bluuebunny/crossref_metadata_2025 huggingface dataset:
Vascular relaxing activity and stability studies of 10,10-difluoro-13,14-dehydroprostacyclin.
Authors: Hatano Y , Kohli J D , Goldberg L I , Fried J , Mehrotra M M
Published: May 2006

10,10-Difluoro-13,14-dehydroprostacyclin was compared with natural prostacyclin (prostaglandin I2, PGI2) for vascular relaxing activity in vitro on helical strips of small canine mesenteric arteries (outside diameter < 1 mm) and bovine coronary arteries (outside diameter 1.5-2.5 mm) partially contracted with prostaglanding F2 alpha as well as in vivo in pentobarbital-anesthetized dogs. The difluoro analog was 3- to 4-fold more active than PGI2 in causing relaxation of both types of strips and appeared at least equipotent to PGI2 in its blood pressure lowering effect in dogs. When incubated with Krebs' bicarbonate solution (pH 7.4) at 37 degrees C, the biological half-life of the difluoro analog was about 24 hr compared to the 10- to 15-min half-life of PGI2 under similar conditions. However, when administered intravenously to dogs, the hemodynamic effects of the difluoro analog were of the same duration as those of PGI2.
Calcitonin, a major gill hormone.
Authors: Milhaud G , Bolis L , Benson A A
Published: May 2006

Isolated salmon gills, under simulated in vivo conditions, transported calcium ion into the ambient seawater when perfused with Ringer's solution containing salmon calcitonin. Similar enhancement of phosphate efflux was observed. Calcitonin caused simultaneous decreases in perfusate flow rates. The effects act synergistically to diminish net plasma calcium concentrations.
Isolation and characterization of overlapping genomic clones covering the chicken alpha 2 (type I) collagen gene.
Authors: Ohkubo H , Vogeli G , Mudryj M , Avvedimento V E , Sullivan M , Pastan I , de Crombrugghe B
Published: May 2006

A series of overlapping recombinant clones, which cover the alpha 2 (type I) collagen gene, have been isolated by stepwise screening of two libraries of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned cDNA containing alpha 2 collagen DNA sequences as hybridization probe. The other clones were obtained by a sequence of screenings using defined fragments of the successive genomic clones as hybridization probes. Several types of experiments indicated that the DNA of these clones are truly overlapping and span 55 kilobase pairs of contiguous DNA sequences in the chicken genome. Sequence analysis of small DNA segments of some of these clones confirm that they contain coding sequences which specify alpha 2 collagen. Electron microscopic analysis of hybrids between type I alpha 2 collagen mRNA and the overlapping genomic clones indicates that the chicken alpha 2 collagen gene has a length of at least 37 kilobases, about 7.4 times longer than the corresponding translatable cytoplasmic mRNA. The coding information for alpha 2 collagen is distributed in more than 50 coding sequences which are interrupted by intervening sequences of various sizes. The structure of the gene implies that the conversion of precursor RNA to mature mRNA for alpha 2 collagen includes at least 50 splicing events.
Structure of the metal clusters in rabbit liver metallothionein
Authors: Otvos James D. , Armitage Ian M.
Published: May 2006

