescent galactoside, 2-(
N
-dansyl)-aminoethyl β-D-thiogalactoside (dansyl-galactoside), competitively inhibits lactose transport by membrane vesicles of
Escherichia coli
, but is not actively transported. An increase in dansyl-galactoside fluorescence is observed upon addition of D-lactate. The fluorescence increase is not observed in membrane vesicles lacking the β-galactoside transport system, and is blocked or rapidly reversed by addition of β-galactosides, sulfhydryl reagents, inhibitors of D-lactate oxidation, or uncoupling agents. The fluorescence increase exhibits an emission maximum at 500 nm and excitation maxima at 345 nm and at 292 nm. The latter excitation maximum is absent unless D-lactate is added, indicating that the bound dansyl-galactoside molecules are excited by energy transfer from the membrane proteins. Titration of vesicles with dansyl-galactoside in the presence of D-lactate demonstrates that the β-galactoside carrier protein represents about 3.3% of the total membrane protein. The data indicate that D-lactate oxidation leads to binding of the fluorescent galactoside to the β-galactoside carrier protein in such a manner that the dansyl group is transferred to a hydrophobic environment within the membrane.
Differentiation in
Nostoc muscorum:
Nitrogenase Is Synthesized in Heterocysts
Authors: Fleming Honorée , Haselkorn Robert
Published: May 2006

Cellular differentiation can be observed in certain filamentous blue-green algae after transfer of the cells from medium containing NH
4
+
or NO
3
-
to nitrogen-free medium. The appearance of differentiated cells (heterocysts) is accompanied by an increase in the activity of nitrogenase, an enzyme complex that reduces N
2
to NH
3
. We have separated vegetative cells from heterocysts in differentiated filaments of
Nostoc muscorum
, and analyzed their proteins by polyacrylamide-gel electrophoresis. The pattern of incorporation of
35
SO
4
-
into proteins of the two cell types during differentiation indicates that the increase in nitrogenase activity is due to the
de novo
synthesis of nitrogenase proteins predominantly, if not exclusively, in heterocysts.
Regulation of RNA Synthesis in Fibroblasts During Transition from Resting to Growing State
Authors: Mauck John C. , Green Howard
Published: May 2006

Addition of serum, containing fibroblast growth factors, to a culture of resting 3T6 cells stimulates a transition to the growing state. Studies of ghost monolayers prepared with the aid of detergent at intervals after stimulation showed an increase in the rate of ribosomal RNA synthesis within 10 min. The rate continued to increase for many hours and reached a level 2.5- to 3.5-fold higher by the time DNA synthesis began. The increasing rate of ribosomal RNA synthesis appeared independent of an increase in the number of ribosomal genes, since it was not affected by prevention of DNA synthesis with cytosine arabinoside.

In contrast to ribosomal RNA, the overall rate of transscription of heterogeneous nuclear RNA was not directly affected by serum growth factors and does not appear to be regulated during the transition from resting to growing state. It seems, instead, to be fixed in relation to the amount of template, for it increases proportionally to DNA content.
Resolution of DL-Tryptophan by Affinity Chromatography on Bovine-Serum Albumin-Agarose Columns
Authors: Stewart Kent K. , Doherty Robert F.
Published: May 2006

Bovine-serum albumin, known to have antipodal specificity in the binding of tryptophan, was selected as the affinity chromatographic matrix for the attempted chromatographic resolution of DL-tryptophan. Complete resolution was accomplished when Dl-tryptophan was chromatographed on bovine-serum albuminsuccinoylaminoethyl-Sepharose.
ATPase and GTPase Activities Associated with a Specific 5S RNA-Protein Complex
Authors: Horne Janet R. , Erdmann Volker A.
Published: May 2006

An RNA-protein complex consisting of 5S RNA and two ribosomal proteins, B-L5 and B-L22, was isolated from
Bacillus stearothermophilus
ribosomes and found to be active in GTP hydrolysis. This activity was not influenced by elongation factor G. Further analysis of this complex showed that it was also able to hydrolyze ATP. Inhibition studies revealed that ATP was a noncompetitive inhibitor of GTP and that GTP was also a noncompetitive inhibitor of ATP, indicating that two different enzymatic sites were involved. Differences in pH optimum and optimal temperature also point to a twosite enzyme complex. Both enzymatic hydrolyses were inhibited by thiostrepton and fusidic acid, which are known inhibitors of protein synthesis.
Regulation of Acetylcholine Receptors in Relation to Acetylcholinesterase in Neuroblastoma Cells
Authors: Simantov Rabi , Sachs Leo
Published: May 2006

