bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction of a general vector for efficient expression of mammalian proteins in bacteria: use of a synthetic ribosome binding site.
Authors: Jay G , Khoury G , Seth A K , Jay E
Published: May 2006

With the premise that mRNAs transcribed in Escherichia coli from cloned eukaryotic DNA inserts do not possess the necessary regulatory signals for recognition by prokaryotic ribosomes, we have constructed a general plasmid vector carrying a chemically synthesized prokaryotic ribosome binding site that will ensure the efficient expression of eukaryotic proteins in E. coli. In addition to the regulatory signals necessary for ribosome recognition, the synthetic segment contains, at one end, a Pst I cleavage site which will direct its insertion to pBR322 DNA and, at the other end, a HindIII site to facilitate attachment of the passenger eukaryotic gene. Using simian virus 40 (SV40) tumor (t) antigen as a model system, we have ligated the SV40 DNA fragment containing the entire t antigen gene in tandem with the synthetic ribosome binding site to pBR322 DNA at the Pst I site, which lies within the coding sequence of the beta-lactamase gene. Initiation of transcription at the beta-lactamase promoter would produce a chimeric mRNA with the synthetic ribosome binding signals and the SV40 sequence flanked by beta-lactamase coding sequences. Utilization of the synthetic regulatory signals for initiation of translation is demonstrated by the efficient synthesis, in bacterial transformants, of authentic SV40 t antigen. Excision of the entire SV40 insert by HindIII from those clones that have retained intact HindIII sites at the junction between the ribosome binding site and the SV40 sequence would allow insertion of other heterologous DNAs by using HindIII linkers. The efficient expression of any DNA insert would require that the entire coding sequence be contiguous and that its termini be randomized by treatment with exonuclease III and nuclease S1 to vary the distance between the translational initiation codon and the synthetic ribosome binding site.
Rapid clonal growth and serial passage of human diploid fibroblasts in a lipid-enriched synthetic medium supplemented with epidermal growth factor, insulin, and dexamethasone.
Authors: Bettger W J , Boyce S T , Walthall B J , Ham R G
Published: May 2006

A serum-free, hormone-supplemented medium, enriched with a mixture of lipids, has been developed that supports rapid clonal growth of human diploid fetal lung fibroblasts (Flow 2000, WI-38, MRC-5, and IMR-90) and of low-passage human foreskin fibroblasts. The medium, which contains less than 1 microgram of total protein per ml, also supports serial passage of Flow 2000 cells under totally serum-free conditions. It provides lipid at a total of 10 micrograms/ml as a liposome prepared from a mixture of soybean lecithin, cholesterol, sphingomyelin, vitamin E, and vitamin E acetate. The soybean lecithin, which contains a variety of naturally occurring phospholipids, can be replaced with a mixture of highly purified phospholipids. Except for possible contaminants in the substances used in its preparation, the serum-free medium is fully defined chemically. It consists of an optimized basal nutrient medium, MCDB 110, supplemented with insulin, epidermal growth factor, dexamethasone, prostaglandins E1 and F2 alpha, phosphoenolpyruvate, dithiothreitol, glutathione, and the lipids listed above.
Transcriptional and translational control of cytoplasmic proteins after serum stimulation of quiescent Swiss 3T3 cells.
Authors: Thomas G , Thomas G , Luther H
Published: May 2006

The synthesis of cytoplasmic proteins from quiescent and serum-stimulated Swiss 3T3 cells was compared by two-dimensional polyacrylamide gel electrophoresis. Four new proteins of Mrs 26,000, 28,000, 45,000, and 47,000 designated N26, N28, N45, and N47, which were not detectable in quiescent cells, appeared 60 min after addition of serum. During the same period, the amount of [35S]methionine incorporated into 10 proteins present in quiescent cells, ranging in Mr from 23,000 to 98,000 and designated Q23-98, increased up to 6-fold, whereas the amount incorporated into three other proteins decreased by a factor of approximately 2. Of the new proteins, N26 was no longer detectable, and the amount of [35S]methionine incorporated into N47 was significantly reduced by 150 min. During this same time, a fifth new protein, N56, appeared, and there was a large increase in the amount of radioactivity incorporated into another protein, Q121. The increases in nine of the proteins were either strongly or completely inhibited by actinomycin D, arguing that the expression of these proteins was under transcriptional control. In contrast, the increases in seven other proteins were unaffected by actinomycin D, suggesting that their expression was under translational control. These proteins will serve as useful markers for determining how cells progress through early lag phase.
Mitotic chromosome loss in a radiation-sensitive strain of the yeast Saccharomyces cerevisiae.
Authors: Mortimer R K , Contopoulou R , Schild D
Published: May 2006

Cells of Saccharomyces cerevisiae with mutations in the RAD52 gene have previously been shown to be defective in meiotic and mitotic recombination, in sporulation, and in repair of radiation-induced damage to DNA. In this study we show that diploid cells homozygous for rad52 lose chromosomes at high frequencies and that these frequencies of loss can be increased dramatically by exposure of these cells to x-rays. Genetic analyses of survivors of x-ray treatment demonstrate that chromosome loss events result in the conversion of diploid cells to cells with near-haploid chromosome numbers.
Thiamine as an integral component of brain synaptosomal membranes.
Authors: Matsuda T , Cooper J R
Published: May 2006