Cadmium-113 nuclear magnetic resonance (
113
Cd NMR) has been used to determine the structures of the multiple cadmium binding sites in the two major isoproteins of rabbit liver metallothionein. The isotopically
113
Cd-labeled metallothionein used in these studies was isolated from the livers of rabbits that had been subjected to repeated injections of
113
CdCl
2
. The native protein isolated from these livers contains an appreciable amount of Zn in addition to Cd, ranging from 2-3 mol per mol of protein out of a total metal content of 7 mol per mol of protein. The
113
Cd NMR spectrum of Cd, Zn-containing metallothionein is quite complex, reflecting the fact that the native protein is a heterogeneous mixture of species containing different relative amounts of Zn and Cd. Replacement of the native Zn with
113
Cd
in vitro
gave a protein whose
113
Cd NMR spectrum was much simpler, containing eight distinct multiplets with chemical shifts ranging from 611-670 ppm. The origin of the multiplet structures has been shown to be
113
Cd-
113
Cd scalar coupling arising from two-bond interactions between
113
Cd ions linked to one another by bridging cysteine thiolate ligands. The size and structures of the metal clusters in the protein were determined by the application of selective homonuclear
113
Cd decoupling techniques. Analysis of these data showed that rabbit liver metallothionein contains two separate metal clusters, one containing four Cd
2+
ions and the other containing three. These two clusters, whose structures are the same in both isoproteins, have been designated “cluster A” and “cluster B,” respectively. Structures for the clusters are proposed that account for the
113
Cd spin coupling data and the participation of all 20 of the cysteine residues in metal ligation, 11 in cluster A and 9 in cluster B. The appearance in the spectrum of eight multiplets rather than the seven that would be expected on the basis of the number of metal binding sites in the protein is an indication of some residual heterogeneity in the
113
Cd-labeled metallothionein sample. The origin of this heterogeneity is suggested to be the presence of a protein species that lacks metal ions at its cluster B binding sites.
Electrophoretic resolution of three major insulin receptor structures with unique subunit stoichiometries.
Authors: Massague J , Pilch P F , Czech M P
Published: May 2006

Plasma membrane insulin receptors, affinity labeled by covalent crosslinking to receptor-bound 125I-labeled insulin, are shown to appear as a heterogeneous population of three major disulfide-linked complexes (Mr 350,000, 320,000, and 290,000) upon electrophoresis in highly porous dodecyl sulfate/polyacrylamide gels in the absence of reductant. This pattern is consistent in all rat and human tissues that were analyzed. Upon reduction of disulfide bonds, each of these receptor structures is dissociated in two successive steps. Low concentrations of dithiothreitol promote a first step of disulfide bond reduction in which the Mr 350,000 species splits into a Mr 210,000 form and the Mr 290,000 species splits into a Mr 160,000 form. In contrast, both the Mr 210,000 and Mr 160,000 receptor fragments are generated from the native Mr 320,000 species upon partial reduction, indicating an asymmetrical structure. The second step of receptor reduction occurs upon treatment of the native disulfide-linked receptor complexes with high concentrations of dithiothreitol. Under these conditions, the Mr 350,000 receptor yields a Mr 125,000 subunit, denoted as alpha, and a Mr 90,000 subunit, denoted as beta, whereas the Mr 290,000 receptor dissociates into the alpha subunit and a Mr 49,000 subunit, denoted as beta 1. The Mr 320,000 receptor band is found to consist of alpha, beta, and beta 1 subunits upon complete reduction. The partially reduced Mr 210,000 receptor fragment is composed of the alpha subunit disulfide-linked to the beta subunit, whereas the Mr 160,000 species consists of the alpha subunit disulfide-linked to the beta 1 subunit. Thus, the stoichiometry of the three ubiquitous native insulin receptor structures of Mr 350,000, 320,000, and 290,000 are (alpha) 2 (beta) 2, (alpha) 2 (beta) (beta 1), and (alpha) 2 (beta 1) 2, respectively.
Covalent binding and hemolytic activity of complement proteins.
Authors: Law S K , Lichtenberg N A , Levine R P
Published: May 2006

We report the inactivation of the third component of complement (C3) by hydroxylamine. C3 hemolytic and covalent binding activities decline with identical kinetics, demonstrating a direct correlation between the two activities. We conclude that covalent, surface-bound C3b is hemolytically active. The inactivation of C3 is first order with respect to hydroxylamine. We also studied C3 inactivation with [14C]methylamine. The inactivation corresponds quantitatively with the labeling of C3 in the C3d domain. The data obtained support the following hypothesis: there is an internal thioester within C3 which becomes highly reactive on activation to C3b, and C3b binds to receptive surfaces by transfer of the acyl function of the thioester to a hydroxyl group on the receptive surface. This proposed model for the reaction of C3 with receptive surfaces also applies to C4, which binds to membrane surfaces covalently and is able to be inactivated by hydroxylamine and methylamine. C5, on the other hand, is not inactivated by treatment with the amines.
Adrenergic regulation of intraocular pressure: identification of beta 2-adrenergic-stimulated adenylate cyclase in ciliary process epithelium.
Authors: Nathanson J A
Published: May 2006

