bluuebunny/crossref_metadata_2025 huggingface dataset:
Escherichia coli mutants defective in dipeptidyl carboxypeptidase.
Authors: Deutch C E , Soffer R L
Published: May 2006

Two independent mutants of Escherichia coli deficient in dipeptidyl carboxypeptidase activity (Dep-) were isolated after mutagenesis with ethyl methanesulfonate. Mating experiments and introduction of specific episomes indicated that the responsible gene was located at approximately 27--31 min on the E. coli chromosome. The Dep- mutants differed from the parental strain in their inability to grow with N-acetylalanylalanylalanine as the sole nitrogen source. Revertants selected for growth on this substrate of the enzyme were found to have reacquired the activity. Enzyme activity was highly sensitive to inhibition by 1-(D-3-mercapto-2-methylpropanoyl)-L-proline (SQ 14225), a potent inhibitor of mammalian dipeptidyl carboxypeptidase (angiotensin-converting enzyme, peptidyl dipeptidase, EC 3.4.15.1). This compound also reduced the rate of growth of the wild type with N-acetylalanylalanylalanine but not with ammonium sulfate. A fraction of the enzyme was released into the medium by osmotic shock, indicating that its presence in the periplasmic space may account for growth with N-acetylated peptides that cannot be taken up by E. coli. In addition to providing information about the specific role of this exopeptidase in E. coli, the Dep- mutants may prove useful for delineating the regulation and cellular function of dipeptidyl carboxypeptidases in higher organisms.
Actinomycin D inhibits initiation of DNA replication in mammalian cells.
Authors: Guy A L , Taylor J H
Published: May 2006

Mammalian (Chinese hamster ovary) cells were synchronized in the division cycle and blocked at the beginning of S phase with fluorodeoxyuridine. Traces of thymidine in the medium allowed cells to enter S phase and initiate DNA replication at some origins. For many hours after the traces of thymidine were depleted new sites for DNA replication accumulated in a small fraction of the DNA. However, these potential origins became active in bidirectional replication only when cells were released by adding [3H]thymidine to the medium. Lysis at 37 degrees C released most of the pulse-labeled DNA as linear double-stranded segments larger than Okazaki fragments and smaller than the unreplicated parental DNA. Release of the newly replicated DNA involves breakage of the template chains at or near each fork. The size of the fragments increased linearly with time of pulse labeling, but the efficiency of their release decreased. The excision could be prevented by lysis at 0 degrees C. When cells were treated with actinomycin D for 3--5 min before release, the new origins failed to function, but chain growth continued from those sites at which initiation had taken place before depletion of thymidine. We interpret these results to indicate that initiation at origins requires an actinomycin D-sensitive step, presumably RNA transcription, while chain elongation, which involves the formation of Okazaki pieces, is relatively insensitive to actinomycin D during growth over long intervals.
Control of DNA synthesis in growing BALB/c 3T3 mouse cells by a fibroblast growth regulatory factor
Authors: Natraj C. V. , Datta Prasanta
Published: May 2006

A cell surface component from quiescent BALB/c 3T3 mouse cells that inhibits DNA synthesis and cell division when added to a culture of growing 3T3 cells has been detected. The inhibition of DNA synthesis by this factor was dependent on concentration and time of incubation; a transient exposure of cells to the factor followed by incubation in its absence for 20 hr was sufficient to elicit its inhibitory effect. The active component appears to be protein in nature, as judged by heat inactivation and trypsin sensitivity. Extracts obtained in an identical manner from quiescent 3T3 cells that had been preincubated
in situ
with uridine diphosphate
N
-acetyl-D-glucosamine (UDP-GlcNAc) did not inhibit DNA synthesis. The effect was specific for UDP-GlcNAc: incubation with three other nucleotide sugars yielded active component. Incubation of the inactive component from UDP-GlcNAc-treated cells with purified
N
-acetyl-β-D-glucosaminidase
in vitro
restored its inhibitory property. Extracts from growing cells failed to inhibit DNA synthesis. These results suggest that reversible glycosylation with
N
-acetyl-D-glucosamine residues may serve as a regulatory signal for the conversion of the active factor to its inactive form. We propose that the onset of quiescence of 3T3 cells is due to a casual relationship between depletion of growth factors in the culture medium and the presence of the active regulatory factor on the cell surface that inhibits DNA synthesis; conversion of the regulatory factor to its inactive form under favorable nutritional status may be viewed as a switch that allows DNA synthesis to resume.
Effect of overall phenotypic selection on genetic change at individual loci.
Authors: Kimura M , Crow J F
Published: May 2006

The selective advantage of an allele Gi (relative to the mean of alleles at this locus) is given by (formula: see text) in which Ai is the average excess of the allele on the character, X; W(X) is the fitness function; F(X) is the frequency function; W is the mean fitness; and the prime denotes differentiation. With truncation selection si = AaF(C)/w in which F(C) is the ordinate at the culling level and w is the proportion saved; this does not depend on any assumption about the distribution of F(X). If the character is normally distributed, si = AiI/sigma2, in which I is the selection differential and sigma2 is the variance of the character distribution. Finally, if the logarithm of the fitness is proportional to the squared deviation from the optimum and the character is distributed normally, si = AiK(Xop--m), in which Xop is the optimum value of the character, m is the mean value, and K is a constant determined by the variances of the fitness function and the frequency function. Truncation is the most efficient form of directional selection in the sense of producing the maximum gene frequency change for a given effect of the gene on the character, but fitness functions can depart considerably from sharp truncation without greatly reducing the efficiency.
A single base-pair change creates a Chi recombinational hotspot in bacteriophage lambda.
Authors: Sprague K U , Faulds D H , Smith G R
Published: May 2006