Purified α-toxin from
Naja nigricollis
snake venom labeled by [
3
H]acetylation binds specifically to the acetylcholine receptors of mouse neuroblastoma cells. Toxin binding was inhibited by inhibitors for nicotinic and muscarinic acetylcholine receptors. Clones of neuroblastoma cells were selected for low acetylcholinesterase (EC 3.1.1.7) activity with antibodies against this enzyme. Selection for an 80-fold decrease in acetylcholinesterase activity was not associated with any decrease in the number of acetylcholine receptors (3.4 × 10
7
per cell). Removal or inactivation of 80% of the acetylcholine receptors by proteolytic enzymes or by compounds that block sulfhydryl groups did not change the activity of acetylcholinesterase on the cell surface. In addition to these results on the separation between acetylcholine receptors and acetylcholinesterase, a common regulation was found in that both the number of acetylcholine receptors and the activity of acetylcholinesterase were increased 5- to 10-fold when the cells stopped to multiply or were induced to differentiate by dibutyryl-cyclic AMP. It is suggested that there are different genes for the acetylcholine receptor and acetylcholinesterase, and that both are regulated during growth and differentiation by a common regulatory gene.
Relation of Detergent HLB Number to Solubilization and Stabilization of D-Alanine Carboxypeptidase from
Bacillus subtilis
Membranes
Authors: Umbreit Jay N. , Strominger Jack L.
Published: May 2006

The ability of various nonionic detergents to solubilize D-alanine carboxypeptidase and other membrane-bound enzymes was correlated to a physical property of the detergent, the HLB number. Only a fairly narrow range of detergents were effective solubilizing agents. Purified carboxypeptidase required either a detergent or detergent plus a lipid fraction for stability. Only those detergents effective in solubilizing the enzyme were effective in stabilizing it.
Cytogenetic Engineering
In Vivo:
Restoration of Biologic Complement Activity to C5-Deficient Mice by Intravenous Inoculation of Hybrid Cells
Authors: Levy Nelson L. , Snyderman Ralph , Ladda Roger L. , Lieberman Rose
Published: May 2006

Splenic macrophages were identified as at least one source of C5 elaboration in normal mice. Hybrid cells were formed from splenic macrophages from C5-deficient mice and either kidney cells from mice with normal amounts of C5 or chicken erythrocytes. These hybrids elaborated C5
in vitro
. C5-Deficient mice inoculated with these hybrid cells developed, in their serum, antigenically active mouse C5, as well as both hemolytic and biologic complement activity. These studies demonstrate the feasibility of genetic “repair” in mammals.
Alteration of Cell-Surface Proteins by Viral Transformation and by Proteolysis
Authors: Hynes Richard O.
Published: May 2006

Putative cell-surface proteins of tissue-culture cells were identified by lactoperoxidase-catalyzed iodination, a technique that attaches label only to proteins outside the cell membrane. Evidence is presented that these proteins are cell derived, not contaminating serum proteins. On “normal” cells, which exhibit density dependence of growth, one protein of high molecular weight was particularly readily iodinated. This protein was easily removed by mild proteolytic digestion, and, in virus-transformed cells, was either absent or unavailable for iodination. The possible relevance of these observations to the control of growth in cell culture is discussed.
Multiple Forms of Elongation Factor 1 from Calf Brain
Authors: Moon Hong-Mo , Redfield Betty , Millard Sara , Vane Floie , Weissbach Herbert
Published: May 2006

Heavy and light forms of elongation factor 1 (EF-1) from calf brain have been partially purified. The heterogeneous heavy species (EF-1
H
) with molecular weights of 2.5 × 10
5
to over 1 × 10
6
appears to be a complex or aggregate of the light form of the enzyme (EF-1
L
); the latter has a molecular weight of between 50,000 and 60,000. EF-1
H
but not EF-1
L
, contains significant amounts of free and esterified cholesterol. Although EF-1
H
and EF-1
L
are both active in aminoacyl-tRNA binding to ribosomes, EF-1
L
reacts with GTP and aminoacyl-tRNA more efficiently than EF-1
H
.
“Private” Genetic Variants and the Frequency of Mutation Among South American Indians
Authors: Neel James V.
Published: May 2006