To determine the site and possible mechanism of action of adrenergic agents in regulating intraocular pressure, experiments were undertaken to identify, localize, and characterize beta-adrenergic receptors associated with adenylate cyclase in various ocular tissues involved in secretion and reabsorption of aqueous humor. The ciliary process epithelium was found to be enriched in an adenylate cyclase with pharmacological characteristics indicative of a predominance of beta 2-adrenergic receptors. The results are consistent with the possibility that the adrenergic nervous system may regulate aqueous humor production through a direct effect on secretion. The data also are relevant to the potential development of drugs that can control increased intraocular pressure.
Dopamine and depolarizing agents regulate the state of phosphorylation of protein I in the mammalian superior cervical sympathetic ganglion.
Authors: Nestler E J , Greengard P
Published: May 2006

The regulation of the state of phosphorylation of protein I, a specific neuronal protein that appears to be associated predominantly with synaptic vesicles, has been studied in intact sections of bovine superior cervical ganglion. For this purpose, a technique was developed that made possible the quantitation of the state of phosphorylation of as little as 5 fmol of protein I. Incubation of ganglion sections in the presence of dopamine, 8-bromo-cyclic AMP, or depolarizing agents (i.e., high K+ concentration or veratridine) increased the state of phosphorylation of protein I relative to that of control ganglion sections. Other results indicated that the effect of dopamine is probably mediated via the activation of a cyclic AMP-dependent protein kinase and that the effect of high K+ concentration is probably mediated via the activation of a calcium-dependent protein kinase.
Kinetics of membrane internalization and recycling during pinocytosis in Dictyostelium discoideum.
Authors: Thilo L , Vogel G
Published: May 2006

Internalization and recycling of plasma membrane during pinocytosis in Dictyostelium discoideum was analyzed quantitatively. A labeling technique was used by which [3H]galactose could be enzymatically bound to and released from the plasma membrane. Label internalized with the plasma membrane was no longer accessible to enzymatic release and could therefore be distinguished quantitatively from label remaining on the cell surface. Internalization of labeled membrane components was measured as a function of pinocytotic uptake. Direct experimental evidence for membrane recycling was obtained by demonstrating that previously internalized label reappeared at the plasma membrane. The experimental data agree with a kinetic model requiring that a shuttle of membrane between two membrane compartments leads to the same surface concentration of label in both. The two compartments consist of the plasma membrane and of cytoplasmic vacuolar membranes; their relative membrane surface areas are 1 and 0.5, respectively. One surface area equivalent of the plasma membrane is internalized during a pinocytotic uptake amounting to 15% of the cell volume. At the observed rate of pinocytosis, this occurred once every 45 min. The average size of the primary pinosomes, as weighted according to their contribution to pinocytotic uptake, was calculated to be about 0.6 microns.
Thrombin increases expression of fibronectin antigen on the platelet surface.
Authors: Ginsberg M H , Painter R G , Forsyth J , Birdwell C , Plow E F
Published: May 2006

Fibronectins (fn) are adhesive glycoproteins which bind to collagen and to fibrin and appear to be important in cellular adhesion to other cells or surfaces. Fn-related antigen is present in human platelets, suggesting a possible role for fn in the adhesive properties of platelets. We have studied the localization of fn in resting and thrombin-stimulated platelets by immunofluorescence and quantitative binding of radiolabeled antibody. In resting fixed platelets, variable light surface staining for fn was observed. When these cells were made permeable to antibody with detergent, staining for fn was markedly enhanced and was present in a punctate distribution, suggesting intracellular localization. Stimulation with thrombin, which is associated with increased platelet adhesiveness, resulted in increased staining for fn antigen on intact platelets. These stimulated cells did not leak 51Cr nor did they stain for F-actin, thus documenting that the increased fn staining was not due to loss of plasma membrane integrity. The thrombin-induced increase in accessible platelet fn antigen was confirmed by quantitative antibody binding studies in which thrombin-stimulated platelets specifically bound 15 times as much radiolabeled F(ab')2 anti-fn as did resting cells. Thus, thrombin stimulation results in increased expression of fn antigen on the platelet surface. Here it may participate in interactions with fibrin, connective tissue, or other cells.
Antigen-specific induction and regulation of antibody synthesis in cultures of human peripheral blood mononuclear cells.
Authors: Hoffmann M K
Published: May 2006