Synaptic plasma membranes were prepared from rat cerebral cortex to determine if thiamine was localized in the membranes. The synaptosomes, prepared by discontinuous sucrose gradient centrifugation, were subjected to osmotic shock at pH 9.5 for 10--15 min and subfractionated on a discontinuous sucrose gradient. The two membrane fractions that were obtained were free of contamination by mitochondrial membrane and soluble fractions. In order to ensure specificity, thiamine was assayed fluorometrically before and after the addition of thiaminase I (thiamin:base 2-methyl-4-aminopyrimidine-5-methenyltransferase, EC 2.5.1.2). The thiamine content of the two membrane fractions was 9 and 10 pmol/mg protein.
Novel sensory adaptation mechanism in bacterial chemotaxis to oxygen and phosphotransferase substrates.
Authors: Niwano M , Taylor B L
Published: May 2006

The involvement of methylation in the chemosensory response of bacteria to many attractants has been clearly established by studies in several laboratories. It has been assumed that adaptation of Salmonella typhimurium and Escherichia coli to all attractants involves methylation of a transmembrane methyl-accepting chemotaxis protein. The methyl donor in this reaction is S-adenosyl-L-methionine, and the protein methyltransferase is the product of the cheR gene. In contrast, adaptation to oxygen and phosphotransferase substrates were found to be independent of this methylation system. In E. coli AW660 (tsr tar trg), which lacks the known methyl-accepting chemotaxis proteins, chemotaxis was normal to oxygen and to substrates of the phosphotransferase system such as D-mannose, D-glucose, and N-acetyl-D-glucosamine. When S-adenosyl-L-methionine was depleted by methionine starvation or by addition of 1-aminocyclopentane-1-carboxylic acid, methylation-dependent adaptation to serine, aspartate, and ribose was defective in wild-type E. coli and S. typhimurium. However, adaptation to oxygen and phosphotransferase substrates was independent of S-adenosyl-L-methionine and the cheR product. These results suggest that there are methylation-independent and methylation-dependent mechanisms for sensory adaptation in bacteria.
A domain of clathrin that forms coats.
Authors: Schmid S L , Matsumoto A K , Rothman J E
Published: May 2006

Triskelions, trimeric complexes of clathrin and associated light chains, are the assembly units of clathrin coats [Ungewickell, E. & Branton, D. (1981) Nature (London) 289, 420-422; Kirchhausen, T. & Harrison, S. C. (1981) Cell 23, 755-761]. We report here that triskelions whose outer arms have been removed by trypsin digestion retain the ability to be assembled into coats. These digested timers contain a 110,000 molecular weight domain of clathrin and lack intact light chains.
Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.
Authors: Leppla S H
Published: May 2006

Anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins individually cause no known physiological effects in animals but in pairs produce two toxic actions. Injection of PA with LF causes death of rats in 60 min, whereas PA with EF causes edema in the skin of rabbits and guinea pigs. The mechanisms of action of these proteins have not been determined. It is shown here that EF is an adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] produced by Bacillus anthracis in an inactive form. Activation occurs upon contact with a heat-stable eukaryotic cell material. The specific activity of the resulting adenylate cyclase nearly equals that of the most active known cyclase. In Chinese hamster ovary cells exposed to PA and EF, cAMP concentrations increase without a lag to values about 200-fold above normal, remain high in the continued presence of toxin, and decrease rapidly after its removal. The increase in cAMP is completely blocked by excess LF. It is suggested that PA interacts with cells to form a receptor system by which EF and perhaps LF gain access to the cytoplasm.
Characterization of external electric field-driven ATP synthesis in chloroplasts
Authors: Vinkler Chana , Korenstein Rafi
Published: May 2006

External electric field-induced ADP phosphorylation (EFP) in lettuce chloroplasts was monitored with a coupled luciferin-luciferase enzymatic assay. This assay made it possible to follow ATP synthesis in a kinetically competent manner. The EFP reaction was found to be a much slower process than the light-driven reaction in the same system. The amount of ATP synthesized after a single electric field pulse corresponds to many turnovers of the ATP synthase.
gamma Heavy chain disease in man: cDNA sequence supports partial gene deletion model.
Authors: Alexander A , Steinmetz M , Barritault D , Frangione B , Franklin E C , Hood L , Buxbaum J N
Published: May 2006

Human gamma heavy chain disease (HCD) is characterized by the presence in serum of a short monoclonal Ig gamma chain unattached to light chains. Although most HCD proteins have internal deletions, in some the defect is NH2-terminal. The OMM gamma 3 HCD serum protein is of the latter type, having undergone an extensive NH2-terminal deletion with a sequence starting within the hinge. A cell line synthesizing the OMM protein has enabled us to study the biogenesis of the abnormal molecule. In vitro translation of isolated mRNA yields a protein containing a hydrophobic NH2-terminal leader sequence. In the intact cell, the precursor molecule is processed normally to yield a protein with an NH2-terminal sequence homologous to the beginning of the variable (V) region. The nucleotide sequence of cDNA prepared from the OMM mRNA encodes a 19-amino acid leader followed by the first 15 residues of the V region. An extensive internal deletion encompasses the remainder of the V and the entire CH1 domain. Immediately following the short V region, there is information in the cDNA for the entire normal hinge. The primary synthetic product is thus an internally deleted molecule that undergoes postsynthetic degradation to yield the NH2-terminally deleted serum protein. The structure of the OMM mRNA suggests that the protein abnormality results from a partial gene deletion rather than defective splicing.
Threshold for lead damage to heme synthesis in urban children.
Authors: Piomelli S , Seaman C , Zullow D , Curran A , Davidow B
Published: May 2006