X4+ mutations, responsible for the Chi phenotype in phage lambda, locally increase the rate of recombination promoted by the Escherichia coli recombination system (Rec). X+ mutations in the cII gene, one of a few sites in lambda at which such mutations arise, were located genetically and physically with overlapping deletions. DNA sequence analysis of the deletion segment containing the X+ C mutations showed that two independent X+ C mutations arose by the same A-T to T-A transversion. Presumably, this change creates a nucleotide sequence recognized by a protein involved in a rate-limiting step of recombination.
H-2 complementation in anti-H-Y cytotoxic T-cell responses can occur in chimeric mice.
Authors: Matsunaga T , Simpson E
Published: May 2006

Cytotoxic T-cell responses against the H-Y antigen in mice are under the control of major histocompatibility complex genes. Not only must cytotoxic T cells recognize both H-Y antigens and "self" H-2K/D molecules to lyse male target cells, but also "appropriate association" between H-Y antigens and H-2K/D antigens is required to induce such cytotoxic responses. Furthermore, it is suggested that appropriate association with H-2-I antigens may also be required to generate H-Y specific helper cells for the cytotoxic response. BALB/c(KdIdDd) mice are nonresponders against syngeneic H-Y antigens, because they lack appropriate associative H-2K/D antigens. This results in the failure of generation of anti H-Y cytotoxic cells, although helper cells may be induced. F1 hybrid mice (BALB/c X C3H/He)F1 or H-2 recombinant mice C3H-OH(KdIdDk) are responders, because H-2Dk (and H-2Kk in the F1) molecules offer appropriate association to H-Y antigens. We here report that allophenic chimeras (H-2d reversible H-2k) and irradiation bone marrow chimeras [H-2d + H-2k leads to F1(H-2d X H-2k)] generate anti-H-Y cytotoxic responses but that cells of the BALB/c(H-2d) genotype comprise most if not all of the cytotoxic cells. A working model is proposed to account for major histocompatibility complex control over anti-H-Y cytotoxic T-cell responses.
Enhanced expression of β
2
-microglobulin and HLA antigens on human lymphoid cells by interferon
Authors: Heron Iver , Hokland Marianne , Berg Kurt
Published: May 2006

Mononuclear cells from the blood of healthy normal humans were kept in cultures under nonstimulating conditions for 16 hr in the presence or absence of human interferon. The relative quantities of HLA antigens and β
2
-microglobulin on the cultured cells were determined by quantitative immunofluorescence (fluorescence-activated cell sorter) and by the capacity of cells to absorb out cytotoxic antibodies against the relevant antigens. Interferons of different origin and purities enhanced the expression of HLA antigens and β
2
-microglobulins, whereas membrane immunoglobulins and antigens recognized by antiserum raised against human brain and T cells were the same on interferon-treated and control cells. Similar interferon effects were observed on an Epstein-Barrvirus-negative Burkitt lymphoma cell line. The enhanced expression of histocompatibility antigen subsequent to intereferon treatment was observed on B- and T-enriched lymphocyte populations and was found to be dose dependent with the optimum with “physiological” concentrations of interferon. Pretreatment of lymphocytes with interferon for 2 hr was found to be as effective as having interferon present during the total culture period. The interferon-induced enhancement of antigen expression on cells was dependent on active protein synthesis.
Inhibition of lymphocyte-mediated cytolysis by 3-deazaadenosine: Evidence for a methylation reaction essential to cytolysis
Authors: Zimmerman Thomas P. , Wolberg Gerald , Duncan Gail S.
Published: May 2006

3-Deazaadenosine (deazaAdo) inhibits lymphocyte-mediated cytolysis
in vitro
at micromolar concentrations and is potentiated markedly in this activity by L-homocysteine thiolactone. DeazaAdo alone causes a rapid, dose-dependent buildup of S-[
3
H]adenosylhomocysteine (AdoHcy) and S-[
3
H]adenosylmethionine in cytolytic lymphocytes labeled with L-[2-
3
H]methionine; smaller amounts of S-3-[
3
H]deazaadenosylhomocysteine (deazaAdoHcy) are also formed in these cells. The simultaneous addition of deazaAdo and L-homocysteine thiolactone to the lymphocytes results in a massive intracellular accumulation of deazaAdoHcy. Both the inhibition of lymphocyte-mediated cytolysis and the cellular accumulation of [
3
H]AdoHcy caused by deazaAdo alone are reversed rapidly by removal of drug from the medium. However, the inhibition of cytolysis and the large cellular buildup of deazaAdoHcy resulting from treatment of the lymphocytes with deazaAdo plus L-homocysteine thiolactone are dissipated more slowly under these same conditions. Unlike adenosine, deazaAdo is not potentiated in its inhibition of lymphocyte-mediated cytolysis by Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone], an inhibitor of cyclic AMP phosphodiesterase, and has little or no effect upon the level of lymphocytic cyclic AMP. DeazaAdo is not metabolized detectably to 5′-nucleotides in the lymphocytes and does not cause a decrease in the pool sizes of CTP, UTP, ATP, or GTP. Both AdoHcy and deazaAdoHcy have been reported to be powerful inhibitors of a variety of
S
-adenosylmethionine-utilizing methyltransferases. The present results, therefore, indicate that the effect of deazaAdo upon lymphocyte-mediated cytolysis is due ultimately to the inhibition of an unidentified but crucial methyltransferase within the cytolytic lymphocytes and provide an insight into the biochemical processes involved in lymphocyte-mediated cytolysis.
gamma-Aminobutyric acid receptors visualized in spinal cord cultures by [3H]muscimol autoradiography.
Authors: Chan-Palay V , Yonezawa T , Yoshida S , Palay S
Published: May 2006