Culture conditions suitable for specific sensitization of human peripheral blood lymphocytes were established. Production of antibody to sheep erythrocytes and to the 2,4,6-trinitrophenyl group conjugated to burro erythrocytes was investigated in terms of cross-sensitization, feedback control by antibody to the immunogen, and regulation by helper and suppressor T cells. Generation of antigen-specific antibody-forming cells was found to be restricted to a narrow concentration range of cultured peripheral blood lymphocytes. The data suggest that antibody formation is limited by suppressor T cells in high-cell-density cultures and by low frequencies of helper T cells in low-cell-density cultures.
Presynaptic inhibition in Aplysia involves a decrease in the Ca2+ current of the presynaptic neuron.
Authors: Shapiro E , Castellucci V F , Kandel E R
Published: May 2006

By voltage clamping presynaptic cell L10 and using pharmacologic separation techniques, we have analyzed the specific ionic currents in the presynaptic cell that correlate with presynaptic inhibition while assaying transmitter release with intracellular recordings from postsynaptic cells. We have found that presynaptic inhibition can be elicited in conditions in which the Na+ and the various K+ channels are pharmacologically blocked and depolarizing current pulses produce only an inward Ca2+ current. Both inward currents and tail currents at and above the K+ reversal potential were always less inward during presynaptic inhibition. The changes in conductance associated with presynaptic inhibition were voltage sensitive and paralleled the voltage sensitivity of the Ca2+ channel. We therefore conclude that presynaptic inhibition is caused by a direct transmitter-mediated decreased of presynaptic Ca2+-channel conductance.
Renaturation and localization of enzymes in polyacrylamide gels: Studies with UDPglucose pyrophosphorylase of
Dictyostelium
Authors: Manrow Richard E. , Dottin Robert P.
Published: May 2006

We have developed procedures for renaturing, in polyacrylamide isoelectric focusing gels, homomeric enzymes (i.e., enzymes with identical subunits) that have been denatured with sodium dodecyl sulfate or urea or both. The renatured enzymes can then be localized as discrete species by conventional histochemical staining. One of these enzymes, uridine diphosphoglucose pyrophosphorylase (UTP:α-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.9) of
Dictyostelium discoideum
, was studied in detail. Conditions have been established for renaturing and localizing this enzyme and for quantitating the amount of activity recovered. Up to 40% of the activity can be recovered after renaturation. This procedure is widely applicable because several enzymes, including alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27), can be localized. It is sensitive enough to resolve isozymes and enzyme variants that differ by a single charged amino acid. It can be used to localize enzymes in crude cell extracts that have been resolved in two-dimensional slab gels by sodium dodecyl sulfate electrophoresis and isoelectric focusing. These methods should allow detailed analysis of genes and their enzyme proteins that, though present in small amounts in eukaryotic cells, perform important metabolic or developmental functions.
Selective oxidation of cysteine and methionine in normal and senile cataractous lenses.
Authors: Garner M H , Spector A
Published: May 2006

The oxidation state of methionine and cysteine in normal and cataractous lenses is reported. In young lenses no oxidation was detected in any protein fraction examined. Only the intrinsic membrane fraction and membrane-related components showed evidence of oxidation in old (60-65 years of age) normal lenses. However, in a similar age group, with the development of cataract, progressive, dramatic changes were observed. With severe cataracts, 60% or more of the methionine in membrane-associated components was found in the methionine sulfoxide form, and methionine sulfone was observed in one case. Most of the cysteine was found oxidized to either the disulfide form or putative cysteic acid. Mixed disulfides with glutathione were observed. Oxidative changes in soluble components as illustrated by alpha-crystallin occurred more gradually. The data clearly support the viewpoint that extensive oxidation of lens proteins occurs with cataract and that it begins at the lens fiber membrane.
RNA primer used in synthesis of anticomplementary DNA by reverse transcriptase of avian myeloblastosis virus.
Authors: Myers J C , Dobkin C , Spiegelman S
Published: May 2006