Although lead has no physiological function and is present in only negligible amounts in the blood of remote populations, it has become customary to accept the usual blood Pb level(s) (BPb) observed in industrialized society as "normal." Pb interferes with many biochemical systems, among them the heme biosynthetic pathway; this is reflected by an exponential increase in erythrocyte protoporphyrin concentration (EP) as PBb increases. The present study estimated the threshold PBb at which an increase of EP occurs in a population of urban children. In the 2,004 children studied, BPbs ranged from 2 to 98 micrograms/dl, with 1,852 having a BPb of less than or equal to 30 micrograms/dl, a value presently considered normal. Preliminary analysis suggested that an exponential increase in the concentration of EP occurred after a threshold BPb (apparently between 12 and 20 micrograms/dl) was reached. Precise definition of the threshold BPb for an increase of EP was next determined by two approaches: segmented line techniques and probit analysis. Whether the entire population was analyzed or only the subset of samples with "normal" BPb (less than or equal to 30 micrograms/dl), both methods yielded a threshold BPb of 15-18 micrograms/dl (average value, 16.5). These studies indicate that the heme synthetic pathway is affected by Pb at a level of exposure commonly observed in urban children, which is well below the limit that is presently too easily accepted as normal.
Temperature dependence of the reduction potential of blue copper in fungal laccase
Authors: Taniguchi Vernon T. , Malmström Bo G. , Anson Fred C. , Gray Harry B.
Published: May 2006

Thin-layer spectroelectrochemical methods have been employed to measure the reduction potentials of the blue copper in
Polyporus versicolor
laccase (EC 1.10.3.2) between 7°C and 41°C (0.2 M sodium phosphate, pH 5.4). Thermodynamic parameters are: Δ
S
° = -13.9 ± 2 cal/mol-K; Δ
H
° = -22.1 ± 0.5 kcal/mol;
E
° (25°C) = 780 ± 3 mV vs. the normal hydrogen electrode. Comparison of the Δ
S
° and Δ
H
° values with those for single-site proteins suggests that the high potential of the blue copper in fungal laccase is attributable mainly to stabilization of the copper (I) center by enhanced ligand binding interactions and that protein solvation effects play a lesser role.
Human papilloma viral DNA replicates as a stable episome in cultured epidermal keratinocytes.
Authors: LaPorta R F , Taichman L B
Published: May 2006

Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.
Prediction of the three-dimensional structure of the leader sequence of pre-kappa light chain, a hexadecapeptide.
Authors: Pincus M R , Klausner R D
Published: May 2006

The three-dimensional structure of the signal sequence for murine kappa light chain has been calculated by using conformational energy calculations. These calculations, based on tested and reliable potential energy functions, employ a novel global search technique to identify the lowest energy structures for the hexadecapeptide signal sequence, Glu-Thr-Asp-Thr-(Leu3-Trp-Val)2-Pro-Gly.l It has been found that the core hydrophobic sequence, Leu3-Trp-Val-Leu3, adopts an alpha-helical conformation that is terminated by chain reversal conformations for the four residues, Trp-Val-Pro-Gly. The amino-terminal four residues adopt low energy conformations that are fully compatible with the succeeding alpha-helix. The immediately neighboring sequence., Asp-Thr, exists in a single lowest energy double-equatorial conformation, whereas the first two residues, Glu-Thr, can adopt a variety of low energy conformations. The calculations arrive at a highly structured and specific model for the conformation of a leader sequence, compatible with recent experimental data.
Poly(ADP-ribosyl)ation of polynucleosomes causes relaxation of chromatin structure.
Authors: Poirier G G , de Murcia G , Jongstra-Bilen J , Niedergang C , Mandel P
Published: May 2006

When rat pancreatic polynucleosomes were poly(ADP-ribosyl)ated with purified calf thymus poly(ADP-ribose) polymerase and examined by electron microscopy, a relaxation of their native zigzag structure was observed. At high ionic strengths control nucleosomes condensed into 250-A-thick fibers, but poly(ADP-ribosyl)ated polynucleosomes did not; they showed a close resemblance to chromatin depleted of histone H1. The relaxed state of poly(ADP-ribosyl)ated polynucleosomes was also confirmed by sedimentation velocity analysis. Histone H1 was found to be the major histone acceptor of poly(ADP-ribose). Poly(ADP-ribose) linked to histone H1 did not seem to cause its dissociation from the chromatin, but it impaired significantly its effect on chromatin condensation.
Binding to selected regions of reovirus mRNAs by a nonstructural reovirus protein.
Authors: Stamatos N M , Gomatos P J
Published: May 2006