Electrophoretic studies were performed on 15 proteins of blood serum and of erythrocytes, from blood specimens from 72 villages of six relatively unacculturated and genetically pure Indian tribes of South America, for a total of 56,237 determinations. At least 10 different “private” variants were encountered, in 131 people. Two previously recognized genetic polymorphisms of these 15 proteins were also encountered. On the assumption that these variants are neutral from the standpoint of natural selection, and that only one-third of amino-acid substitutions in proteins result in electrophoretically detectable variants, the mutation rate is estimated from a formulation of Kimura and Ohta to be about 8 × 10
-5
per locus per generation. The calculation involves several approximations which can be improved by further investigations; if confirmed, then for this class of mutations Indian mutation rates are roughly an order of magnitude higher than commonly envisioned.
The Presence of Ribonucleotides in Mature Closed-Circular Mitochondrial DNA
Authors: Grossman Lawrence I. , Watson Robert , Vinograd Jerome
Published: May 2006

Mitochondrial DNA from mouse and HeLa cells is nicked by alkali and chick-embryo ribonuclease H. It has been concluded that ribonucleotides are present in the closed-circular duplex. A comparative study of the scission rates at pH 13.0 of RNA and of mitochondrial DNAs indicates that there are no more than 10 ribonucleotides in the major fraction of the mitochondrial DNAs.
Both the Fast and Slow Refolding Reactions of Ribonuclease A Yield Native Enzyme
Authors: Garel Jean-Renaud , Baldwin Robert L.
Published: May 2006

The fast reaction (

T̄2

≃ 50 msec) observed previously in the refolding of thermally unfolded ribonuclease A (disulfide bonds intact) has now been studied by two properties indicative of enzyme function: binding of a competitive inhibitor (2′CMP) and hydrolysis of a substrate (CpA → C > p + A). Both the binding and catalytic reactions are fast (<2 msec) compared to refolding. Binding of 2′CMP occurs during both fast and slow refolding reactions, and the protein folded in the fast reaction has a normal binding constant for 2′CMP. Recovery of enzymatic activity during the fast refolding reaction, as measured by the rate of CpA hydrolysis, parallels the kinetic curve for 2′CMP binding. When the kinetics of refolding are measured by the burying of exposed tyrosine groups, no difference is found. The presence of 2′CMP has no effect on the kinetics of refolding.

The results show that the fast refolding reaction does not yield an intermediate in the refolding of RNase A. Instead, both fast and slow refolding reactions have a common product, fully active RNase A. Although they show a 100-fold difference in rates of refolding, the starting materials for the fast and slow refolding reactions have similar properties, as regards: (
a
) the molar absorbancy at 286 nm, reflecting the state of exposed tyrosine groups, and (
b
) their apparent failure to bind 2′CMP.
Direct Evidence of the C-Like Form of Sodium Deoxyribonucleate
Authors: Brahms J. , Pilet J. , Lan Tran-Thi Phuong , Hill L. R.
Published: May 2006

From infrared linear dichroism studies, direct evidence is given for a new Na DNA form, which is similar to the C form of Li DNA. This C-like form in Na DNA occurs under conditions of very low NaCl content (at high relative humidity), high NaCl content (at low relative humidity), and low G + C content of the DNA (at low relative humitiy). The C form of Li DNA was originally discovered by x-ray diffraction, but has not previously been characterized by infrared spectroscopy. The C form is primarily characterized by the orientation of the O2- -O3 line of the phosphate group, which is about 48° (±2°) with respect to the helical DNA axis and which is different from that of the B and A forms. Na DNA molecules rich in A + T may entirely or partially adopt the C form at low relative humidity. Since from infrared evidence, the C-like form is found not only in Li DNA at low relative humidity but also in Na DNA under different conditions, the C form may be biologically important. A possible role for recognition by proteins is suggested.
Production of a Noncovalently Bonded Pentamer of Immunoglobulin M: Relationship to J Chain
Authors: Tomasi T. B
Published: May 2006