When either the homologous RNA (avian myeloblastosis virus RNA) or a heterologous RNA (poliovirus RNA) was used as a template, the anticomplementary DNA synthesized in vitro by avian myeloblastosis virus reverse transcriptase (RNA-directed DNA nucleotidyltransferase, EC 2.7.7.7) was primed by fragments of the original RNA template that usually had adenosine at their 3' ends. When we used phage T/ RNA ligase (EC 6.5.1.3) to label the 3' end of the RNA template fragments contained in the RNA . cDNA hybrid intermediate, adenosine was found to be the principal nucleoside carrying the label. We infer from these results that the ribonuclease H (hybrid nuclease) activity of the reverse transcriptase creates fragments of the original RNA template with adenosine as the principal 3' terminus and that these fragments serve as primers for the synthesis of anticomplementary DNA.
Characterization of globin domains: Heme binding to the central exon product
Authors: Craik Charles S. , Buchman Steven R. , Beychok Sherman
Published: May 2006

We have prepared and isolated the peptide fragments coded for by the three exons of the human β-globin gene, using the arginine-specific protease clostripain (EC 3.4.22.8). The region encoded by the central exon (amino acid residues 31-104) contains an arginine at position 40. This site was less susceptible to cleavage than the two sites that correspond to the exon-intron boundaries, and the isolated central fragment was an approximately equimolar mixture of the entire central fragment, β
o
31-104
, and the somewhat smaller fragment contained within it, β
o
41-104
. This central fragment mixture bound heme stoichiometrically and tightly at micromolar concentrations, generating a strong Soret absorption band as well as a characteristic absorption band in the visible spectrum. The Soret band occurred at the same wavelength and had the same shape as in hemoglobin, exhibiting an intensity greater than ⅔ that achieved when native intact β globin is reconstituted with heme. Nearly the full intensity was regained when an equivalent of heme was added to the unfractionated digest, suggesting that the noncovalently associated side fragments add precision to the fit of the heme pocket. Three controls were used in establishing the specificity of heme binding to the central fragment mixture. Similar, but preliminary, experiments have also been undertaken with α globin. A clostripain digest containing α
o
1-31
and α
o
32-141
bound heme, yielding a Soret band identical to that observed in α subunits reconstituted from the native globin chains and heme. Measurements of circular dichroism spectra as indices of secondary structure suggested a role for the side exon products in the acquisition of the native three-dimensional structure of hemoglobin. These experiments confirm a prediction of W. Gilbert that the product of the central exon of the globin gene is a complete functional domain that binds heme tightly and specifically.
Glyoxylate cycle in toad urinary bladder: possible stimulation by aldosterone.
Authors: Goodman D B , Davis W L , Jones R G
Published: May 2006

A homogenate of the toad urinary bladder epithelial cell layer has the two enzymatic activities unique to the glyoxylate cycle--isocitrate lyase (threo-Ds-isocitrate glyoxylate-lyase, EC 4.1.3.1) and malate synthase [L-malate glyoxylate-lyase (CA-acetylating), EC 4.1.3.2]--as well as the capacity to carry out CN-insensitive palmitoyl-CoA oxidation. When tissue is incubated in the presence of a fatty acid substrate, tissue glycogen levels increase. Additionally, in the presence of aldosterone, glycogen levels are higher. These data demonstrate the presence of glyoxylate cycle enzymes in tissue of a higher animal and raise the possibility that such tissue can convert lipid to carbohydrate.
Benzodiazepine receptor protein identified and visualized in brain tissue by a photoaffinity label.
Authors: Möhler H , Battersby M K , Richards J G
Published: May 2006

Flunitrazepam, a potent benzodiazepine, reversibly binds to the benzodiazepine receptor with high affinity. When irradiated with UV light, flunitrazepam was irreversibly linked to brain tissue. Incorporation of [3H]flunitrazepam was inhibited by other benzodiazepines with a potency corresponding to their affinity for the benzodiazepine receptor. Photolabeling with flunitrazepam reduced the number of benzodiazepine receptors determined by reversible binding of benzodiazepines, whereas the apparent affinity of the remaining receptors was unchanged. Half-maximal incorporation of flunitrazepam occurred at a concentration similar to the apparent dissociation constant of flunitrazepam. Thus, flunitrazepam appears to be a photoaffinity label for the benzodiazepine receptor. The receptor component photolabeled with flunitrazepam was a protein of molecular weight 50,000. Its location in cerebral and cerebellar cortex slices could be visualized by electron microscopic autoradiography. A predominant localization of benzodiazepine receptors in regions of synaptic constants, including those formed by GABAergic neurons (GABA is gamma-aminobutyric acid), was observed.
Regulation of protein synthesis during spore germination in Dictyostelium discoideum.
Authors: Dowbenko D J , Ennis H L
Published: May 2006