gamma-Aminobutyric acid (GABA) receptors were visualized in mouse spinal cord explant cultures by [3H]muscimol autoradiography over certain small neurons of the dorsal horn grey matter, in the interneuronal neuropil of dorsal and ventral horns, and (rarely) in small clusters on processes of anterior horn cells. Specificity was indicated in control experiments by inhibition of binding by [3H]muscimol after pretreatment with GABA or a GABA analogue, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, isoxazole 16), or with receptor antagonists bicuculline and picrotoxin. Unlabeled muscimol exchanged effectively with [3H]muscimol and produced autoradiographic label in locations indistinguishable from those found in experiments with [3H]muscimol alone. Pretreatment with the GABA uptake and transport inhibitors (-)-nipecotic acid and guvacine did not affect binding with [3H]muscimol. These experiments indicate that explant culture systems can be used for demonstration of functional receptors.
Nonequivalence of alpha-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons.
Authors: Carbonetto S T , Fambrough D M , Muller K J
Published: May 2006

alpha-Bungarotoxin binds selectively to chick sympathetic neurons that are responsive iontophoretically applied acetylcholine. alpha-Bungarotoxin (125 nM) does not affect the response of cultured neurons to acetylcholine, nor does it affect a cholinergic synaptic potential recorded from sympathetic ganglia. d-Tubocurarine (100 muM) inhibits alpha-bungarotoxin binding and blocks acetylcholine receptor function in both preparations, but alpha-bungarotoxin does not protect acetylcholine receptors against d-tubocurarine blockade of acetylcholine responses. The receptor for alpha-bungarotoxin can be extracted from neuronal membranes with nonionic detergents and, when assayed by velocity sedimentation in sucrose gradients, sediments at a rate faster than that of skeletal muscle acetylcholine receptors. Treatment of alpha-bungarotoxin-receptor complexes with glutaraldehyde (0.1%, wt/vol) increases their stability from a half-time for dissociation of 3.5 hr to greater than 6 days at 23 degrees. This permits a quantitative assay of alpha-bungarotoxin-receptor complexes after relatively long periods of velocity sedimentation. It is concluded that alpha-bungarotoxin does not bind to the acetylcholine-binding site of neuronal acetylcholine receptors. These results compel a reevaluation of studies that assume that alpha-bungarotoxin is a specific ligand for neuronal acetylcholine receptors.
Stimulation of bilirubin catabolism in jaundiced Gunn rats by an inducer of microsomal mixed-function monooxygenases
Authors: Kapitulnik Jaime , Ostrow J. Donald
Published: May 2006

The homozygous, jaundiced Gunn rat compensates for its inability to form bilirubin conjugates by production of polar bilirubin metabolites which can be excreted in the bile without conjugation. To assess whether microsomal mixed-function monooxygenases might be involved in formation of these metabolites, a potent inducer of these enzymes, 2,3,7,8-tetrachlorodibenzo-
p
-dioxin, was administered to Gunn rats in a single intraperitoneal dose of 10 μg/kg. Four to 6 days after treatment, plasma bilirubin levels had declined by a mean of 61%, whereas no significant change was observed in control Gunn rats that received only the dioxane vehicle. Use of tracer [
14
C]bilirubin showed that the decline in plasma bilirubin was the result of a 7-fold increase in fractional bilirubin turnover which reduced the bilirubin pool to an average of 11% of the control values. In the new steady state, total bilirubin turnover was unaltered. The accelerated fractional bilirubin turnover was associated with augmented biliary excretion of polar bilirubin metabolites and a 16-fold increase in hepatic benzo[
a
]-pyrene hydroxylase activity. Hepatic bilirubin-UDPglucuronate transferase was not induced, and no bilirubin conjugates appeared in the bile. This chemical simulation of the alternate pathways of bilirubin catabolism by the inducer suggests that microsomal cytochrome
P
448
-dependent monooxygenases may be involved in the formation of the polar bilirubin metabolites excreted by the Gunn rat.
Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.
Authors: Nguyen-Huu M C , Sippel A A , Hynes N E , Groner B , Schütz G
Published: May 2006

The synthesis of ovalbumin mRNA sequences was studied in isolated nuclei from hen oviduct. Two different methods of analysis were used to distinguish in vitro synthesized from preexisting mRNA sequences: (i) Mercurated ribonucleotides were used for in vitro RNA synthesis, and the newly synthesized RNA was purified by chromatography on sulfhydryl-agarose and hybridized to radioactive ovalbumin cDNA. (ii) [3H]UTP was used to label the in vitro synthesized RNA. Hybridization to unlabeled mercurated cDNA, RNase A digestion, and subsequent purification of the hybrids on SH-agarose allowed the quantitation of newly synthesized ovalbumin mRNA sequences. Approximately 0.1% of the newly synthesized RNA was identified as ovalbumin RNA by both methods. The synthesis of ovalbumin RNA progressed during the incubation of nuclei and was sensitive to actinomycin D and low concentrations of alpha-amanitin. The preferential in vitro transcription of the ovalbumin gene (1000-fold over random transcription of the chicken genome) by RNA polymerase B (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) suggests that the specificity of in vitro RNA synthesis is retained in isolated nuclei.
Discrete length classes of DNA depend on mode of dehydration.
Authors: Vollenweider H J , James A , Szybalski W
Published: May 2006