Human immunoglobulin M was reduced with concentrations of 2-mercaptoethylamine chosen so that approximately 40% of the immunoglobulin M was reduced to 7S subunits. Under these conditions, which selectively cleave intersubunit disulfides, J chain was released. The 7S subunits of immunoglobulin M so produced did not contain J chain. The high-molecular-weight immunoglobulin M remaining after treatment with 2-mercaptoethylamine had a sedimentation coefficient of 18.0 S and molecular weight of 1 million, and dissociated in 4 M guanidine into subunits similar in size to the 7S subunits. J chain was found in the 18.0S, noncovalently linked immunoglobulin M from which it was released only after more complete reduction with 10 mM dithiothreitol. The results are consistent with the hypothesis that J chain participates in the formation of the intersubunit linkages. However, it need not necessarily be directly involved in all of the intersubunit disulfides. It may play an important role in modulating the assembly of immunoglobulin M subunits, perhaps by inducing conformational changes that lead to noncovalent interactions between the subunits.
Amino-Acid Sequence of Porcine Pepsin
Authors: Tang J. , Sepulveda P. , Marciniszyn J. , Chen K. C. S. , Huang W-Y. , Tao N. , Liu D. , Lanier J. P.
Published: May 2006

As the culmination of several years of experiments, we propose a complete amino-acid sequence for porcine pepsin, an enzyme containing 327 amino-acid residues in a single polypeptide chain. In the sequence determination, the enzyme was treated with cyanogen bromide. Five resulting fragments were purified. The amino-acid sequence of four of the fragments accounted for 290 residues. Because the structure of a 37-residue carboxyl-terminal fragment was already known, it was not studied. The alignment of these fragments was determined from the sequence of methionyl-peptides we had previously reported. We also discovered the locations of activesite aspartyl residues, as well as the pairing of the three disulfide bridges. A minor component of commercial crystalline pepsin was found to contain two extra amino-acid residues, Ala-Leu-, at the amino-terminus of the molecule. This minor component was apparently derived from a different site of cleavage during the activation of porcine pepsinogen.
Circular Dichroism of Chromaffin Granule Proteins
In Situ:
Analysis of Turbidity Effects and Protein Conformation
Authors: Rosenheck Kurt , Schneider Allan S.
Published: May 2006

The circular dichroism spectra for proteins
in situ
in catecholamine secretory vesicles (chromaffin granules) is presented together with an analysis of protein conformation and turbidity effects on the spectra. The calculational analysis has resolved scattering and absorption effects in the turbid suspension spectra using a coated-sphere scattering model which allows for different materials in its shell and core. The intrinsic conformation of the proteins
in situ
was estimated by an iterative procedure with various trial protein conformations, for the chromaffin granules both intact and after release of their contents. The resulting average secondary structures (within about 10%) are: (25% α helix, 15% β structure) for the membrane proteins and (15% α helix, 5% β structure) for the soluble contents. The protein conformation did not change with osmotic release of the granule's contents. Consequently, if chromogranins are involved in a catecholamine storage complex, this is not reflected in any detectable change in their average secondary structure.
In Vitro
Binding of Retinol to Rat-Tissue Components
Authors: Bashor Mark M. , Toft David O. , Chytil Frank
Published: May 2006

The high-speed supernatant fraction of rat liver, lung, kidney, testis, and intestinal mucosa contains a component capable of binding [
3
H]retinol
in vitro
when binding is analyzed by sucrose density gradient centrifugation or gel filtration. This binding component can be distinguished from one identified in rat serum. Whereas the tissue component sediments in the 2S region of sucrose gradients, the serum component sediments in the 4.6S region. Molecular weight estimations by gel filtration indicate molecular weights of 16,000 and 67,000 for the tissue and serum binding components, respectively. Unlabeled retinol, but not retinoic acid, competes for the binding of [
3
H]retinol in tissue cytosols. Competition for the binding of [
3
H]retinol by unlabeled retinal has also been observed in tissue cytosols, but may result from the
in vitro
reduction of retinal to retinol. Unlabeled retinol, retinal, and retinoic acid fail to compete for the binding of [
3
H]retinol in serum under the conditions used. The tissue binding component (testis) is sensitive to digestion with Pronase, but not with RNase or DNase, indicating a protein nature for this component.
In Vitro
Transcription of the Viral-Specific Sequences Presnet in the Chromatin of Cells Transformed by Simian Virus 40
Authors: Shih Thomas Y. , Khoury George , Martin Malcolm A.
Published: May 2006