Spore germination in the slime mold Dictyostelium discoideum is a particularly suitable paradigm for studying the regulation of gene expression because developmentally regulated changes in both protein and mRNA synthesis occur during the synchronous transition from dormant spore to growing ameba. To investigate the regulation of protein synthesis during germination, we labeled activated spores with [35S]methionine at 1-hr intervals during germination, until amebas emerged (at 3 hr). The labeled proteins were resolved by two-dimensional polyacrylamide gel electrophoresis. Six classes of proteins were distinguished, depending on the time of onset and duration of their synthesis: (i) proteins made only during the first hour of germination, (ii) proteins made during the second hour, (iii) proteins made during the third hour, (iv) those synthesized only between 1 and 3 hr after activation, (v) peptides made only between 0 and 2 hr after activation, and (vi) proteins that were made throughout germination, mRNA isolated from dormant spores and from spores at different stages of germination was translated in a wheat germ cell-free protein-synthesizing system, and the proteins made in vitro were compared to those synthesized in vivo. The majority of the changes in the pattern of protein synthesis that occurred during the different stages of germination were attributable to the presence or absence of translatable mRNA. It is concluded that the synthesis of a majority of the proteins during spore germination is transcriptionally controlled.
Sendai virus receptor: proposed recognition structure based on binding to plastic-adsorbed gangliosides.
Authors: Holmgren J , Svennerholm L , Elwing H , Fredman P , Strannegård O
Published: May 2006

The binding of Sendai virus to polystyrene petri dishes to which various gangliosides of defined structures had been adsorbed was determined. The ganglioside-bound virus was visualized either by a water vapor condensation method or by a hemadsorption method. By either assay, specific virus binding of high affinity was demonstrated to the gangliosides GT1a, GQ1b, and GPlc which have a common end sequence in the oligosaccharide moiety: NeuAc alpha 2 leads to 8NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc leads to. Binding also occurred to the GD1a and GT1b gangliosides, which have the same end carbohydrate sequence except for the terminal N-acetylneuraminic acid, but the affinity was only 1-9% of that of the gangliosides with a terminal disialosyl linkage. It is proposed that the structure NeuAc alpha 2 leads to 8NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc is the recognition-specific structure of the receptor for Sendai virus that is present on cell membrane gangliosides and possibly also glycoproteins. Binding tests to plastic-adsorbed glycolipids are suggested to be a useful tool for identification of the receptor recognition structure.
Methotrexate-induced misincorporation of uracil into DNA
Authors: Goulian M. , Bleile B. , Tseng B. Y.
Published: May 2006

A line of human lymphoid cells was tested for the presence of dUMP in DNA with or without treatment with the dihydrofolate reductase inhibitor, methotrexate. Cells treated with methotrexate and labeled with [
3
H]dUrd contained dUMP in DNA in readily detectable amounts (≈0.8 pmol of dUMP per μmol of total DNA nucleotide), and this was increased ≈3-fold if the cells were also treated with Ura at the same time. No dUMP (<1 fmol/μmol of DNA) could be detected by these methods in DNA from cells not treated with methotrexate, regardless of whether Ura was present or absent. The presence of dUMP in DNA from cells treated with methotrexate is a result of the great increase in intracellular concentration of dUTP and the fall in dTTP that accompany inhibition of thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP
C
-methyltransferase; EC 2.1.1.45) by the drug. These changes are apparently sufficient to overcome the normal mechanisms that exclude dUMP from DNA, and the enhancement by Ura reflects suppression of one of the mechanisms, Ura removal from DNA by the enzyme Ura-DNA glycosylase. The results suggest an active lesion of DNA in cells in which thymidylate synthetase is inhibited. Under these conditions there appears to be a cyclic incorporation and removal of dUMP resulting from reinsertion of dUMP during gap repair at sites of Ura removal. This consequence of the normal excision-repair process, which occurs when intracellular levels of dUTP approach those of dTTP, may have effects related to the cytotoxicity of drug inhibitors of thymidylate synthetase, clinical deficiencies of folate and vitamin B-12, and thymineless death, in general.
Rate enhancement of reconstitution of glyceraldehyde-3-phosphate dehydrogenase by a covalently bound coenzyme analog.
Authors: Jaenicke R , Krebs H , Rudolph R , Woenckhaus C
Published: May 2006