The length of double-stranded coliphage lambda DNA, as determined by electron microscopy using the benzyldimethylalkyl ammonium chloride technique, depends on the mode of dehydration. The freeze-dried DNA form is the longest (16.5 micron), whereas dehydration in methanol (15.9 micron) or in ethanol (three forms: 15.2 micron, 13.9 micron, and 12.4 micron) results in progressively shorter molecules. These measured lengths of the freeze-dried, methanol-dehydrated, and shortest ethanol-dehydrated forms correspond to the axial rise per nucleotide pair in the B, C, and A forms of DNA, respectively. The remaining forms of ethanol-dehydrated DNA seem to represent novel intermediary conformations of DNA. In agreement with the predicted increment, DNA exposed to ethidium bromide and freeze-dried is elongated by 39% (22.9 micron). All size classes show the same relative distribution pattern of bound Escherichia coli RNA polymerase molecules (nucleoside triphosphate:RNA nucleotidyltransferase, EC2.7.7.6), used as intramolecular markers, indicating that the dehydration-caused transitions are uniform.
Alteration of the physicochemical properties of triphosphoinositide by nicotinic ligands.
Authors: Cho T M , Cho J S , Loh H H
Published: May 2006

The concentrations of nicotinic drugs required to effect a 50% transfer of triphosphol[3H]isositide from an aqueous phase to a nonaqueous phase closely approximated their concentrations for both in vivo neuromuscular blocking activity and binding to purified nicotinic receptors, and correlated well (correlation coefficient = 0.95)) with their biological activities measured by other workers in an Electrophorus electroplax preparation. The triphospho[3H]inositide transfer induced by nicotinic ligands was dependent on the lipid concentration and was potentiated by Ca2+. The affinity constants of 45Ca2+ for triphosphoinositide were similar to those for the purified nicotinic receptor. These and other findings suggest the possibility that triphosphoinositide (phosphatidylinositol bisphosphate) is a binding site of the nicotinic receptor.
Theoretical study of genetic variability, assuming stepwise production of neutral and very slightly deleterious mutations.
Authors: Maruyama T , Kimura M
Published: May 2006

Mathematical treatments are presented that enable us to compute the amount of genetic variability maintained in a finite population, assuming that mutations occur in stepwise fashion and that both selectively neutral and slightly deleterious alleles are involved. Two numerical examples show that, if very slightly deleterious mutations are prevalent, the amount of genetic variability increases much more slowly as the population number increases than is the case when all the mutations are strictly neutral.
Murine complement component 3: genetic variation and linkage to H-2.
Authors: Da Silva F P , Hoecker G F , Day N K , Vienne K , Rubinstein P
Published: May 2006

Two electrophoretic variants of murine complement component 3 (C3) were detected by using high-voltage electrophoresis of fresh mouse serum in agarose gels. Most of the inbred strains tested were homozygous for the S allele (for the slow-migrating variant); only four out of 46 strains had the alternative F allele (fast variant). Pen-bred Swiss-Webster animals belonged to one of three phenotypes--S, F, or SF--and the genes responsible for this variation segregated in a strictly Mendelian manner. In three such crosses, with 5* offspring, C3 segregated with H-2 in 46 instances, corresponding to a recombination frequency of approximately equal to 0.12.
Limit theorems for weighted sums and stochastic approximation processes
Authors: Lai T. L. , Robbins Herbert
Published: May 2006

By establishing certain limit theorems for martingale transforms and weighted sums, a Marcinkiewicz—Zygmund strong law together with weak and strong invariance principles is developed for stochastic approximation processes, under minimal conditions on the errors.
Common methionine-tryptic peptides near the amino-terminal end of primate papovavirus tumor antigens
Authors: Simmons Daniel T. , Martin Malcolm A.
Published: May 2006

The tumor antigens directed by human papovaviruses BK and JC and the monkey papovavirus simian virus 40 have two methionine-containing tryptic peptides in common. These peptides are constituents of the small forms of papovavirus tumor antigen (17,000 daltons) which are present in lytically infected and transformed cells and which are believed to share some amino acid sequences with the amino-terminal portion of the larger tumor antigen species (97,000 daltons). In addition to the two peptides, which are present in all three papovavirus tumor antigens, the larger forms of the tumor antigens specified by simian virus 40 and BK virus share four other methionine-containing tryptic peptides, two of which are also present in the smaller (17,000 daltons) species of antigen. The occurrence of common peptides at the amino-terminal portion of tumor antigens of primate papovaviruses suggests that these conserved regions may play a fundamental role in the function of these proteins and in the propagation of these viruses in nature. The tryptic peptides of the small forms of papovavirus tumor antigen were examined and compared to those present in the large species. Out of a total of nine and ten methionine-containing peptides in the 17,000-dalton tumor antigens of simian virus 40 and BK virus, seven and nine peptides, respectively, are constituents of the corresponding larger (97,000 daltons) forms of the antigen.
ATP-dependent Ca
2+
uptake into plant membrane vesicles
Authors: Gross Joachim , Marmé Dieter
Published: May 2006

