bluuebunny/crossref_metadata_2025 huggingface dataset:
Superiority of interconvertible enzyme cascades in metabolic regulation: Analysis of multicyclic systems
Authors: Chock P. B. , Stadtman E. R.
Published: May 2006

Escherichia coli
glutamine synthetase and glycogen phosphorylase are prototypes for models of “closed” and “opened” bicyclic cascade systems. Steady-state functions relating the fractional activation of interconvertible enzymes to the concentrations of allosteric effectors and to the catalytic constants of the several converter enzymes in such cascades were determined. The study shows that when the active form of an interconvertible enzyme in one cycle catalyzes the covalent modification of the interconvertible enzyme in a second cycle, the two cycles become coupled, such that the fractional activity of the second interconvertible enzyme is a multiplicative function of all parameters in both cycles, i.e., of 14 and 18 parameters for the closed and the opened bicyclic cascade, respectively. Therefore, from the standpoint of cellular regulation, bicyclic cascades are superior to the monocyclic cascades analyzed previously [E. R. Stadtman & P. B. Chock (1977)
Proc. Natl. Acad. Sci. USA
74, 2761-2765], because: (
i
) they can respond to a greater number of allosteric effectors; (
ii
) they can achieve much greater amplification of responses to primary stimuli (e.g., with only 2-fold changes in each parameter the amplification factors of one-cycle and two-cycle cascades are 320 and 102,400, respectively); (
iii
) they can generate a sigmoidal response (Hill numbers of >2) of interconvertible enzyme activity to increasing concentrations of an allosteric effector. This is because there are more steps in a bicyclic cascade at which a given effector can interact. A similar analysis of multicyclic cascade systems shows that the capacity for amplification increases exponentially as the number of cycles in the cascade increases. In addition, regulation by cyclic cascades can achieve enormous variability of the fractional activity of the interconvertible enzyme by shifting the steady-state distribution between active and inactive forms. One equivalent of ATP is consumed in each interconversion cycle to provide the energy needed to maintain the steady-state activity of the modified enzyme at a metabolically required level. Therefore, the decomposition of ATP associated with the cyclic cascade is not a wasteful process.
Extensive disulfide bonding at the mammalian cell surface.
Authors: Hynes R O , Destree A
Published: May 2006

Cell surface proteins of cultured cells are disulfide bonded to a greater degree than are total cellular proteins. In particular, the "large external transformation-sensitive" (LETS) protein, a major surface protein, is present almost exclusively in disulfide-bonded complexes including homodimers and also higher aggregates held together by disulfide bonds or concovalent interactions. Other cell surface proteins also appear to be involved in disulfide bonding, both intramolecular and intermolecular. In virally transformed cells, LETS protein and its disulfide complexes are absent and certain other disulfide-bonded proteins are also not observed.
Regulation of DNA synthesis: age-dependent cooperation among G1 cells upon fusion.
Authors: Rao P N , Sunkara P S , Wilson B A
Published: May 2006

The object of this study was to determine whether the inducer(s) of DNA synthesis in mammalian cells accumulates gradually throughout the G1 period or becomes available suddenly at the G1-S transition. HeLa cells, synchronized at various points in the G1 period, were fused by using UV-inactivated Sendai virus. Early G1 cells were fused with mid-G1 or late G1 cells and late G1 cells were fused with mid-G1 cells. The G1 traverse of mono-, bi-, and trinucleated cells was studied. The bi- and trinucleated cells of mid-G1 and late G1 parents traversed the G1 period significantly faster than did their mononucleated counterparts. The reduction in the duration of the G1 period was proportional to the number and age of nuclei at the time of fusion. There was no significant difference between the mono- and binucleated cells of the early G1 parent in their rates of entry into S period. In light of these findings, a model is proposed in which the inducer(s) of DNA synthesis accumulates gradually throughout the G1 period, reaching a critical level at the G1-S boundary when DNA replication is initiated; after reaching a peak during early or mid-S period, it declines to below the critical level when DNA synthesis ceases.
Glycolipid-bound sialic acid in serum: increased levels in mice and humans bearing mammary carcinomas.
Authors: Kloppel T M , Keenan T W , Freeman M J , Morré D J
Published: May 2006

In mice bearing transplantable mammary carcinomas, serum levels of sialic acid-containing glycolipids were elevated 2.5-fold in pooled serum samples from which gangliosides were purified by column chromatography. A method is also described by which ganglioside content was estimated on as little as 1.0 ml of whole blood to permit studies with individual tumor-bearing mice and age- and litter-matched controls. Using this method, we observed similar elevations in ganglioside levels that were independent of age and sex of the animal and appeared in advance of palpable tumors. Following excision of the tumors, the glycolipid sialic acid values dropped below control levels and remained there. Serum sialic acid of the glycolipid fraction was elevated nearly 2-fold in human carcinoma patients and appeared to decline after surgery.
Independent expression of the gene coding for the constant domain of immunoglobulin light chain: Evidence from sequence analyses of the precursor of the constant region polypeptide
Authors: Burstein Yigal , Zemell Ronald , Kantor Frida , Schechter Israel
Published: May 2006