Separated strands of simian virus 40 (SV40) DNA fragments were used in hybridization experiments to study the RNA transcribed by
Escherichia coli
RNA polymerase from the chromatin of cells transformed by SV40. The template activity of chromatin of the transformed cell line 11A8 (mouse-embryo cells) examined is about 17% that of purified DNA, suggesting that most of the chromatin DNA is repressed by chromosomal proteins. The SV40-specific RNA present in the RNA transcribed
in vitro
from 11A8 chromatin hybridizes specifically with the minus strand of SV40 DNA. Little or no reaction occurs with the plus strand of viral DNA. The SV40-specific RNA transcribed
in vitro
from chromatin of transformed cells shares sequences with the RNA produced during the early phase of SV40 lytic infection, and is similar to that present in the 11A8 cell line
in vivo
. Although the influence of chromosomal proteins on this pattern of transcription was not definitely determined, preliminary evidence indicates that an asymmetric pattern of transcription may also occur when 11A8 DNA is transcribed by
E. coli
RNA polymerase.
Regulation of Nucleoside Cyclic 3′:5′-Monophosphate Phosphodiesterase Activity from Rat Brain by a Modulator and Ca
2+
Authors: Kakiuchi Shiro , Yamazaki Reiko , Teshima Yoshiko , Uenishi Kunihiro
Published: May 2006

Gel filtration of the 40,000 rpm supernatant fraction of a homogenate of rat cerebral cortex on a Sepharose 6B column yielded two fractions: fraction II with the “Ca
2+
plus Mg
2+
-dependent” phosphodiesterase activity and fraction III containing its modulator. The activity of fraction II was stimulated by micromolar concentrations of Ca
2+
and the modulator when present together; the modulator stimulated the activity of fraction II only when the Ca
2+
concentration was above a threshold value (about 2 μM with 0.4-1 μM substrate), and the stimulatory effect of Ca
2+
was dependent upon the presence of the modulator. A possibility is discussed that the modulator may reversibly bind to the enzyme, which by itself is inactive, to form an active enzyme-modulator complex and that Ca
2+
stimulates the activity of phosphodiesterase by shifting the equilibrium between these three species towards the formation of the active enzyme-modulator complex. Although fraction II hydrolyzed both cyclic AMP and cyclic GMP, hydrolysis of the latter was more significantly influenced by Ca
2+
and the modulator than that of the former, and the “Ca
2+
plus Mg
2+
-dependent” phosphodiesterase is likely to be a cyclic GMP enzyme. This conclusion is based on the following evidence: (
a
) Ca
2+
stimulated hydrolysis of cyclic GMP by fraction II more than that of cyclic AMP. (
b
) In the presence of Ca
2+
and the modulator, fraction II hydrolyzed cyclic GMP about 8 times faster than cyclic AMP when incubated with 0.4 μM substrate. (
c
) Half-maximal stimulation of hydrolysis of cyclic GMP was attained at a lower concentration of Ca
2+
(4 μM) than that of cAMP (8 μM). (
d
) Increase in the concentration of Ca
2+
from 0.06 μM to 12 μM in the presence of the modulator caused a decrease in the
K

m

value of cyclic GMP hydrolysis by fraction II from 20 μM to 2 μM accompanied by 4-fold increase in the
V
max
value. Under similar conditions, there was only a slight decrease in the
K

m

value of cylic AMP hydrolysis (90 μM → 50 μM), although the
V
max
value increased 7-fold. The anomalous shape of the kinetic plot of cyclic GMP hydrolysis became linear when the Ca
2+
concentration was increased in the presence of the modulator. The modulator seems to be a protein, but it is heat stable. It is probably identical to the protein activator of phosphodiesterase first described by Cheung.
Structure of Hemoglobin S Fibers: Optical Determination of the Molecular Orientation in Sickled Erythrocytes
Authors: Hofrichter James , Hendricker David G. , Eaton William A.
Published: May 2006

Possible orientations of deoxyhemoglobin S molecules within sickle-cell fibers are delimited by polarized absorption measurements on single sickled cells and single crystals of deoxyhemoglobin A. The polarization ratio of cells provides a lower limit for that of an individual fiber and, coupled with the absorption properties of the deoxyhemoglobin molecule, restricts the orientation of the long molecular (
x
) axis to within 22° of the fiber axis. Adopting the stacked ring model of Finch
et al.
for the molecular positions and the additional constraint that at least one mutated (β6) site is part of an intermolecular contact, our optical result requires that the true molecular dyad (
y
) axis pass through some part of an adjacent molecule in the same ring. This range of orientations for the
y
axis is approximately perpendicular to those described in existing models and places at least one β6 residue in position to be part of a contact between molecules in the same ring.
Changes in Rabbit-Liver Lysosomes and Fructose 1,6-Bisphosphatase Induced by Cold and Fasting
Authors: Pontremoli S. , Melloni E. , Salamino F. , Franzi A. T. , Flora A. De. , Horecker B. L.
Published: May 2006