Membrane vesicles were extracted from etiolated and light-grown plants, a plant cell suspension culture, and an alga. Upon addition of ATP and Mg
2+
, active Ca
2+
uptake into the vesicles against a concentration gradient was shown. The dependence of this uptake on ATP and Mg
2+
concentrations, pH, and temperature is described. In the absence of oxalate, equilibrium between Ca
2+
uptake and efflux was reached after about 30 min. In the presence of oxalate, Ca
2+
accumulation continued for at least 120 min. Ca
2+
efflux from preloaded vesicles did not depend on ATP. Addition of the ionophores A23187 and Ro 20-0006/006 caused an immediate release of accumulated free Ca
2+
. ATP-dependent Ca
2+
uptake was not inhibited by 10 μM oligomycin.
Evidence for splicing of avian sarcoma virus 5′-terminal genomic sequences onto viral-specific RNA in infected cells
Authors: Krzyzek R. A. , Collett M. S. , Lau A. F. , Perdue M. L. , Leis J. P. , Faras A. J.
Published: May 2006

The 5′-terminal nucleotide sequences of the avian sarcoma virus (ASV) genome are transcribed by the reverse transcriptase
in vitro
into a DNA transcript that represents the entire distance (∼100 nucleotides) between the tRNA
Trp
primer molecule and the 5′ terminus. We have used these DNA
100
transcripts in hybridization reactions with ASV-specific RNA from infected avian cells and find nucleotide sequences complementary to these transcripts on all of the various size classes of viral mRNA identified. Similar hybridization results were obtained with a specific DNA transcript complementary to viral genomic nucleotide sequences between the tRNA
Trp
primer molecule and up to, but not including, the terminal redundant sequences (DNA
70
), indicating that the observed hybridization of DNA
100
to all size classes of viral RNA in infected cells did not reflect hybridization of DNA
100
to the terminal redundant sequences at the 3′ end of the viral genome.
Escherichia coli
RNase H hydrolysis of RNA·DNA hybrids consisting of genomic 35S RNA obtained from virus and DNA
100
transcripts indicated that viral genomic sequences complementary to these DNA transcripts were not present at sites distal to the ends of the RNA genome and therefore not adjacent to the corresponding gene sequences representing the various species of viral mRNA from infected cells. These studies suggest that the 5′-terminal genomic nucleotide sequences, or a portion thereof, are somehow added or “spliced” onto each ASV-specific mRNA species in infected cells either during or after transcription of proviral DNA for some as yet undetermined purpose.
Composition of vacuoles and sarcoplasmic reticulum in fatigued muscle: electron probe analysis.
Authors: Gonzalez-Serratos H , Somlyo A V , McClellan G , Shuman H , Borrero L M , Somlyo A P
Published: May 2006

Electron probe analysis, cryo-ultramicrotomy, and freeze-substitution were used to determine the nature of vacuolation and the subcellular composition in fatigued frog skeletal muscle fibers. The vacuoles caused by fatigue were part of the T-tubule system and contained high concentrations of NaCl. The calcium concentration in the terminal cisternae was higher than previously measured normal resting values. Mitochondrial calcium content was relatively low (mean +/- SEM, 2 +/- 2 mmol/kg dry weight). Fiber NaCl was increased. It is concluded that fatigue is not due to the depletion of calcium stores from the terminal cisternae or to uncoupling of mitochondria due to calcium loading but may be caused by multiple mechanisms including failure of the T-tubule action potential.
Regulation of glutamine synthetase in cultured 3T3-L1 cells by insulin, hydrocortisone, and dibutyryl cyclic AMP
Authors: Miller Richard E. , Hackenberg Richard , Gershman Howard
Published: May 2006

The 3T3-L1 mouse fibroblast cell line develops morphological and biochemical characteristics of adipocytes when maintained at confluence. This conversion to adipocytes is accelerated by addition of insulin to the culture medium [Green, H. & Kehinde, O. (1975)
Cell
5, 19-27]. During the course of the insulin-mediated adipocyte conversion, the specific activity (units/mg of protein) of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] increases more than 100-fold. The specific activities of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) and glucose-6-
P
dehydrogenase (D-glucose-6-phosphate:NADP
+
1-oxidoreductase, EC 1.1.1.49) also increase but less dramatically (1.5- to 3-fold). In contrast, confluent cells maintained in the absence of insulin for the same time (12-20 days after confluence) display only minimal increases in the activity of these enzymes. Maintenance of confluent cells in culture medium lacking added L-glutamine has little, if any, effect on glutamine synthetase activity in either control or insulin-treated cultures. Treatment of confluent 3T3-L1 cultures with hydrocortisone (1 μg/ml) for 3 days prior to harvesting results in an increase in glutamine synthetase specific activity of 12-fold for control cultures maintained for 13 days in the absence of insulin and 1.4-fold for adipocyte cultures maintained for 13 days in the presence of insulin (10 μg/ml). Treatment of 3T3-L1 control cells and adipocytes with dibutyryl cyclic AMP (1 mM) plus theophylline (1 mM) decreases the glutamine synthetase specific activity and almost completely reverses the insulin- and hydrocortisone-mediated increases in enzyme activity. In contrast, treatment with dibutyryl cyclic AMP plus theophylline has relatively little effect on the specific activities of hexokinase or glucose-6-
P
dehydrogenase or on the protein content of the cultures. These data indicate that glutamine synthetase activity is hormonally regulated in 3T3-L1 cells.
Influence of homoeologous chromosomes on gene-dosage effects in allohexaploid wheat (Triticum aestivum L.).
Authors: Aragoncillo C , Rodríguez-Loperena M A , Salcedo G , Carbonero P , García-Olmedo F
Published: May 2006