The mRNA coding for the κ-type constant region (C
κ
) was purified from two clones derived from the MPC-11 mouse myeloma. This mRNA directs the cell-free synthesis of a C
κ
precursor (molecular weight, about 15,000) in which an extra piece, 17 residues long, precedes the NH
2
-terminal residue (Ala
109
) of the C
κ
region. The partial sequence of the extra piece is: Met-X-Thr-Asp-Thr-Leu-Leu-Leu-Trp-Val-Leu-Leu-Leu-Trp-Val-Pro-X- (X is unknown). Met
1
was shown to be the initiator methionine. The sequence of the C
κ
extra piece is completely different from any known sequence preceding residue Ala
109
in whole light (L) chains, thus establishing that the C
κ
-region mRNA could not have originated from mRNA coding for the whole L chain. The structural features of the C
κ
extra piece (marked hydrophobicity, size, and a methionine at the NH
2
-terminus) are identical to those characteristic of the NH
2
-terminal extra piece linked to the variable (V) region of whole L-chain precursors. In addition, the C
κ
extra piece and the extra piece linked to the V region of MOPC-321 L chain have 70% sequence homology. These findings can be explained by the two genes-one Ig chain hypothesis, if we assume that the DNA coding for the extra piece (xp-DNA) is a constitutive part of the
V
gene. According to this model, the C
κ
-region mRNA could have originated from: (
i
) translocation of this
V
gene to the
C
gene, deletion of the entire mature
V
gene, and “end-to-end” repair of the remaining xp-DNA to the
C
gene; (
ii
) translocation to the
C
gene only of the xp-DNA portion of the
V
gene. Alternatively, we may assume that the xp-DNA is not covalently linked to the mature
V
gene at all times, as might be the case for the DNA of hypervariable regions presumed to be in episomes. This raises the intriguing speculation that the xp-DNA represents a third distinct gene, designated
xp
-gene. The presumed
xp
-gene may be involved in the regulation of gene transcription: when linked to the mature
V
gene it initiates a chain of events leading to whole L-chain mRNA formation; when attached to the
C
gene it leads to its transcription to provide the C-region mRNA.
Stimulation of RNA synthesis in isolated nuclei by partially purified preparations of simian virus 40 T-antigen.
Authors: Ide T , Whelly S , Baserga R
Published: May 2006

T-Antigen was partially purified from nuclei of cells transformed by simian virus 40 (SV 40). When nuclei isolated from either rat liver or quiescent hamster cells were preincubated with T-antigen preparations, there was a marked stimulation of RNA synthesis in an in vitro assay, up to 150% above control levels. The stimulation of RNA synthesis was inhibited by hamster antiserum against T-antigen but not by normal hamster serum. When the T-antigen preparations were fractionated on glycerol gradients, the fractions containing complement-fixing activity with antiserum to T-antigen also had the highest stimulatory activity on nuclear RNA synthesis. T-Antigen was also partially purified from nuclei of cells transformed by a temperature-sensitive A mutant of SV40. When preincubated up to 2 hr at 50 degrees, the T-antigen preparation from these temperature-sensitive A mutants was rapidly inactivated, in terms of both complement-fixing activity and ability to stimulate RNA synthesis in isolated rat liver nuclei. Under the same conditions of preincubation, T-antigen preparations from cells transformed by wild-type SV40 maintained their complement-fixing activity and ability to stimulate RNA synthesis. These results suggest that the biological action of T-antigen may be exerted at the level of transcription.
Identification and cloning of the chloroplast gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase from Chlamydomonas reinhardi.
Authors: Gelvin S , Heizmann P , Howell S H
Published: May 2006

mRNA coding for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from Chlamydomonas reinhardi has been isolated from small polyribosomes immunoadsorbed to column-bound anti-LS antibody. 32P-Labeled LS mRNA was used as a hybridization probe to detect LS genes. The probe hybridized to C. reinhardi chloroplast DNA and at hybridization saturation revealed that there are approximately 75 LS genes per chloroplast. When chloroplast DNA was digested with the restriction endonuclease EcoRI and the fragments were transferred to a nitrocellulose filter, the LS mRNA probe hybridized to a DNA fragment of molecular weight 3.2 X 10(6). This same fragment codes (in part) for 16S and 23S chloroplast rRNAs, which are also coded (in part) by fragments of molecular weights 9.0, 2.3, and 0.4 X 10(6). The restriction fragment containing the LS gene has been cloned in the Escherichia coli plasmid pMB9.
Membrane assembly
in vitro
: Synthesis, glycosylatio, and asymmetric insertion of a transmembrane protein
Authors: Katz Flora N. , Rothman James E. , Lingappa Vishwanath R. , Blobel Günter , Lodish Harvey F.
Published: May 2006

Membrane assembly was observed to proceed in cell-free extracts. Specifically, the membrane glycoprotein of vesicular stomatitis virus was synthesized in crude extracts of wheat germ in the presence of membrane vesicles derived from pancreatic endoplasmic reticulum. The resulting glycoprotein spans the lipid bilayer asymmetrically, is glycosylated, and is indistinguishable in these respects from the form of the glycoprotein found in the rough endoplasmic reticulum of virus-infected cells. Both glycosylation and asymmetric transmembrane insertion of the glycoprotein into membranes
in vitro
require protein synthesis in the presence of membranes. The carboxyl-terminal 5% of the polypeptide chain is located on the external surface of vesicles, corresponding to the cytoplasmic surface of the endoplasmic reticulum in cells. Most, or all, of the amino-terminal portion of the glycoprotein, as well as the protein-bound carbohydrate, appears to be located within the lumen of the membrane vesicles. These findings demonstrate that insertion of this membrane protein occurs during or immediately after protein synthesis. The results are consistent with the concepts that the growing membrane protein is extruded across the endoplasmic reticulum membrane amino terminus first and that glycosylation is restricted to the lumenal surface of the membrane. The cell-free system reported here should prove valuable for studying these processes.
Intermediate role of adenosine 3′:5′-cyclic monophosphate and protein kinase during gonadotropin-induced steroidogenesis in testicular interstitial cells
Authors: Dufau Maria L. , Tsuruhara Tsuneo , Horner Kathleen A. , Podesta Ernesto , Catt Kevin J.
Published: May 2006