When assembled into 13--19S particles, the reovirus nonstructural protein sigma-NS selectively binds single-stranded RNAs. Sedimentation analyses combined with binding to nitrocellulose membrane filters showed that 1--2 pmol of reovirus mRNAs from the large, medium, or small size classes saturated in vitro the binding site(s) on 13--19S particles containing 100 pmol of sigma-NS. All mRNA segments in each size class bound to particles, and no mRNAs in one size class excluded the binding of mRNAs in any other class. In competition experiments, the maximal binding of all reovirus mRNAs to particles of sigma-NS was achieved when medium and small mRNAs were bound before the large mRNAs. This preferred order of addition of mRNAs to sigma-NS resulted in a marked increase in the size of some of the complexes. This finding suggests that the addition of large mRNAs last to particles promoted the formation of complexes with more than one RNA segment bound per particle. The 13--19S particles of sigma-NS protected 20- to 40-nucleotide RNA fragments from nuclease digestion. At least one of the protected fragments from mRNAs of each size class included the 3' terminus; the remaining were from internal regions of the mRNAs. The protected RNA fragments rebound to particles during a second or third cycle of binding in a configuration in which they were fully protected from nuclease digestion. We conclude that binding of particles of sigma-NS to reovirus mRNAs was not at random sites but was to specific regions unique for members of each size class.
Dose--response curve for ethylnitrosourea-induced specific-locus mutations in mouse spermatogonia.
Authors: Russell W L , Hunsicker P R , Raymer G D , Steele M H , Stelzner K F , Thompson H M
Published: May 2006

The extreme mutagenic effectiveness of N-ethyl-N-nitrosourea in the mouse has permitted the accumulation of the most extensive dose--response data yet obtained for chemical induction of specific-locus mutations in spermatogonia. In the lower portion of the curve, below a dose of 100 mg/kg, the data fall statistically significantly below a maximum likelihood fit to a straight line. Independent evidence indicates that, over this dose range, ethylnitrosourea reaches the testis in amounts directly proportional to the injected dose. It is concluded that, despite the mutagenic effectiveness of ethylnitrosourea, the spermatogonia are apparently capable of repairing at least a major part of the mutational damage when the repair process is not swamped by a high dose. This finding is important both in basic studied on the mutagenic action of chemicals in mammals and in risk estimation.
Hepatic uptake of chylomicron remnants in WHHL rabbits: a mechanism genetically distinct from the low density lipoprotein receptor.
Authors: Kita T , Goldstein J L , Brown M S , Watanabe Y , Hornick C A , Havel R J
Published: May 2006

Homozygous Watanabe hereditary hyperlipidemic (WHHL) rabbits have a near-complete deficiency of low density lipoprotein (LDL) receptors in liver and other tissues. As a result, these rabbits clear LDL from plasma at an abnormally slow rate. In the current studies we show that WHHL rabbits clear chylomicrons from plasma at a normal rate. Chylomicrons are cleared by a two-step process: (i) hydrolysis of triglycerides in extrahepatic tissues to yield cholesteryl ester-rich remnant particles and (ii) rapid uptake of the remnants by liver. Normal and WHHL rabbits were given intravenous injections of rat chylomicrons labeled either in the lipid portion with [3H]cholesterol and [14C]palmitate or in the protein portion with [125]iodine. All radiolabeled components were removed from plasma at comparable rates in normal and WHHL rabbits. Comparable amounts of radioactivity accumulated in livers of animals from both genotypes. In vitro assays showed that liver membranes from WHHL rabbits were markedly deficient in the binding of 125I-labeled chylomicron remnants as well as 125I-labeled LDL, implying that chylomicron remnants can bind to the hepatic LDL receptor. We conclude that the rabbit liver normally has at least two genetically distinct lipoprotein uptake mechanisms, both of which recognize chylomicron remnants: (i) the LDL receptor and (ii) a specific chylomicron remnant uptake mechanism that is not measured adequately by current in vitro membrane binding assays. WHHL rabbits possess a normal chylomicron remnant uptake mechanism that allows them to clear chylomicrons from plasma at a rapid rate despite their genetic deficiency of LDL receptors.
Phenotyping of cytochromes
P
-450 in human tissues with monoclonal antibodies
Authors: Fujino Tadahiko , Park Sang Shin , West Donna , Gelboin Harry V.
Published: May 2006

Cytochrome
P
-450 (
P
-450)-dependent aryl hydrocarbon hydroxylase (AHHase) and 7-ethoxycoumarin deethylase (ECDEtase) in human tissues were differentially inhibited by monoclonal antibodies (MAbs) that were prepared to inhibit and completely inhibited the activity of 3-methylcholanthrene-induced rat liver
P
-450. The AHHase and ECDEtase of placentas from individual women who smoked were inhibited by the MAbs by 83-90% and by 34-74%, respectively. Benz[
a
]anthracene (B
a
A)-induced AHHase and ECDEtase in lymphocytes were inhibited 18-65% and 30-78%, respectively. The enzymes in both control and B
a
A-induced human cells in culture were inhibited to different extents. Both the AHHase and ECDEtase in control and B
a
A-induced monocytes and in normal liver were largely unaffected by the MAb. Thus, we have with the MAbs: (
i
) identified
P
-450s with a common antigenic site in placenta, lymphocytes, and human cells in culture; (
ii
) identified two forms of hydrocarbon-induced
P
-450s in lymphocytes, at least one of which is common with the induced
P
-450s of placenta and with a
P
-450 form present in uninduced lymphocytes; (
iii
) identified two forms of
P
-450 responsible for smoking-induced ECDEtase activity in placenta, one of which is also responsible for AHHase activity; (
iv
) shown that the
P
-450s of liver, basal, and B
a
A-induced monocytes are different from the MAb-sensitive
P
-450s of placenta and lymphocytes; (
v
) quantitated in several human tissues the percentages of control and inducible AHHase and ECDEtase that are dependent on the MAb-sensitive
P
-450; and (
vi
) defined by HPLC the contribution of the MAb-sensitive
P
-450 to the formation of specific benzo[
a
]-pyrene metabolites. The results demonstrate the value of MAbs for defining antigenic site relatedness for different enzymatic functions of
P
-450s and for identifying and quantifying the amount of a specific enzyme activity in a tissue dependent on specific
P
-450s. This study may be a prototype for the use of MAbs for phenotyping and mapping of
P
-450s responsible for specific metabolic reactions and, thus, may be useful in determining the relationship of
P
-450 phenotype to individual differences in drug metabolism and carcinogen susceptibility.
Nickel-containing factor F430: chromophore of the methylreductase of Methanobacterium.
Authors: Ellefson W L , Whitman W B , Wolfe R S
Published: May 2006