Gene-dosage responses for a group of six wheat endosperm proteins have been investigated by using compensated nulli-tetrasomic lines of cv. Chinese Spring. Practically linear dosage responses have been observed for all the proteins. However, for two of the proteins (and probably for a third one), the net output of protein, at each dosage of its structural gene, was 30-80% higher when the chromosome carrying an active homoeogene was absent. The possible significance of this effect in connection with the loss of gene redundancy undergone by polyploids is discussed.
Analysis of malignancy in human cells: malignant and transformed phenotypes are under separate genetic control.
Authors: Stanbridge E J , Wilkinson J
Published: May 2006

Human cell hybrids derived from malignant HeLa and normal fibroblast parental cells expressed many of the transformed properties of the HeLa parent but their tumor-producing capability was suppressed. Hybrids derived from HeLa/HeLa fusions retained both their transformed and malignant phenotypes. Thus, an apparent separation of the control of the transformed versus malignant phenotype is indicated. Furthermore, several transformed properties--including lack of density-dependent inhibition of growth, lectin agglutination, lowered requirement for serum growth factors, and anchorage independence--are expressed coordinately in the nontumorigenic hybrids. This finding suggests that none of these properties by themselves, or in concert, endows a cell with tumorigenic potential.
Development of a spin assay for reserve bilirubin loading capacity of human serum.
Authors: Hsia J C , Kwan N H , Er S S , Wood D J , Chance G W
Published: May 2006

The capacity of human serum albumin to bind bilirubin is of paramount importance in the prevention of neurological damage due to hyperbilirubinemia in the neonate. Currently, there is no rapid, reliable method to determine the reserve bilirubin loading capacity of serum albumin. A method using electron spin resonance spectroscopy with a dianionic spin-label molecular probe [1-N-(2,2,6,6-tetramethyl-1-oxyl-4-piperidinyl)-5-N-(1-aspartate)-2,4-dinitrobenzene] is presented which yields the reserve binding capacity of the bilirubin high-affinity binding site on human serum albumin and reserve bilirubin loading capacity of serum in mg/dl. Clinical validation of this spin assay may improve the neonatal care of jaundiced newborns.
Calcium leakage as a cause of the high resting tension in vascular smooth muscle from the spontaneously hypertensive rat.
Authors: Noon J P , Rice P J , Baldessarini R J
Published: May 2006

Aortic strips from spontaneously hypertensive (SH) rats relax in calcium-free physiological medium and contract to approximately 60% of maximum when calcium is again restored to the medium. In vivid contrast, the resting tension of aortic strips from normal rats is unaffected by manipulation of the calcium concentration of the bathing medium. These findings, as well as the reduced sensitivity of aortic strips from SH rats to norepinephrine and the observation that aortic strips from SH rats relax at a faster rate in calcium-free medium in comparison with aortic strips from normal rats, are consistent with the hypothesis that vascular smooth muscle membranes from SH rats leak calcium at a rate that is only partially compensated by the calcium pump.
A simple model of DNA superhelices in solution.
Authors: Camerini-Otero R D , Felsenfeld G
Published: May 2006

Closed circular DNA molecules in aqueous solution take the form of interwound superhelices over a wide range of superhelix densities. We describe a very simple model of such a superhelix in which twisting and bending forces are in balance, subject both to topological constraints and to a limitation on the distance of closet approach of the interwound duplexes of the superhelix. The model is consistent with some of the observed physical properties of closed circular DNA, and suggests that there may be severe limits to the range of allowable geometries for the superhelix structure of minimum energy.
Regulation of adenylate cyclase activity mediated by muscarinic acetylcholine receptors.
Authors: Nathanson N M , Klein W L , Nirenberg M
Published: May 2006

Carbachol, an activator of muscarinic acetylcholine receptors of NG108-15 hybrid cells, inhibits adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] rapidly and reversibly and slowly evokes a 200-300% increase in adenylate cyclase activity over a period of 24-30 hr. Both the inhibition of adenylate cyclase and the gradual increase in enzyme activity are dependent on muscarinic acetylcholine receptors and the receptor activator. Withdrawal of carbachol results in a gradual return of adenylate cyclase activity to control levels over a period of 6 hr; the half-life for decay of enzyme activity is 1.6 hr. These results show that muscarinic acetylcholine receptors mediate both transient and long-lived effects on adenylate cyclase activity that resemble those of opiates.
Founder effect and number of private polymorphisms observed in Amerindian tribes.
Authors: Neel J V , Thompson E A
Published: May 2006

In studies extending over the past dozen years, we have observed eight examples of "private" genetic polymorphisms in 12 Amerindian tribes surveyed for electrophoretic variants of an average of 25 proteins. Each of these is presumed to trace to a single mutation. In a preceding communication [Thompson, E.A. & Neel, J.V. (1978) Proc. Natl. Acad. Sci. USA 75, 1442-1445] the statistical theory was developed for estimating the likelihood of such a founder effect in a tribal population of this type. In this paper that theory is applied to the distribution defined by these eight variants. It is demonstrated that on the assumption that the phenotypes in question are selectively neutral, such findings are most compatible with a mutation rate of 7 X 10(-6)/locus per generation. This figure applies only to variants that can be detected by the electrophoretic technique.
Regulation by membrane sialic acid of β1H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway
Authors: Fearon Douglas T.
Published: May 2006