Discrepancies between adenosine 3′:5′-cyclic monophosphate (cAMP) and steroid production have been frequently observed in isolated target cells stimulated by low concentrations of trophic hormone. This dissociation is particularly marked in the interstitial cells of the testis, where testosterone production is elicited by gonadotropin concentrations in the picomolar range. Because of these observations, and a disparity between steroidogenesis and protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activation in Leydig cells, the role of cAMP as a mediator of the acute steroidogenic response has been questioned. This problem has been further analyzed by assay of free and occupied cAMP-binding sites of the regulatory subunit of protein kinase in basal and hormone-stimulated cells. Free sites were measured by a [
3
H]-cAMP-binding assay, and occupied sites were measured by radioimmunoassay of endogenous cAMP eluted from receptor protein.

After stimulation of purified Leydig cells with 0.1-10 pM human chorionic gonadotropin, a dose-dependent decrease in available [
3
H]cAMP-binding sites was observed, with no change in binding affinity. The reduction in cAMP-binding sites was equivalent to the increase in occupancy of cAMP receptors by endogenous nucleotide formed during gonadotropin action. Fractional occupancy of cAMP receptors rose progressively from basal values of 0.2-0.40 to full saturation as intracellular cAMP rose 10- to 30-fold during hormone stimulation. The testosterone dose-response curve was coincident with the initial part of the cAMP-receptor occupancy curve. These changes in endogenous cAMP binding to the regulatory subunit were accompanied by a significant increase in protein kinase activity in gonadotropin-stimulated Leydig cells. These observations provide direct evidence for the role of cAMP and protein kinase during hormonal activation of steroidogenesis in the Leydig cell by low concentrations of gonadotropin.
Vasoactive intestinal polypeptide in brain: localization in and release from isolated nerve terminals.
Authors: Giachetti A , Said S I , Reynolds R C , Koniges F C
Published: May 2006

The vasoactive intestinal polypeptide was present in synaptosomal (nerve ending) preparations from cerebral cortex, hypothalamus, and striatum of rat brain in higher concentrations than in these tissues as a whole. The total content and relative specific activity of the peptide increased with progressive purification of the synaptosomal fractions and generally followed the distribution of known synaptosomal constituents--dopamine, norepinephrine, and lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27). The peptide was also released from synaptosomal pellets with increased K+ concentration, and this release was Ca2+-dependent. The findings suggest a role for vasoactive intestinal polypeptide as a transmitter or modulator of synaptic function.
Changes in surface properties of normal and transformed cells caused by tunicamycin, an inhibitor of protein glycosylation.
Authors: Duksin D , Bornstein P
Published: May 2006

Normal and virally transformed mouse (3T3) and human (WI-38) cells were treated with tunicamycin, an inhibitor of lipid-carrier-dependent glycosylation of proteins. Incubation of cells with tunicamycin (1 microgram/ml) caused detachment and death of simian virus 40- and polyoma-transformed cells within 24 hr; these effects were not seen with nontransformed cell lines. However, the proliferation of 3T3 cells was inhibited by tunicamycin and, after a few days, a distinct change from an epithelioid to an abnormally elongated shape was observed. Both inhibition of growth and the morphological changes were reversible. A marked decrease in concanavalin A agglutinability was observed in virally transformed cells treated with tunicamycin (0.5 microgram/ml), but agglutination by wheat germ agglutinin or soybean agglutinin was unaffected. Analysis of biosynthetically labeled proteins showed that a high-molecular-weight protein, presumed to be related to fibronectin, is markedly reduced in the medium of cells cultured in the presence of tunicamycin. These results suggest that tunicamycin interferes with the insertion or function of one or more cell-surface glycoproteins. Such cell-surface changes could affect a number of cellular properties, including attachment, cell shape, and agglutinability by some lectins.
Large-scale automated isolation of Escherichia coli mutants with thermosensitive DNA replication.
Authors: Sevastopoulos C G , Wehr C T , Glaser D A
Published: May 2006

We have screened about 1.4 million colonies of Escherichia coli K-12 for their ability to grow on nutrient agar at 30 degrees and 41 degrees. Among the 2266 temperature-sensitive mutants found, 110 were defective in DNA synthesis but not in protein synthesis at 41 degrees. Three of these dna mutations mapped at two previously undescribed loci on the E. coli genetic map and may represent new genes involved in DNA replication in E. coli. The mutant isolation was aided by novel automatic machinery that inoculated agar-filled petri dishes with mutagenized E. coli cells laid down in square arrays of evenly spaced rows and columns on the agar. Time-lapse photographs taken before and after a temperature shift were used to find colonies of temperature-sensitive mutants. These mutations were mapped by interrupted conjugation and viral cotransduction methods, and the mutants were divided into three classes according to the kinetics of DNA synthesis at the restricted temperature. Some of the mutants exhibited mutator activity at partially restrictive temperatures. It is argued that some genes involved in DNA synthesis remain to be discovered.
Conformational requirements for dopamine-induced vasodilation.
Authors: Volkman P H , Kohli J D , Goldberg L I , Cannon J G , Lee T
Published: May 2006