The yellow chromophore of the methyl-coenzyme M methylreductase of Methanobacterium thermoautotrophicum has been found to be the nickel-containing factor F430. Treatment of 63Ni-labeled methylreductase with 80% aqueous methanol released the radiolabel as well as the yellow chromophore; both properties were associated with a single compound that was found to be identical to F430, the stoichiometry being 1 mol of nickel per mol of F430 and 2 mol of F430 per mol of methylreductase.
Type I osteogenesis imperfecta: a nonfunctional allele for pro alpha 1 (I) chains of type I procollagen.
Authors: Barsh G S , David K E , Byers P H
Published: May 2006

Type I osteogenesis imperfecta (OI) is a dominantly inherited disease characterized clinically by bone fractures during childhood, blue sclerae, and frequent hearing loss accompanied by a decreased content of type I collagen in bone and skin. Cultured skin fibroblasts from three individuals affected with the disease produce half-normal levels of type I procollagen, a disulfide-bonded trimer that contains two pro alpha 1(I) chains and one pro alpha 2(I) chain. In normal cells, pro alpha 1(I) and pro alpha 2(I) are synthesized in a 2:1 ratio and only assembled molecules are secreted. In contrast, the OI cells contain equimolar amounts of pro alpha 1(I) and pro alpha 2(I), which suggests that trimer assembly and secretion are limited by the level of pro alpha 1(I) synthesis. The "extra" pro alpha 2(I) in the OI cells is in a nondisulfide-bonded configuration and is not secreted but apparently contributes to an increased level of intracellular degradation. Thus, decreased production of type I procollagen in these patients is the result of decreased synthesis of pro alpha 1(I). These results suggest that the stoichiometry of pro alpha chains in type I procollagen is determined by the conformation of the chains rather than the ratio in which they are synthesized, that molecules containing more than a single pro alpha 2(I) chain are not assembled, and that the production of this heteropolymeric molecule may be effectively regulated by controlling the synthesis of only one of the subunits.
Possible metabolic basis for the different immunodeficient states associated with genetic deficiencies of adenosine deaminase and purine nucleoside phosphorylase.
Authors: Carson D A , Wasson D B , Lakow E , Kamatani N
Published: May 2006

An inherited deficiency of adenosine deaminase (Ado deaminase; adenosine aminohydrolase, EC 3.5.4.4) causes severe combined immunodeficiency disease in humans. A similar deficiency in purine nucleoside phosphorylase (Puo phosphorylase; purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) engenders a selective cellular immune deficit. To elucidate the possible metabolic basis for the contrasting immunologic phenotypes, we compared the toxicity toward mature resting human lymphocytes of the Ado deaminase substrates deoxyadenosine and adenosine and the Puo phosphorylase substrate deoxyguanosine. When Ado deaminase was inhibited, micromolar concentrations of deoxyadenosine progressively killed nondividing helper and suppressor-cytotoxic T cells, but not B cells. The toxicity required phosphorylation, with subsequent dATP formation. The deoxyadenosine analogs 2-chlorodeoxyadenosine, 2-fluorodeoxyadenosine, and adenine arabinonucleoside also killed resting T cells. Cell death was unrelated to inhibition of adenosylhomocysteinase (EC 3.3.1.1) but was preceded by a gradual decline in ATP levels. As much as 1 mM deoxyguanosine did not impair resting lymphocyte viability, despite the synthesis of dGTP. The combination of 200 microM adenosine plus 500 microM homocysteine thiolactone killed dividing lymphocytes but had no discernible toxic effect toward resting T cells, which accumulated adenosylhomocysteine over a 4-hr period but thereafter excreted the nucleoside into the culture medium. The different clinical syndromes associated with genetic deficiencies of Ado deaminase and Puo phosphorylase may be explained by the ability of dATP to kill mature resting T lymphocytes by depleting ATP levels.
Marking the polarity of RNA molecules for electron microscopy by covalent attachment of psoralen-DNA restriction fragments.
Authors: Wollenzien P L , Cantor C R
Published: May 2006

We have developed a general procedure for covalently attaching complementary restriction fragments to RNA molecules and visualizing the fragment in the electron microscope after denaturation. This is accomplished by creating psoralen monoadducts on the restriction fragment by irradiation at 390 nm before hybridization to the RNA and subsequently converting the monoadducts to RNA . DNA crosslinks by irradiation at 360 nm. Using this procedure, we have determined the location of a crosslink placed in Escherichia coli 165 rRNA by a previous irradiation. A consistent assignment is obtained by marking the crosslinked RNA with restriction fragments complementary to the 5' end or the 3' end of the 165 rRNA molecule.
Involvement of outside DNA sequences in the major kinetic path by which EcoRI endonuclease locates and leaves its recognition sequence.
Authors: Jack W E , Terry B J , Modrich P
Published: May 2006