Sheep erythrocytes in their native state did not activate the alternative complement pathway, as measured by lysis in dilutions of normal human serum containing [ethylenebis(oxyethylenenitrilo)] tetraacetic acid but acquired this capacity after membrane sialic acid residues had been removed (by sialidase) or modified (by NaIO
4
). Activation of the alternative pathway by sheep erythrocytes required removal or modification of at least 40% of the membrane sialic acid to reach threshold, and it increased proportionately when larger amounts of sialic acid had been affected. Studies with isolated proteins of the alternative pathway demonstrated that the altered erythrocyte membranes resembled natural activators in protecting bound C3b from inactivation by C3b inactivator and β1H and protecting bound amplification C3 convertase (C3b,Bb) from decay-dissociation by β1H. A 1% decrease in intact sialic acid was associated with a 1% decrease in β1H activity in decay-dissociation of membrane bound C3b,Bb. Because removal of the C8 and C9 carbon atoms from the polyhydroxylated side chain of sialic acid by oxidation with NaIO
4
was functionally equivalent to removal of the entire sialic acid moiety, secondary effects of the latter reaction, such as diminution of the negative charge of the membrane or exposure of penultimate galactose residues, were not considered to be responsible for the altered activity of β1H. These studies suggest that facilitation, by membrane sialic acid residues, of the interaction between bound C3b and β1H is essential to prevent the particle from effectively activating the alternative pathway.
Human bronchus-mediated mutagenesis of mammalian cells by carcinogenic polynuclear aromatic hydrocarbons.
Authors: Hsu I C , Stoner G D , Autrup H , Trump B F , Selkirk J K , Harris C C
Published: May 2006

Cultured human bronchial explants activated benzo[alpha]pyrene (BzaP) into electrophilic metabolites that bind to DNA in bronchial epithelial cells. Promutagenic and mutagenic metabolites of BzaP were also released into the culture medium. An increase in mutation frequency for ouabain resistance was found in Chinese hamster V-79 cells when they were cocultivated with bronchial explants in the presence of BzaP. The proximate carcinogenic form of BzaP, the 7,8-diol [(+/-)-r7,t8-dihyroxy-7,8-dihydrobenzo[a]pyrene], was 5-fold more potent as a promutagen than the parent compound. Neither BzaP nor the 7,8-diol increased the mutation frequency in V-79 cells when they were cultured without bronchial explants. The mutation frequency was directly related to the binding levels of BzaP to bronchial DNA and the concentratin of either BzaP or the 7,8-diol in the medium.
Biosynthesis of reverse transcriptase from Rauscher murine leukemia virus by synthesis and cleavage of a
gag-pol
read-through viral precursor polyprotein
Authors: Kopchick J. J. , Jamjoom G. A. , Watson K. F. , Arlinghaus R. B.
Published: May 2006

Reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase) from Rauscher leukemia virus is synthesized in infected cells by way of a read-through poly- rotein of 200,000 molecular weight. This polyprotein (Pr200

gag-pol

) was precipitated by antiserum to RT; in a previous study all the monospecific antisera to
gag
proteins recognized Pr200

gag-pol

. Pr200

gag-pol

contains both p30 and RT peptide sequences. Intermediate RT-related precursors of 145,000 (Pr145

pol

), 135,000 (Pr135

pol

), and 125,000 (Pr125

pol

) molecular weights were specifically recognized by precipitation from infected cell extracts by antiserum to RT. These proteins shared methionine-containing tryptic peptide sequences with a virion polypeptide of 80,000 molecular weight (p80

pol

) precipitate by antiserum to RT. Purification of active RT enzyme from virions labeled with [
3
H]methionine showed that p80

pol

was the major component, based on analysis by gel electrophoresis and tryptic peptide mapping experiments. A polypeptide (Pr80

pol

), similar in size to mature viral p80

pol

, was also precipitated from infected cells by antiserum to RT. Its peptide map was nearly identical to that of virion p80

pol

. Pulse-chase studies showed that Pr80

pol

, Pr125

pol

, and Pr135

pol

were stable polypeptides, whereas Pr200

gag-pol

and Pr145

pol

were unstable precursors. Pulse-chase studies with the protein synthesis inhibitor, cycloheximide, showed that the processing of Pr200

gag-pol

occurred for a short time in the absence of protein synthesis.
Regulation of cholesterol biosynthesis in HeLa S3G cells by serum lipoproteins: Dexamethasone-mediated interference with suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase
Authors: Cavenee Webster K. , Johnston Dean , Melnykovych George
Published: May 2006

Depression of the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase [mevalonate:NADP
+
oxidoreductase (CoA-acylating); EC 1.1.1.34] was elicited by the removal of serum from the growth medium of HeLa S3G cells with a concomitant expected increase in cellular sterol biosynthesis; if dexamethasone (9α-fluoro-11β,17α,21-trihydroxy-16α-methyl-1, 4-pregnadiene-3,20-dione) was present in the serumless medium, there was an augmentation of HMG-CoA reductase activity but a suppression of sterol biosynthesis. When human serum, human low density lipoprotein, or calf serum was present in the medium, there was a reduction of both the enzyme activity and sterol biosynthesis, but the presence of dexamethasone resulted in an increase in HMG-CoA reductase activity as compared to the controls containing human serum, low density lipoprotein, or calf serum alone. In contrast, either low density lipoprotein or whole serum supplementation eliminated the differences in acetate incorporation into sterols between glucocorticoid-treated and untreated cells. Human high density lipoproteins had little effect on the enzyme activity and abolished the difference in sterol biosynthesis only at relatively high concentrations. Addition of low density lipoproteins to cells after preincubation in serumless medium elicited the same rate of decay of HMG-CoA reductase (
t
1/2
3.8-4.2 hr) regardless of the presence of glucocorticoids in the medium, but there was an exaggerated lag before the onset of suppression in the hormone-treated cells. If free cholesterol was present in the medium, the dexamethasone augmentation of HMG-CoA reductase was maintained, but the addition of either 7-ketocholesterol or 25-hydroxycholesterol abolished the difference between glucocorticoid-treated and control cells. These observations suggest that, under certain physiological conditions, HMG-CoA reductase activity no longer accurately reflects cellular sterol biosynthesis.
Mapping of linear and circular forms of mouse mammary tumor virus DNA with restriction endonucleases: evidence for a large specific deletion occurring at high frequency during circularization.
Authors: Shank P R , Cohen J C , Varmus H E , Yamamoto K R , Ringold G M
Published: May 2006