The availability of a series of semi-rigid analogs of dopamine and epinine has made it possible to investigate the conformational requirements for action on dopamine and beta2-adrenergic vascular receptors. The analogs were screened for dopamine-agonist action by intra-arterial injections into the renal vascular bed and for beta2-adrenergic activity by similar injections into the femoral vascular bed in dogs pretreated with phenoxybenzamine. 2-Amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (A-6,7-DTN) and its N-methyl derivative (analogous to the trans beta rotamer of dopamine) exhibited pronounced dopamine-agonist activity and minimal beta2-adrenergic activity. In contrast, the semi-rigid analog of the trans alpha rotamer of dopamine (2-amino-5,6-dihydroxy-1,2,3,4-tetrahydronaphthalene; A-5,6-DTN) and its N-methyl derivative exerted pronounced beta2-adrenergic activity but were inactive as dopamine agonists.6,7-Dihydroxytetrahydroisoquinoline, a semi-rigid analog of the cis beta rotamer of dopamine, did not produce renal vasodilation. These results indicate that a conformation of dopamine similar to that found in A-6,7-DTN is required for dopamine-vascular activity, while the conformation found in A-5,6-DTN is preferred for interaction with beta2-adrenergic receptors.
Theoretical study of the effect of enzyme-enzyme interactions on steady-state enzyme kinetics.
Authors: Hill T L
Published: May 2006

Equilibrium statistical mechanics is much concerned with problems involving intermolecularinteractions, either in lattices or in pure fluids or solutions. The possibility of enzyme-enzyme interactions suggests that the same problems might be studied profitably at steady state as well as at equilibrium. In the systems we consider, each of the identical enzyme molecules of the system undergoes steady-state stochastic cycling among states i equal 1,....,n. But the molecules do not cycle independently. Two neghboring molecules, in states i and j, interact with a free energy wij (a function of the distance r in the solution case).The instantaneous transition probabilities between states for a given molecule will depend on the instantaneous interactions between the molecule in question and its neighbors. The primary question of interest is how the enzyme flux is influenced by the interactions. The general problem is outlined here and some simple special cases are treated. The discussion will be continued in a following paper [Hill, T. L. (1977) Proc. Natl. Acad. Sci. USA 74, in press]
Evidence for pro-β-nerve growth factor, a biosynthetic precursor to β-nerve growth factor
Authors: Berger Edward A. , Shooter Eric M.
Published: May 2006

The biosynthesis of β-nerve growth factor (βNGF) was studied in mouse submaxillary glands incubated with L-[
35
S]cystine. βNGF was isolated from tissue extracts by the addition of antiserum against βNGF and the washed immunoprecipitates were analyzed by sodium dodecyl sulfate gel electrophoresis. With short labeling periods (10 and 25 min) there is a major labeled species with an apparent molecular weight of 22,000 and a smaller peak comigrating with purified βNGF chains (13,260). As time proceeds, the radioactivity in the 22,000 molecular weight peak plateaus, while the label in βNGF continues to increase, until by 4 hr it greatly exceeds the radioactivity of the 22,000 molecular weight species. When glands incubated for 10 min are transferred to medium containing a large excess of unlabeled L-cystine, the 22,000 molecular weight peak gradually declines, and there is a corresponding increase in radioactivity at the βNGF position. The 22,000 molecular weight species isolated from sodium dodecyl sulfate gels possesses all the cystine-containing peptides of βNGF, and possibly two additional ones. When immunoprecipitates from submaxillary glands labeled for 25 min are incubated with the γ subunit (a specific arginyl-esteropeptidase associated with βNGF in the 7S NGF complex), the radioactivity in the 22,000 molecular weight species is converted to the βNGF position. The results suggest that the 22,000 molecular weight species is a biosynthetic precursor to βNGF, and that the γ subunit may function as a specific protease in the processing event.
Single-stranded DNA from oncornavirus-infected cells enriched in virus-specific DNA sequences
Authors: Leibovitch Serge A. , Tapiero Haim , Harel Jacques
Published: May 2006

We previously found that a minor fraction of single-stranded DNA (ss-DNA) isolated from native nuclear DNA of normal chicken embryonic cells and cells of other species hybridized with bulk nuclear DNA or cellular RNA in great excess. At least one-third of ss-DNA belonging to the nonrepetitious part of the cell genome could be hybridized to homologous RNAs. In the present work, similar results were obtained with ss-DNA from cells of chickens infected by avian myeloblastosis virus (AMV). To investigate whether this enrichment of ss-DNA in transcribed DNA sequences involves provirus DNA, radioactive AMV RNA and cDNA copies of AMV RNA were used. Most of the 70S AMV RNA hybridized much faster to ss-DNA from productively infected leukemic cells than to bulk DNA. cDNA, either double-stranded or single-stranded, made in the presence of actinomycin D hybridized to total nuclear DNA with similar kinetics. In contrast, about half of the double-stranded cDNA molecules hybridized 40-50 times faster to ss-DNA than to total DNA, indicating that only one of the provirus DNA strands seems to be present in ss-DNA. This was confirmed by the fact that relatively insignificant amounts of the ss-cDNA molecules made in the presence of actinomycin D could be annealed to ss-DNA as compared with bulk DNA. These results indicate that actively transcribed DNA sequences can be selectively distributed in the ss-DNA fraction, probably because of single strand breaks in the vicinity of transcription sites.
Inhibition of human platelet thromboxane synthetase by 9,11-azoprosta-5,13-dienoic acid.
Authors: Gorman R R , Bundy G L , Peterson D C , Sun F F , Miller O V , Fitzpatrick F A
Published: May 2006