We have examined the kinetics of the interaction between endodeoxyribonuclease EcoRI (EC 3.1.23.13) and nine linear DNA fragments that range in size between 34 and 6,200 base pairs and contain the EcoRI site of plasmid pBR322 in a central location. The kinetic parameters governing both formation and decay of specific endonuclease . DNA complexes increase 8-fold with increasing chain length over this size range. In contrast, equilibrium competition experiments demonstrated that the intrinsic affinity of endonuclease for its recognition sequence is independent of DNA chain length over this range. Thus, DNA sequences outside the recognition site enhance the rate at which EcoRI endonuclease locates or leaves its recognition site without affecting the intrinsic thermodynamic parameters of site-specific interaction. These results are consistent with a facilitated diffusion mechanism for specific DNA site location by this enzyme.
Genes encoding Escherichia coli aspartate transcarbamoylase: the pyrB-pyrI operon.
Authors: Pauza C D , Karels M J , Navre M , Schachman H K
Published: May 2006

In both Escherichia coli and Salmonella typhimurium there is approximately balanced synthesis of the six catalytic and six regulatory polypeptide chains of the regulatory enzyme aspartate transcarbamoylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2). This control is achieved by the contiguous pyrB and pyrI genes, which encode the catalytic and regulatory chains, respectively. Evidence for this single transcriptional unit was obtained by a study of various deletion mutations and from the DNA sequence of the intercistronic region between pyrB and pyrI. One pyrB deletion, pyrB748, produced a normal level of regulatory chains even though it removed a substantial portion of the pyrB gene. Another deletion, pyrB740, shares a similar terminus at one end within pyrB, but the promoter region is removed. In this deletion mutation, there is no production of the regulatory polypeptide, indicating that a single region adjacent to pyrB controls transcription of pyrI as well. Molecular cloning and subsequent DNA analysis demonstrated that the pyrB and pyrI genes are contiguous with pyrI as the distal gene in the operon. The cistrons are separated by a 15-nucleotide untranslated region containing a sequence capable of interacting with the 16S ribosomal RNA and allowing translation of the pyrI cistron.
Insertion of a movable genetic element, 297, into the T-A-T-A box for the H3 histone gene in Drosophila melanogaster.
Authors: Ikenaga H , Saigo K
Published: May 2006

From a cloned library of Drosophila DNA, a recombinant phage was isolated which contained a nucleotide sequence homologous to both the histone gene and a movable genetic element, 297 [Saigo, K., Millstein, L. & Thomas, C.A., Jr. (1981) Cold Spring Harbor Symp. Quant. Biol. 45, 815-827]. We have determined the nucleotide sequences of the termini of 297 as well as its insertion site in the histone gene in the clone. Our results strongly suggest that (i) although 297 ends with typical long terminal repeats, integrated 297 is not bounded by 5' T-G...C-A 3', dinucleotides commonly found at the very ends of integrated forms of other similar genetic elements, and (ii) the insertion of 297 occurred exactly within the T-A-T-A box for the H3 histone gene containing T-A-T-A-T-A. The structures of the insertion sites of 297 were also examined by using two other recombinant clones of the histone gene. A 6-base-pair-long segment, T-A-T-A-T-A, was again found to be present at the insertion sites of 297 in both clones. Based on these results, it was concluded that, at least within the histone gene cluster, the insertion of 297 was carried out in a site-specific fashion with T-A-T-A-T-A as the target sequence.
Regulation of the
in vitro
antibody response by neuroendocrine hormones
Authors: Johnson Howard M. , Smith Eric M. , Torres Barbara A. , Blalock J. Edwin
Published: May 2006

Treatment of lymphocytes with inducers of interferon α (IFN-α) results in the production of corticotropin (ACTH) and endorphin-like activities. The pro-opiomelanocortin-derived hormones ACTH and α-, β-, and γ-endorphin and the structurally related hormones [Leu]- and [Met]enkephalin were therefore tested for their effects on the
in vitro
antibody response of mouse spleen cells. ACTH and α-endorphin were potent inhibitors (≥80% suppression) of the antibody response to the T-cell-dependent antigen sheep erythrocytes at a concentration of 0.5 μM. [Met]- and [Leu]enkephalin were moderate inhibitors (approximately 60% suppression) at 0.2-2 μM, and β- and γ-endorphin were minimal inhibitors (approximately 20% suppression) at 5-6 μM. At higher concentrations ACTH also inhibited the antibody response to the T-cell-independent antigen dinitrophenyl-Ficoll, suggesting that T-cell function was more sensitive to blockage by these hormones than was B-cell function. ACTH and IFN had similar suppression properties; thus, the hormone-like activities associated with IFN-α may play a role in IFN-induced immunosuppression. α-Endorphin immunosuppression was blocked by naloxone, which suggested that α-endorphin exerted its effects through binding to opiate-like receptors on the spleen cells. The failure of β-endorphin to suppress the immune response significantly was not due to its failure to bind to the opiate-like receptors because it blocked α-endorphin-induced suppression. Direct evidence for both opiate and ACTH receptors on the spleen cells was obtained in binding studies with labeled enkephalin and ACTH. Such studies revealed the presence of both high- and low-affinity receptors. The data show that neuroendocrine polypeptide hormones can regulate the immune response.
Ethanol disordering of spin-labeled mouse brain membranes: correlation with genetically determined ethanol sensitivity of mice.
Authors: Goldstein D B , Chin J H , Lyon R C
Published: May 2006