Kinetic analysis of the in vitro reconstitution of glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] from yeast showed that both oxidized and reduced coenzyme enhance the transconformation reaction, which is rate limiting in the sequential folding-association process at high enzyme concentrations (Krebs, H., Rudolph, R. & Jaenicke, R. (1979) Eur. J. Biochem. 100, 359-364). In the present study the reconstitution of the enzyme has been analyzed after covalent modification with the coenzyme analog 3-[(3-bromoacetylpyridinio)-propyl]adenosine pyrophosphate. Reconstitution of the modified enzyme, as determined by the regain of the native tryptophan fluorescence, is found to be more then 10 times faster than refolding of the unmodified apoenzyme and more than 5 times faster than that of the unmodified holoenzyme. Various degrees of denaturation and the presence of up to 0.4 M guanidine . HCl do not affect the rate of reconstitution of the modified enzyme. The kinetic effect of free or covalently bound coenzyme is discussed in terms of a decrease in free energy of the native or native-like structure or in terms of a decreased activation energy of rate-limiting steps in the process of reconstitution. Stabilization of the dimeric intermediate or acceleration of its transformation seems to be the most likely explanation for the observed effect of free or covalently bound coenzyme on the rate of reconstitution.
DNA alkylation and unwinding induced by benzo[a]pyrene diol epoxide: modulation by ionic strength and superhelicity.
Authors: Gamper H B , Straub K , Calvin M , Bartholomew J C
Published: May 2006

Superhelical and partially relaxed DNAs of simian virus 40 were allowed to react in vitro with (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP diol epoxide). The modified DNA contained N2 guanine and N6 adenine hydrocarbon adducts in the ratio 86:14. Superhelical simian virus 40 DNA was approximately 6% more susceptible to modification than was partially relaxed viral DNA. Counterions inhibited DNA alkylation by up to 90%, Mg2+ being 50-fold more effective than Na+. The sensitivity of covalent binding to helix stability is consistent with a reaction complex in which BaP diol epoxide is intercalated. The superhelical density of the modified DNA substrates was determined electrophoretically relative to partially relaxed standards, and an unwinding angle for the hydrocarbon adducts was calculated. The angle was dependent upon the superhelicity of the DNA molecule and ranged from 330 degrees to 30 degrees. These data indicate that the modified base pairs are disrupted and, in the presence of torsional strain, act as centers for the further denaturation of up to eight adjacent base pairs. In the absence of such strain the alkylation sites have an ordered structure, with the attached hydrocarbon probably oriented in the minor or major groove of the helix.
Exon shuffling generates an immunoglobulin heavy chain gene.
Authors: Maki R , Traunecker A , Sakano H , Roeder W , Tonegawa S
Published: May 2006

From endonuclease EcoRI partial libraries of DNAs from mouse embryo and MOPC 141, a gamma 2b-producing myeloma, clones were isolated by using a DNA fragment carrying the gamma 2b constant (C) region gene as a hybridization probe. One clone from MOPC 141 contained a heavy chain variable (V) gene and the C gamma 2b gene, as demonstrated by R-loop mapping. The V gene and C gene in this clone were separated by a 3.9-kilobase intron. The characterization of this clone as well as the embryonic clones suggest that at least two recombination events occurred to create the gamma 2b gene in MOPC 141. One of the events is analogous to the V-J joining previously demonstrated in the light chain genes, which brings the major part of the V gene next to a short coding sequence (J). The other event we refer to as "C mu-C gamma 2b switch recombination" because a portion of the intron between the V gene and C gene of the rearranged gamma 2b gene is derived from the 5' flanking sequence of the embryonic C mu gene. A model suggesting how the phenomenon of switch seen in lymphocytes may occur is presented.
H-2 restriction: Independent recognition of H-2 and foreign antigen by a single receptor
Authors: Siliciano Robert F. , Zacharchuk Charles M. , Shin Hyun S.
Published: May 2006