Exposure of rabbits to cold or fasting results in marked increases in the number and size of liver lysosomes, associated with increases in the total and “free” proteolytic activity. Changes in lysosomal morphology also indicate increased activity and fragility. These effects of cold and fasting are correlated with decreases in the levels of liver fructose bisphosphatase (EC 3.1.3.11) activity and changes in the molecular properties of the purified enzyme, including the loss of a small peptide containing the only tryptophan residue in the peptide chain.
DNA Polymerase III Star Requires ATP to Start Synthesis on a Primed DNA
Authors: Wickner William , Kornberg Arthur
Published: May 2006

DNA polymerase III star replicates a ϕX174 single-stranded, circular DNA primed with a fragment of RNA. This reaction proceeds in two stages. In stage I, a complex is formed requiring DNA polymerase III star, ATP, spermidine, copolymerase III
*
, and RNA-primed ϕX174 single-stranded, circular DNA. The complex, isolated by gel filtration, contains ADP and inorganic phosphate (the products of a specific ATP cleavage) as well as spermidine, polymerase III star, and copolymerase III star. In stage II, the chain grows upon addition of deoxynucleoside triphosphates; ADP and inorganic phosphate are discharged and chain elongation is resistant to antibody to copolymerase III star. Thus ATP and copolymerase III star are required to initiate chain growth but not to sustain it.
Characterization of a Feedback-Resistant Phosphoribosylpyrophosphate Synthetase from Cultured, Mutagenized Hepatoma Cells That Overproduce Purines
Authors: Green Christopher D. , Martin David W.
Published: May 2006

A clone of cells in which the regulation of purine metabolism is genetically altered was selected and isolated from chemically mutagenized HTC cells (a line of rat hepatoma cells in continuous culture). The clone, designated MAU V, was selected for increased ability to salvage exogenous purines by isolating it in medium containing methylmercaptopurine ribonucleoside, adenine, and uridine, in which medium wild-type cells cannot divide. We have characterized these cells as having an increased rate of
de novo
purine biosynthesis, apparently as the result of an altered phosphoribosylpyrophosphate (PRPP) synthetase. The altered enzyme has normal catalytic properties but an altered sensitivity to feedback inhibition by purine and pyrimidine nucleotides. The types of inhibitions (competitive and uncompetitive) exerted by AMP, ADP, and TDP on the wild-type enzyme have been maintained in the altered enzyme, but values for

K
i

have been increased by factors of 10, 17.5, and 5, respectively. The specific catalytic activities of AMP: pyrophosphate phosphoribosyltransferase and IMP:pyrophosphate phosphoribosyltransferase are normal. The mutant cell may serve as a model for a specific human disease, one type of dominantly inherited overproduction hyperuricemia.
Detection of a Free Radical in the Primary Reaction of Chloroplast Photosystem II
Authors: Malkin Richard , Bearden Alan J.
Published: May 2006

Electron paramagnetic resonance (EPR) spectroscopy has revealed a new free-radical signal produced by illumination of spinach chloroplasts at 77°K. This signal is observed only when an oxidant (ferricyanide) is added to the chloroplast suspension in the dark before illumination. The EPR signal is produced at 77°K by illumination with 645-nm monochromatic light capable of activating Photosystem II but not with 715-nm illumination capable of activating Photosystem I. Furthermore, since the signal shows a relative increase in chloroplast fragments enriched in Photosystem II but is absent in chloroplast fragments enriched in Photosystem I, we conclude that this new EPR signal is associated with the primary photoact of Photosystem II in chloroplasts. On the basis of the measured EPR parameters (
g
= 2.0026 ± 0.0002, linewidth = 8 G), it is suggested that the signal may be associated with the reaction-center chlorophyll of Photosystem II.
A Stable Solid Model for Protein Sulfenyl Iodides
Authors: Field Lamar , White Jerry E.
Published: May 2006

The first simple sulfenyl iodide that is a pure stable crystalline solid is reported. This product, ISCMe
2
CH(NHCbz)CONH-Ph-
p
-Cl(10), contains linkages that simulate the two peptide linkages vicinal to the -SI moiety in a protein sulfenyl iodide. Unlike typical simple sulfenyl iodides, solid 10 remains unchanged at ambient conditions for more than ten weeks; however, it is quite reactive in solution. Under proper circumstances, therefore, a sulfenyl iodide is proved to be a stable but highly reactive species. The model 10 and related compounds should afford prospects for better understanding of the unusual stabilities of certain protein sulfenyl iodides and of ways in which other such compounds may function in mediating iodination reactions in the thyroid gland.
Expression of Differentiated Functions in Hepatoma Cell Hybrids: Alanine Aminotransferase
Authors: Sparkes Robert S. , Weiss Mary C.
Published: May 2006