Disordering of brain and erythrocyte membranes by ethanol in vitro was measured by ESR using 5-doxylstearic acid as spin label. Synaptosomal plasma membranes and erythrocyte membranes were isolated from two lines of mice developed, by selective breeding, for differential sensitivity to hypnotic effects of ethanol. Membranes taken from alcohol-sensitive "long-sleep" mice were more strongly disordered by ethanol in vitro than were membranes from alcohol-resistant "short-sleep" mice. Furthermore, within a population of genetically heterogeneous mice, the most ethanol-sensitive animals had the most ethanol-sensitive synaptosomal plasma membranes. In vivo sensitivity of the individual mice was evaluated by measuring brain ethanol levels at a precise behavioral end point, recovery from ataxia. The data extend our previous observations of correlations between in vitro and in vivo effects of ethanol and suggest that membrane disordering may be a primary mechanism of acute effects of ethanol.
Estrogen induces a demethylation at the 5' end region of the chicken vitellogenin gene.
Authors: Wilks A F , Cozens P J , Mattaj I W , Jost J P
Published: May 2006

The two isoschizomeric restriction endonucleases Msp I and Hpa II have been used to study changes in the methylation pattern of the vitellogenin gene after estrogen treatment. In the liver of the both estrogen-treated and nontreated chickens the vitellogenin gene is heavily methylated. However, estradiol causes a demethylation of a Hpa II site(s) at the 5' end region of the gene. The same Hpa II restriction site(s) is also unmethylated in the liver of egg laying hens but is methylated in the liver of roosters or in erythrocyte DNA. In the mature oviduct where no vitellogenin is synthesized but where several egg white proteins are under the control of estrogen, there is also a demethylation of the 5'end region of the vitellogenin gene. This demethylation does not precede the initiation of transcription of the gene but persists, and increases, even after vitellogenin transcription has ceased in the liver. Taken together, the results strongly suggest that in estrogen-responsive tissues the vitellogenin gene becomes demethylated at its 5' end region after estrogen stimulation whether the gene is expressed or not.
The primary ultrastructural defect caused by anophthalmic white (Wh) in the Syrian hamster.
Authors: Asher J H , James S C
Published: May 2006

Anophthalmic white (Wh) of the Syrian hamster is a highly pleiotropic dominant spotting color mutation causing homozygotes to be deaf, blind, and white. An ultrastructural analysis of embryonic tissue reveals that Wh causes the retention of cilia by cells of opposing layers of the embryonic retina and by the lens fiber cells. Previous ultrastructural analyses indicate that Wh also causes the retention of cilia by secretory cells of the anterior lobe of adult pituitaries. We propose that the primary ultrastructural defects caused by Wh is the retention of cilia by embryonic cells. These retained cilia are hypothesized to interfere with normal cell-cell interactions and subsequent cell differentiation.
Effects of adrenal cortical steroids and osmotic blood-brain barrier opening on methotrexate delivery to gliomas in the rodent: the factor of the blood-brain barrier.
Authors: Neuwelt E A , Barnett P A , Bigner D D , Frenkel E P
Published: May 2006

The effect of adrenal cortical steroids and osmotic blood-brain barrier modification on methotrexate delivery to normal and glioma-bearing rats was studied. In animals with the avian sarcoma virus-induced glioma, osmotic blood-brain barrier modification resulted in significantly increased delivery of methotrexate to the tumor-bearing hemisphere (including the tumor, the brain around the tumor, and the brain distant to the tumor), compared to the nonmodified hemisphere or to control animals. The administration of adrenal steroids, followed by intracarotid methotrexate, resulted in slightly decreased chemotherapeutic agent (methotrexate) delivery to the tumor, the brain around the tumor, and the brain distant to the tumor. When adrenal steroids were given prior to barrier modification and methotrexate therapy, the level of methotrexate was significantly less in the tumor. These studies provide evidence that the blood-brain barrier exists in tumors and is a factor in drug delivery to tumors. Steroid administration greatly interferes with the enhancement of drug delivery to tumors that can be achieved with osmotic blood-brain barrier modification.
Expression of the structural proteins of Semliki Forest virus from cloned cDNA microinjected into the nucleus of baby hamster kidney cells.
Authors: Kondor-Koch C , Riedel H , Söderberg K , Garoff H
Published: May 2006

The three structural proteins of Semliki Forest virus--i.e., the capsid, p62, and E1 proteins--were expressed in baby hamster kidney cells from cloned DNA transcribed from the virus-specific 4.1-kilobase mRNA. The cDNA was engineered into an expression vector developed by others [Mulligan, R. C. & Berg, P. (1980) Science 209, 1422--1427] downstream from the simian virus 40 early promoter and was introduced into cell nuclei by microneedle injection. Immunofluorescence analysis of injected cells showed that the capsid protein was located in the cell cytoplasm, whereas the membrane proteins were associated with cellular membranes. The p62 protein was shown to be transported from the rough endoplasmic reticulum to the plasma membrane, whereas the E1 protein remained in the rough endoplasmic reticulum.
Three-dimensional structural model of eubacterial 5S RNA that has functional implications.
Authors: Pieler T , Erdmann V A
Published: May 2006