The synthetic prostaglandin analog 9,11-azoprosta-5,13-dienoic acid (azo analog I) has been found to be a potent inhibitor of human platelet thromboxane synthetase by three independent analytical methods: electron-capture gas chromatography, radioisotopic thin-layer chromatography, and radioimmunoassay. In the presence of azo analog I, human platelet aggregation induced by either the prostaglandin endoperoxide PGH2 or arachidonic acid was antagonized. The addition of azo analog I shifted the transformation of endoperoxides away from thromboxane synthesis and toward prostaglandin E2 synthesis. The specificity of azo analog I is demonstrated by its selective inhibition of the second wave of either ADP- or epinephrine-induced platelet aggregation. These data indicate that PGH2 must be converted to thromboxane A2 in order to induce human platelet aggregation.
Role of the host in virus assembly: cloning of the Escherichia coli groE gene and identification of its protein product.
Authors: Hendrix R W , Tsui L
Published: May 2006

Correct assembly of the heads of bacteriophages lambda and T4 requires the function of the groE gene of the Escherichia coli host. We have isolated a transducing derivative of lambda, called lambda gt-Ec.groE, that carries a functional copy of the groE gene. Unlike wild-type lambda, this phage is able to form plaques on hosts with a mutant groE gene. We have isolated an amber mutation in the groE gene carried by the phage, and this has made it possible to identify the groE product as a protein of molecular weight 65,000. In the phage, the groE gene is under the control of an early phage promoter.
Uracil incorporation: A source of pulse-labeled DNA fragments in the replication of the
Escherichia coli
chromosome
Authors: Tye Bik-Kwoon , Chien Janice , Lehman I. R. , Duncan Bruce K. , Warner Huber R.
Published: May 2006

Uracil is incorporated into newly synthesized DNA by mutants of
Escherichia coli
with reduced levels of dUTPase (dUTP nucleotidohydrolase; EC 3.6.1.23). Excision-repair of the incorporated uracil results in the generation of labeled DNA fragments that appear after brief pulses with [
3
H]thymidine [Tye, B-K., Nyman, P.-D., Lehman, I. R., Hochhauser, S. & Weiss, B. (1977)
Proc. Natl. Acad. Sci. USA
74, 154-157]. Uracil is also incorporated into the newly synthesized DNA of strains of
E. coli
that contain normal levels of dUTPase. DNA fragments generated by the postreplication excision-repair of uracil may therefore contribute to the pool of nascent DNA (Okazaki) fragments that normally appear in wild-type strains. Discontinuous DNA replication has been examined in the absence of uracil excision by comparing Okazaki fragments in strains that are defective in DNA polymerase I (
polA
-
) and
polA
-
strains that are also defective in uracil
N
-glycosidase, an enzyme required for the excision-repair of uracil in DNA (
polA
-
ung
-
). Little or no difference was detected in the level of Okazaki fragments in the
polA
-
strain as compared with the
polA
-
ung
-
strain. Thus, the uracil-induced cleavage of DNA cannot be the sole mechanism for the generation of Okazaki fragments. Mutants that are defective both in dUTPase and in uracil
N
-glycosidase incorporate uracil into their DNA with a high frequency (up to 1 per 100 nucleotides). These uracil residues, once incorporated, persist in the DNA without an adverse affect on the growth of the cells.
Estrogenic activity
in vivo
and
in vitro
of some diethylstilbestrol metabolites and analogs
Authors: Korach Kenneth S. , Metzler Manfred , McLachlan John A.
Published: May 2006

The diethylstilbestrol (DES) metabolite (β-dienestrol), which had been identified in mouse, rat, monkey, and human urine, and two proposed metabolic intermediates (diethylstilbestrol α,α′-epoxide and α,α′-dihydroxy DES) were synthesized and their estrogenic activities determined. In addition, three DES analogs, α-dienestrol, DES-dihydroxy diethyl phenanthrene (DES-phenanthrene), and 1-(α-ethyl, 4α-hydroxyphenyl)indanyl-5-ol (indanyl-DES), were studied. Estrogenic activities of the compounds
in vivo
were determined by the immature mouse uterine weight bioassay;
in vitro
, their estradiol receptor binding activity (competitive equilibrium binding, sucrose gradient analysis, and association rate inhibition assays) was determined. Results of the mouse uterine weight bioassay gave the following order of estrogenicity: DES > α-dienestrol ≥ DES-epoxide > indanyl-DES > dihydroxy DES > β-dienestrol > DES-phenanthrene. Results of competitive equilibrium binding analyses of these compounds with estradiol-17β for the mouse uterine cytosol receptor followed the same order seen for the bioassay, except for indanyl-DES. DES, indanyl-DES, and α-dienestrol had the greatest affinities (
K
a
values approximately 0.5-19.1 × 10
10
M
-1
), while DES-phenanthrene had the lowest (
K
a
= 3.5 × 10
7
M
-1
± 1.2). Sucrose gradient analysis of the above competition preparations illustrated the displacement of [
3
H]estradiol from the receptor peak. This displacement was receptor specific and concentration dependent and correlated with the equilibrium binding concentrations. In addition, the most hormonally active substances demonstrated the greatest rate inhibition in the estradiol cytosol receptor association rate reaction (
V
0
). The rank order of estrogenicity of the compounds determined in this study should be useful in evaluating alternative metabolic pathways of DES as well as distinguishing biologically active metabolites from relatively inactive ones.
alpha-Lactalbumin mRNA in 4-day lactating rat mammary gland.
Authors: Qasba P K , Nakhasi H L
Published: May 2006