The expression of alanine aminotransferase (EC 2.6.1.2), an enzyme that is inducible in the liver, has been examined in somatic hybrid cells formed by crossing well-differentiated rat hepatoma cells with rat diploid epithelial cells, the former characterized by high activity and inducibility of the enzyme, and the latter by the absence of detectable activity. The hybrid cells that contain essentially complete chromosomal sets of the two parents show only very low activity and little inducibility. Among numerous “segregated” hybrid subclones, which have lost up to 40% of the chromosomes initially present, several show expression of intermediate levels of enzyme activity and very little inducibility, and two independent subclones are characterized by full re-expression of both baseline and inducible enzyme activity. The electrophoretic mobility of the enzyme from the latter hybrids, from the hepatoma parental cells, and from rat liver is identical. The absence of a correlation between total chromosome number of the hybrid cells and re-expression of alanine aminotransferase suggests that the loss of specific chromosomes is required for re-expression. In these hybrid cells, the re-expression of alanine aminotransferase baseline and inducibility is independent of that of tyrosine aminotransferase inducibility.
Effect of Stimulation of the Host Defense System by Coenzyme Q
10
on Dibenzpyrene-Induced Tumors and Infection with Friend Leukemia Virus in Mice
Authors: Bliznakov Emile G.
Published: May 2006

Members of the coenzyme Q group increase the phagocytic activity in rats, as measured by the carbon clearance technique, and increase the hemolytic antibody formation in mice. In addition, prior treatment with low doses of chloroquine hydrochloride combined with coenzyme Q
10
results in increased numbers of survivors, prolonged survival time, and reduced parasitemia in blood-transferred
Plasmodium berghei
infection in mice

In an extension of these studies, using emulsions of coenzyme Q
10
, I demonstrated the following effects on two tumor systems in mice: (
i
) Treatment with coenzyme Q
10
decreased splenomegaly and hepatomegaly and increased the number of surviving mice infected with Friend leukemia virus. (
ii
) Treatment with coenzyme Q
10
reduced the percentage of mice with tumors, increased the number of survivors, and reduced the tumor size in mice with tumors induced by 3,4,9,10-dibenzpyrene. The effect on both tumor systems was dose-dependent. These studies support the hypothesis that the host defense system plays a definitive role in the defense of the host against invasion by various agents, including neoplasia.
RNA-Linked Short DNA Fragments During Polyoma Replication
Authors: Magnusson Göran , Pigiet Vincent , Winnacker Ernst L. , Abrams Richard , Reichard Peter
Published: May 2006

During
in vitro
incubation, nuclei from polyoma-infected cells elongate the daughter strands of the replicative intermediate of polyoma DNA. This process is now shown to involve the transient formation of short fragments (4-5 S), a process that is stimulated by the addition of ribonucleoside triphosphates. The presence of stretches of RNA at the 5′-end of short DNA chains was determined from Cs
2
SO
4
equilibrium centrifugation and from the finding that isotope from α-
32
P-labeled deoxynucleoside triphosphates was recovered in 2′(3′)-ribonucleotides after alkaline hydrolysis. Transfer occurred preferentially with [α-
32
P]dCTP as substrate. Starvation for deoxynucleotides by
in vivo
treatment with hydroxyurea resulted in the accumulation of short fragments that are deficient in RNA. Our results suggest that a late step during the discontinuous synthesis of polyoma DNA is selectively inhibited when deoxynucleotides are in short supply.
Evidence for Boundary Lipid in Membranes
Authors: Jost Patricia C. , Griffith O. Hayes , Capaldi Roderick A. , Vanderkooi Garret
Published: May 2006