Escherichia coli 5S RNA and its specific protein complexes were hydrolyzed with the single-strand-specific nuclease S1. Based on the results, a tertiary structural model for E. coli 5S RNA is proposed in which ribosomal proteins E-L5, E-L18, and E-L25 influence the conformation of the RNA. This may be of significance for ribosomal function. Comparison of the proposed E. coli 5S RNA structure with those of 18 other prokaryotic 5S RNAs led to a generalized eubacterial 5S RNA tertiary structure in which the majority of the conserved nucleotides are in non-base-paired regions and several conserved "looped-out" adenines (in E. coli, adenines -52, -53, -57, -58, and -66) are implied to be important for protein recognition or interaction or both.
Assignment of the structural gene for the third component of human complement to chromosome 19.
Authors: Whitehead A S , Solomon E , Chambers S , Bodmer W F , Povey S , Fey G
Published: May 2006

The third component of complement (C3) is synthesized and secreted by cultured human primary fibroblasts. A monoclonal antibody having specificity for an antigenic determinant carried by human but not mouse C3 was used to study the continued expression of human C3 in three panels of independently derived human-mouse somatic cell hybrids. Expression of the human product was shown to segregate with human chromosome 19 and with no other chromosome or group of chromosomes. A unique-sequence human genomic C3 DNA clone was isolated and used as a probe in DNA hybridization experiments with DNA prepared from appropriate human-mouse somatic cell hybrids to confirm assignment of the human C3 gene to chromosome 19.
T-cell suppression and contrasuppression induced by histamine H2 and H1 receptor agonists, respectively.
Authors: Siegel J N , Schwartz A , Askenase P W , Gershon R K
Published: May 2006

The intensity of Lyl+T helper and delayed type hypersensitivity effector cell activities is governed, in part, by an interplay between two classes of immunoregulatory T cells: suppressor cells and contrasuppressor cells. We asked whether histamine, at concentrations and duration of exposure that we calculated might be achieved at local sites of inflammation, could activate either or both of these classes of regulatory cells in vitro. To answer this question we used spleen cells from mice treated in vivo with the toleragen trinitrobenzenesulfonic acid as regulators of in vitro generation of primary anti-trinitrophenyl self-cytotoxic T lymphocytes. Under the conditions used, these spleen cells had no major regulatory effects. However, if these cells were preincubated with histamine at 0.1 mM for 30-60 min, suppressor activity was induced, but this occurred inconsistently and with nonstoichiometric results. The use of synthetic histamine agonists revealed that histamine may activate both suppressor and contrasuppressor cell subsets. A histamine H1 receptor agonist [2-(2-pyridyl)-ethylamine dihydrochloride] had a propensity to activate contrasuppression, whereas an H2 receptor agonist (dimaprit) tended to activate suppressor cells. Thus, histamine may have opposing actions that obscure suppression. This duality was shown by treatment of pyridylethylamine-induced contrasuppressor cells with complement and anti-I-J antibody that kills contrasuppressor cells. This treatment revealed a high level of suppressor cell activity that was not expressed until the opposing contrasuppressor cells were removed. Because histamine is released at local sites of delayed type hypersensitivity, these results indicate that histamine may serve as an inducer of microenvironmental immunomodulation by activating regulatory T cells at sites where immune responses are taking place.
Isolation of 16L virus: a rapidly transforming sarcoma virus from an avian leukosis virus-induced sarcoma.
Authors: Neel B G , Wang L H , Mathey-Prevot B , Hanafusa T , Hanafusa H , Hayward W S
Published: May 2006

We have isolated a replication-defective rapidly transforming sarcoma virus (designated 16L virus) from a fibro-sarcoma in a chicken infected with td107A, a transformation-defective deletion mutant of subgroup A Schmidt-Ruppin Rous sarcoma virus. 16L virus transforms fibroblasts and causes sarcomas in infected chickens within 2 wk. Its genomic RNA is 6.0 kilobases and contains sequences homologous to the transforming gene (fps) of Fujinami sarcoma virus (FSV). RNase T1 oligonucleotide analysis shows that the 5' and 3' terminal sequences of 16L virus are indistinguishable from (and presumably derived from) td107A RNA. The central part of 16L viral RNA consists of fps-related sequences. These oligonucleotides fall into four classes: (i) oligonucleotides common to the putative transforming regions of FSV and another fps-containing avian sarcoma virus, UR1; (ii) an oligonucleotide also present in FSV but not in UR1; (iii) an oligonucleotide also present in UR1 but not in FSV; and (iv) an oligonucleotide not present in either FSV, UR1, or td107A. Cells infected with 16L virus synthesize a protein of Mr 142,000 that is immunoprecipitated with anti-gag antiserum. This protein has protein kinase activity. These results suggest that 16L virus arose by recombination between td107A and the cellular fps gene.
Two-fluid theory and thermodynamic properties of liquid mixtures. Application to hard-sphere mixtures
Authors: Brandani V. , Prausnitz J. M.
Published: May 2006

This paper is the second of three describing a two-fluid theory of binary liquid mixtures. The general theory presented in the preceding paper is used to derive a model for calculating thermodynamic properties of hard-sphere mixtures. Calculations indicate that desirable boundary conditions are satisfied.