Rat alpha-Lactalbumin (alpha-LA) mRNA has been purified from the total RNA of 4-day lactating mammary gland by poly(U)-Sepharose chromatography and by fractionation of the poly(A)+ RNA on sucrose gradients. alpha-LA mRNA was homogeneous as judged by electrophoresis in urea/agarose gel and in 1.5% agarose gel under glyoxal-denaturation conditions, which gave a molecular weight of 210,000. The chymotryptic fingerprints of the protein synthesized with this mRNA, in a translational system derived from wheat germ, were similar to those of purified rat alpha-LA. In rat milk or lactating gland, the content of Mr 42,000 casein is 6 times greater than that of Mr 29,000 casein and 10 times greater than that of alpha-LA. However, alpha-LA mRNA (Mr 210,000) and two casein mRNas (Mr 460,000 and 390,000), coding for Mr 42,000 and 29,000 caseins, were present in equal proportions when measured with cDNA probes in the total RNA of 4- to 5-day lactating gland. Moreover, alpha-LA and total casein were synthesized in a ratio fo 1:2.5 in a wheat-germ translational system using the total RNA of the lactating gland. The results suggest post-transcription controls in the synthesis of these proteins.
Interaction of RNA polymerase and rho in transcription termination: coupled ATPase.
Authors: Das A , Merril C , Adhya S
Published: May 2006

We have previously described temperature sensitive rho mutants of Escherichia coli (e.g., rho15) that are defective in transcription termination at various signals, including an IS2 DNA insertion in the gal operon [Das, A., Court, D. & Adhya, S. (1976) Proc. Natl. Acad. Sci. USA, 73, 1959-1963]. In this paper, we report the isolation of mutants altered in the beta subunit of RNA polymerase (a class of Rifampicin-resistant mutants), which restore gal IS2 polarity in the rho 15 strain. It has been shown that one of these suppressor RNA polymerases (rpoB101) requires rho to terminate transcription of phage lambda mRNA. In contrast to the wild type RNA polymerase, the suppressor RNA polymerase also terminates lambda mRNA transcription in the presence of rho15 protein. We have isolated new rho mutants (e.g., rho112) that are defective in transcription termination in the rpoB101 strain. These results strongly support the notion that rho and RNA polymerase interact functionally during transcription termination. We have shown that rho15 catalyzes ATP hydrolysis during transcription with rpoB101 RNA polymerase, but not with wild-type RNA polymerase. Because rho 15 protein hydrolyzes ATP in the presence of free RNA, we suggest that rho may recognize the 3'-OH end of RNA. During transcription, this recognition involves an interaction with RNA polymerase, resulting in the displacement of the polymerase and the release of the nascent mRNA.
Laser Raman evidence for new cloverleaf secondary structures for eukaryotic 5.8S RNA and prokaryotic 5S RNA.
Authors: Luoma G A , Marshall A G
Published: May 2006

Neither of the two previously proposed secondary structures for eukaryotic 5.8S RNA is consistent with the present laser Raman results. A new, highly stable "cloverleaf" secondary structure not only fits the Raman data but also accounts for previously determined enzymatic partial cleavage patterns, base sequence and pairing homologies, and G-C and A-U base pair numbers and ratios. The new cloverleaf model also conserves several structural features (constant loops, bulges, and stems) consistent with known 5.8S RNA functions. Finally, we propose a similar new cloverleaf secondary structure for Escherichia coli 5S RNA, consonant with many known properties of prokaryotic 5S RNA.
Effect of anesthetics and pressure on the thermotropic behavior of multilamellar dipalmitoylphosphatidylcholine liposomes.
Authors: Mountcastle D B , Biltonen R L , Halsey M J
Published: May 2006

The effects of gaseous anesthetics and pressure on the thermotropic behavior of multilamellar dipalmitoyl-phosphatidylcholine liposomes were studied by using a high-sensitivity differential scanning calorimeter. It was found that halothane and enflurane decreased the transition temperature and increased the width of the transition without affecting the enthalpy change for the main gel-to-liquid crystalline transition. This demonstrated that the anesthetics decreased the degree of cooperative interaction between phospholipid molecules within the bilayer. Increasing the pressure increased the transition temperature but did not affect the transition width or enthalpy change. However, the increase in pressure reversed the effect of anesthetic on both the transition temperature and transition width. It is suggested that an understanding of the effect of anesthetics on the degree of cooperative interaction between phospholipids may be a key to understanding anesthetic action.
Does 3'-terminal poly(A) stabilize human fibroblast interferon mRNA in oocytes of Xenopus laevis?
Authors: Sehgal P B , Soreq H , Tamm I
Published: May 2006

Polynucleotide phosphorylase (polyribonucleotide:orthophosphate nucleotidyltransferase, EC 2.7.7.8) purified from Escherichia coli was used enzymatically to deadenylate polyadenylated human fibroblast interferon mRNA preparations obtained from human diploid fibroblasts (FS-4 strain) induced by poly(I)-poly(C) (20 microgram/ml) in the presence of cycloheximide (50 microgram/ml, 4 hr). Both the polyadenylated and the deadenylated interferon mRNA preparations were translated into biologically active human interferon when injected into oocytes of Xenopus laevis. In the oocytes the functional stability of deadenylated interferon mRNA was indistinguishable from that of polyadenylated interferon mRNA.
Extent of genetic variation at a highly polymorphic esterase locus in Drosophila pseudoobscura.
Authors: Coyne J A , Felton A A , Lewontin R C
Published: May 2006