Rat hepatoma cells infected with mouse mammary tumor virus contain multiple forms of unintegrated viral DNA when grown in the presence of glucocorticoids. Using the DNA transfer procedure of Southern, we have prepared restriction endonuclease fragment maps of these forms of viral DNA. The maps indicate that: (i) the major species of viral DNA is a linear molecule of 5.9 X 10(6) Mr located in the cytoplasm; (ii) the nuclei contain covalently closed circular viral DNA of two distinct sizes (5.1 X 10(6) and 5.9 X 10(6) Mr) in addition to linear molecules (5.9 X 10(6) Mr); (iii) the linear molecule has specific termini; (iv) there is extensive homology between regions at or near termini of the linear molecule; (v) the predominant form of circular DNA lacks 1.2 kilobase pairs present in both the larger circular molecule and the linear molecule; and (vi) the sequences deleted from the majority of the circular DNA molecules are located at the ends of the linear DNA that are joined during circularization.
A general method to assess similarity of protein structures, with applications to T4 bacteriophage lysozyme.
Authors: Remington S J , Matthews B W
Published: May 2006

A method is proposed that permits the structural similarity between any pair of proteins to be analyzed in a completely general manner. In the proposed procedure, all possible structural segments of a given length from one protein are compared with all possible segments from the other protein. This set of comparisons reveals any structural similarities between the two proteins being compared, and also provides a basis for estimating the probability that a particular degree of structural homology could have occurred by chance. Application of the method to the comparison of T4 bacteriophage lysozyme and carp calcium-binding protein suggests that the previously reported structural similarity between parts of these two proteins [Tufty, R. M.& Kretsinger, R. H. (1975) Science 187, 167-169] is no better than would be expected by chance. On the other hand, the structural correspondence between phage lysozyme and hen egg-white lysozyme [Rossman, M.G. & Argos, P. (1976) J. Mol. Biol. 105, 75-96] does appear to be significant.
Synapse turnover: a mechanism for acquiring synaptic specificity.
Authors: Ruffolo R R , Eisenbarth G S , Thompson J M , Nirenberg M
Published: May 2006

Neurons are generated in chick retina that are able to form synapses with striated muscle cells for only a brief period during embryonic development. The ability to form synapses is lost with a half-life of 21 hr. Retina neuron-myotube synapses form rapidly but soon are terminated. Chick embryo spinal cord neurons also form synapses with muscle cells for only a limited time during development, but these synapses are long lived. These results show that different classes of synapses turn over at different rates and suggest that part of the specificity of synaptic circuits may be acquired during development by a process of selection based on synapse termination rates.
Fe-O2 bonding and oxyheme structure in myoglobin.
Authors: Makinen M W , Churg A K , Glick H A
Published: May 2006

In the polarized electronic absorption spectrum of oxymyoglobin in single crystals, charge-transfer states involving orbitals of the iron and dioxygen ligand are defined as probes of oxyheme orbital structure and coordination geometry. The spectrum of sperm whale oxymyoglobin is diagnostic of a bent (formula: see text) oxheme coordination geometry with totally spin-paired, ground-state electronic configurations of the iron and of the dioxygen ligand. In contrast, Aplysia myoglobin is distinguishably different in oxyheme structure, indicating that the geometry of Fe-O2 bonding in heme proteins can be altered by the protein environment.
Interactions between adenovirus, a tumor promoter, and chemical carcinogens in transformation of rat embryo cell cultures.
Authors: Fisher P B , Weinstein I B , Eisenberg D , Ginsberg H S
Published: May 2006

The tumor-promoting agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA) caused a 2- to 3-fold enhancement of transformation of secondary rat embryo cells that had been injected with a temperature-sensitive mutant of adenovirus type 5(H5ts 125). In addition, transformed foci appeared earlier and were larger in cultures grown in the presence of TPA. The addition of TPA could be delayed until up to 7 days after viral injection and still enhancement was observed. Exposure of the cells to 7,12-dimethylbenz[a]pyrene prior to H5ts125 infection also resulted in a 2- to 4-fold enhancement of transformation, and this enhancement was further augmented 2- to 3-fold when cells were grown in TPA after virus infection. Whereas TPA did not enhance the cloning efficiency of normal rat embryo cells, it did enhance the cloning efficiency of isolated colonies of adenovirus-transformed cells when these were grown alone or cocultured with a 100-fold excess of normal rat embryo cells. The enhancement of adenovirus transformation by TPA appears to be due to its ability to facilitate expression of the transformed state rather than an effect on virus uptake or integration.