Cytochrome oxidase (EC 1.9.3.1) isolated from beef-heart mitochondria with an appropriate phospholipid content forms vesicular structures. Lipid-protein interactions in this model membrane system were studied with the lipid spin label, 16-doxylstearic acid. As the phospholipid/protein ratio is varied, two spectral components are observed. At low phospholipid/protein ratios (≤0.19 mg of phospholipid per mg of protein) the lipid spin label is highly immobilized. At higher phospholipid content an additional component characteristic of fluid lipid bilayers is evident. By summation of digitalized spectra and subsequent integration it was shown that all composite spectra could be approximated by assuming only two components are present, and that the amount of phospholipid bound to the protein is independent of the extent of the fluid bilayer region. The experimentally determined amount of phospholipid for maximum occupancy of protein-bound sites is about 0.2 mg of phospholipid per 1.0 mg of protein. Calculations show that this ratio is consistent with a single layer of phospholipid surrounding the protein complex. The data are interpreted as evidence for a boundary of immobilized lipid between the hydrophobic protein and adjacent fluid bilayer regions in this membrane model system.
Isolation of a New RNA Polymerase-Binding Protein from Sporulating
Bacillus subtilis
Authors: Greenleaf Arno L. , Linn Thomas G. , Losick Richard
Published: May 2006

RNA polymerase was precipitated from extracts of radioactively labeled vegetative and sporulating
Bacillus subtilis
with antiserum prepared against vegetative core polymerase. The precipitates were solubilized and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antiserum added to an extract of vegetative
B. subtilis
precipitated only the known subunits of core RNA polymerase, but antiserum added to an extract of sporulating cells precipitated a new polypeptide of 70,000 daltons in addition to the subunits of core enzyme. The 70,000-dalton polypeptide precipitated from an extract of a mixture of vegetative and sporulating
B. subtilis
, separately labeled with two different radioisotopes, contained only the radioisotope characteristic of the sporulating cells.

The 70,000-dalton protein has been freed of core RNA polymerase and extensively purified by chromatography on phosphocellulose. Precipitation of the purified 70,000-dalton protein by the anti-polymerase serum requires the prior addition of vegetative or sporulation core RNA polymerase. The reaction is specific since the purified protein is not precipitated during antibody precipitation of either phage λ repressor or bovine serum albumin.

The RNA polymerase-binding protein appears during the third hour of sporulation and is apparently not synthesized by the sporulation-defective mutant
rfr
10.
Immunoglobulin Synthesis and Secretion by Cells in the Mouse Thymus That Do Not Bear θ Antigen
Authors: Vitetta Ellen S. , Uhr Jonathan W. , Boyse Edward A.
Published: May 2006

The mouse thymus contains cells that synthesize and secrete minute amounts of immunoglobulin (Ig). These cells were studied by a combination of cytotoxic tests (with alloantisera prepared in congenic strains of mice) followed by pulse labeling of the surviving cell population with [
3
H]tyrosine. The immunoglobulin-synthesizing cells constitute less than 2% of the total cell population and are θ
-
, TL
-
, Ly-B
-
, Ig
+
, H-2
+
, and PC
+
. Since this phenotype is not characteristic of cells of the thymocyte lineage, the Ig
+
cells are probably plasma cells and/or bone marrow-derived (B) lymphocytes.

Similar studies of spleen cells indicate that immunoglobulin-synthesizing and secreting cells are θ
-
.
Inactive Enzyme Molecules in Aging Mice: Liver Aldolase
Authors: Gershon Harriet , Gershon David
Published: May 2006

Evidence is presented that there is a considerable accumulation of inactive fructose-1,6-diphosphate aldolase (EC 4.1.2.7) in the liver of senescent mice. Liver aldolase was purified from 3-month-old mice and used to immunize rabbits. It was demonstrated with the monospecific antibody thus produced that the liver aldolase of young adult (3 month) and aged (31 month) mice are antigenically identical. With the antibody, inactive enzyme molecules (crossreacting material) in liver homogenate of old mice were detected. The liver aldolase of senescent mice had half as much active enzyme per mg of protein, as well as per antigenic unit, as did the liver aldolase of young adult mice. The accumulation of faulty enzyme molecules may be one of the causes of debilitation leading to senescence and death.
Development of Locomotor Patterns in the Absence of Peripheral Sense Organs and Muscles
Authors: Davis William J.
Published: May 2006

The role of peripheral sense organs and muscles in specifying the circuitry of the central nervous system during ontogeny was tested in larval lobsters. Presumptive locomotor appendages, the abdominal swimmerets, were extirpated before their differentiation. Electrophysiological recordings made 2-4 weeks later from the corresponding motor nerves showed that, despite the absence of the target muscles and sense organs, normal reflexes and normal patterns of rhythmic locomotor output appeared in the swimmeret motoneurons at the usual developmental stage. Therefore, target muscles and sense organs are unnecessary to the differentiation of normal motor output patterns in this simple invertebrate locomotor system.