The esterase-5 locus of Drosophila pseudoobscura has been screened for variation by using a wide variety of electrophoretic conditions and by testing heat sensitivity. Among 50 isogenic lines from 17 populations, 21 genetic variants were found at the locus, 18 by electrophoresis and 3 by heat denaturation. Including alleles previously known, there are a total of 30 alleles at this locus in natural populations. This represents a doubling of the previous estimates of polymorphism at this locus. The study confirms that, in contrast to monomorphic loci, loci judged polymorphic under one detection procedure show large increases in polymorphism when examined extensively.
Rescue by coenzyme Q10 from electrocardiographic abnormalities caused by the toxicity of adriamycin in the rat.
Authors: Folkers K , Choe J Y , Combs A B
Published: May 2006

The administration of adriamycin to rates increased (P less than 0.01) the interval, measured in msec, of the electrocardiographic QRS traces in rats, and the magnitude of the increase was ca. 50%. The administration of coenzyme Q10 to such adriamycin-treated rats allowed "rescue" or restoration of a normal QRS complex after 7 days of administration of coenzyme Q10. The QRS complex then remained normalized during the subsequent period of 21-30 days, by which time the cumulative dose of adriamycin had reached 24 mg/kg. Also, the QRS interval was lower (P less than 0.01) on day 33 than it was for rats treated to the same day with adriamycin alone. Coenzyme Q10 offers promise of rescue from at least some of the cardiotoxicity occurring in adriamycin-treated cancer patients, probably by a similar mechanism to that of the clinical rescue from toxicity of methotrexate by a cofactor of folic acid (citrovorum factor).
Sensitivity of fatty acid cyclooxygenase from human aorta to acetylation by aspirin.
Authors: Burch J W , Baenziger N L , Stanford N , Majerus P W
Published: May 2006

The rate of acetylation of fatty acid cyclooxygenase (prostaglandin synthase, EC 1.14.99.1) by [acetyl-3H]-aspirin was measured in microsomes from human aortas and coronary arteries and intact and disrupted human platelets. We also measured the inhibition by aspirin of prostacyclin generation from exogenous arachidonic acid in shredded human aorta. Cyclooxygenase in human aorta and coronary artery microsomes is approximately 1/250th as sensitive to aspirin as enzyme in intact platelets, and 1/60th as sensitive to aspirin as enzyme measured in a platelet microsomal preparation. On the basis of the in vitro data presented, we predict that small oral doses of aspirin are sufficient to inhibit platelet prostaglandin production but are not sufficient to substantially affect aorta or coronary artery prostaglandin production.
beta-Endorphin induces nonconvulsive limbic seizures.
Authors: Henriksen S J , Bloom F E , McCoy F , Ling N , Guillemin R
Published: May 2006

The endogenous opioid peptide, beta-endorphin, induces nonconvulsive limbic epileptiform activity when administered intraventricularly to rats. Epileptiform activity is elicited by beta-endorphin in doses that are devoid of analgesic or behavioral signs. Equimolar intraventricular doses of morphine or of the enkephalin analog [DAIa2,Met5]enkephalin-NH2 fail to elicit this limbic epileptiform activity. These observations, together with the recent immunohistochemical localization of beta-endorphin to midline limbic structures, suggest that beta-endorphin may have an important role in the regulation of limbic excitability.
Pulvomycin, an inhibitor of protein biosynthesis preventing ternary complex formation between elongation factor Tu, GTP, and aminoacyl-tRNA.
Authors: Wolf H , Assmann D , Fischer E
Published: May 2006

Pulvomycin and the synonymous antibiotics labilomycin and 1063-Z are shown to inhibit prokaryotic protein synthesis by acting on elongation factor Tu (EF-Tu): in the presence of the antibiotic, the affinity of EF-Tu for guanine nucleotides is altered, the EF-Tu.GDP/GTP exchange is catalyzed, and the formation of the EF-Tu.GTP complex is stimulated. Hydrolysis of GTP by EF-Tu, induced by aminoacyl-tRNA, ribosomes, and mRNA or by kirromycin, is inhibited by pulvomycin. As shown by Millipore filtration, chromatographic analysis, and hydrolysis protection experiments, pulvomycin prevents interaction between aminoacyl-tRNA and EF-Tu.GTP to yield the ternary complex aminoacyl-tRNA.EF-Tu.GTP. Thus, enzymatic binding of aminoacyl-tRNA to ribosomes is blocked.
Molecular analysis of cloned human 18S ribosomal DNA segments.
Authors: Wilson G N , Hollar B A , Waterson J R , Schmickel R D
Published: May 2006

A fraction of DNA from the human fetal lung fibroblast line IMR-90, 30-fold enriched for ribosomal DNA, was cloned in the lambda phage vector Charon 16A. Of 978 clones assayed by hybridization to a mixture of 125I-labeled 18S and 28S ribosomal RNA, 11 recombinants containing a 3.8-megadalton segment of human 18S ribosomal DNA were identified. Restriction endonuclease analysis of these clones demonstrated variation only in orientation of the human gene segment within the phage vector. Restriction sites that we had previously detected from analysis of restriction products of unfractionated human DNA by using the Southern transfer method were also present in the cloned DNA segment. Recombinant DNA technology thus provides a valid and efficient means to define structural conservation or variation within families of human genes.
Immunoreactivity and receptor binding of mixed recombinants of human growth hormone and chorionic somatomammotropin.
Authors: Burstein S , Grumbach M M , Kaplan S L , Li C H
Published: May 2006

The behavior in six radioligand assays of the recombinant obtained by the noncovalent complementation of the reduced and carbamoylmethylated 134-residue amino-terminal fragment of human growth hormore with the reduced and carbamoylmethylated 51-residue carboxyl-terminal fragment of human chorionic somatomammotropin was compared to that of the analogous recombinant of the 133-residue amino-terminal fragment of human chorionic somatomammotropin and the 51-residue carboxyl-terminal fragment of human growth hormone. The determinants for the hepatic growth hormone receptor binding and for the lactogenic receptor binding of human growth hormone are on the amino-terminal fragments. The antigenic determinants for both a monospecific antiserum to human growth hormone and a monospecific antiserum to human chorionic somatomammotropin also are on the amino-terminal fragments of their respective antigens. The mixed recombinant of the amino-terminal fragment of human growth hormone with the carboxyl-terminal fragment of human chorionic somatomammotropin retains full radioimmuno- and radioreceptor activity after lyophilization.
Interactive computer surface graphics approach to study of the active site of bovine trypsin
Authors: Feldmann Richard J. , Bing David H. , Furie Barbara C. , Furie Bruce
Published: May 2006

A descriptive medium for the presentation of protein structure has been developed and used to evaluate the structure of the active site of bovine trypsin (EC 3.4.21.4). This technique, involving advanced computer graphics technology, permits the facile display of a representation of the molecular surface of proteins of known structure and employs color to code the structural or chemical features of this surface. Benzamidine derivatives were inserted into the benzamidine-binding site of trypsin and the binary inhibitor-trypsin complex was evaluated by using the computer-generated structure. On the basis of qualitative assessments of the contribution of electrostatic and hydrophobic forces to the binding energy associated with complex formation, we made predictions concerning the effects of interaction of benzamidine substituents and amino acid side chains upon the binding energy associated with inhibitor-protein binding. The computer display of the molecular surfaces of the binary complex of substituted benzamidines and trypsin permitted unique insight into the identity and chemical properties of the atoms that participate at the interface of the molecular surfaces of the inhibitor and the protein. The computer-generated molecular surface display can potentially be combined with quantitative definition of the physical forces involved in the interaction of molecular surfaces. This technology should facilitate the study of the structure-activity relationship of substrates, inhibitors, and drugs that bind to proteins of known three-dimensional structure.
Diamagnetic anisotropy and orientation of alpha helix in frog rhodopsin and meta II intermediate.
Authors: Chabre M
Published: May 2006

The diamagnetic anisotropy of retinal rod outer segments, and its variation upon bleaching, have been measured with a rotating field device. A large molar diamagnetic asymmetry is found for rhodopsin. This cannot be explained by an anisotropy of the aromatic side chains of the protein, nor by the orientation of the retinal chromophore. However, it can be accounted for by an orientation perpendicular to the disc membrane of a major proportion of the alpha-helical segments of the protein. Upon bleaching a decrease of 9 +/- 2% of the diamagnetic asymmetry is observed when going to the meta II intermediate. This change is not mainly due to a reorientation of the retinal, since it is practically insensitive to detachment of the chromophore by addition of NH2OH. Comparison with recent UV linear dichroism results indicate that it may be due to the rotation of a trytophan residue in the bleaching sequence.
Conformational features of distamycin-DNA and netropsin-DNA complexes by Raman spectroscopy.
Authors: Martin J C , Wartell R M , O'Shea D C
Published: May 2006

The binding of distamycin and netropsin to duplex DNA has been studied by Raman spectroscopy. Several changes occur in the Raman spectra of these drugs upon binding DNA. These changes were analyzed by assigning specific motions to the observed Raman bands through the use of molecular subunits of the drugs and normal mode calculations. Our analysis indicates that pyrrole ring and peptide group vibrations are altered upon binding to DNA. The environments of the pyrrole ring methyl groups are not affected by the binding. These data provide physical evidence consistent with a binding model in which the methyl groups on the pyrroles project away from the DNA and the peptide N-H groups form hydrogen bonds with the DNA.
Topographic study of the cell surface of micrococcus radiodurans.
Authors: Baumeister W , Kübler O
Published: May 2006

The paracrystalline outer membraneous layer (HPI layer) of Micrococcus radiodurans has been investigated by negative- and positive-staining electron microscopy and subsequent digital image processing. The subunit structure of the major HPI layer protein complex and the lipid-protein distribution in the plane of the membrane have been determined. The HPI layer was found to be highly asymmetric in a transmembrane direction, with the major protein complex only partly penetrating into a lipid-containing backing layer intimately associated with it.
An amino-terminal fragment of
lac
repressor binds specifically to
lac
operator
Authors: Ogata Ronald T. , Gilbert Walter
Published: May 2006

We have examined the effect of an aminoterminal peptide from the
lac
repressor (residues 1-59) on the methylation of purines in the
lac
operator with dimethyl sulfate. The peptide perturbs the methylation of the operator, and the peptide-induced pattern of inhibition and enhancement of methylation, across the operator, closely resembles the pattern induced by intact repressor. This demonstrates that this small amino-terminal peptide binds specifically to the
lac
operator and that the mechanism of recognition and binding is basically the same as that of intact repressor.