We describe two situations in which the recognition of hapten can compensate for the lack of recognition of appropriate
H-2
gene products in hapten-specific, H-2 restricted, T lymphocyte-mediated cytolysis. First, we show that although recognition of appropriate
H-2
gene products is essential for the lysis of target cells bearing a low hapten density, significant hapten-specific lysis of H-2 inappropriate target cells is observed at high levels of target cell derivatization. Secondly, we show that hapten-conjugated anti-H-2 antibody inhibits cytolysis poorly even though its binding to target cell H-2 antigens is equivalent to that of underivatized antibody. These results suggest that hapten and H-2 are recognized independently and are therefore inconsistent with the altered-self model. Although our data do not exclude the dual-recognition model, we prefer to interpret them within the framework of a single-receptor model in which hapten and H-2 are recognized independently by receptors of identical idiotype on the T cell. We postulate that the affinity of these receptors for the relevant
H-2
gene product is low enough so that the T cell is not activated by encounters with normal-self cells expressing that
H-2
gene product. However, when self cells express in addition a foreign antigen that can also be recognized by the same receptor, then the force of T cell-target cell interaction may be increased sufficiently to activate T cell effector function.
Highly efficient induction of type C retroviruses by a human tumor in athymic mice.
Authors: Gautsch J W , Knowles A F , Jensen F C , Kaplan N O
Published: May 2006

We have found that 1 of 20 human tumors transplanted and passaged in nude mice was associated with a massive induction of endogenous murine leukemia virus (MuLV). Separation and growth of these viruses on various substrates indicated that both ecotropic and xenotropic MuLV were present in the induced mixture. Tryptic peptide fingerprints of the p30 and gp70 structural elements of the viruses indicated that all of the known endogenous muLVs of BALB/c mice were present in the mixture. In addition, a new xenotropic MuLV was identified. The human tumor that induced the viruses was an oat cell carcinoma. The oat cell carcinoma possibly produced a specific hormone or factor that acts as a potent inducer of endogenous type C retroviruses.
Three-dimensional structure of the "long" neurotoxin from cobra venom.
Authors: Walkinshaw M D , Saenger W , Maelicke A
Published: May 2006

The three-dimensional structure of alpha-cobra-toxin, the "long" neurotoxin from the venom of Naja naja siamensis, has been determined at 2.8-A resolution. Crystals grown as hexagonal needles have space group P6522 with unit cell parameters a = b = 74.59 A, c = 42.89 A; one molecule per asymmetric unit. Phases were determined with a single isomorphous derivative with HgI2 by using the anomalous scattering of the single-site HgI2 molecule to resolve the phase ambiguity. The polypeptide chain folds into three major loops and one tail emerging from a globular head. The protruding long central loop (residues 21-40) is flanked on either side by two shorter loops (residues 4-13 and 44-55); the tail piece (residues 63-71) hangs behind this loop. The molecular conformation is determined by four disulfides in the head and one at the tip of the long loop, by a triple-stranded beta-pleated sheet involving this loop, and by hydrophobic interactions stabilizing the other two loops. The structure of alpha-cobratoxin is compared to that described for the "short" erabutoxin b which shows similar arrangement of structurally and functionally invariant groups.
Calcium translocation mechanism in sarcoplasmic reticulum vesicles, deduced from location studies of protein-bound spin labels.
Authors: Champeil P , Rigaud J L , Gary-Bobo C M
Published: May 2006

Sarcoplasmic reticulum vesicles were exposed to various thiol-directed spin labels, and the position of the label on the inner or outer vesicle surface was investigated as a function of the ATPase (adenosinetriphosphatase; ATP phosphohydrolase, EC 3.6.1.3) chemical state. Previous measurements of label accessibility to externally added ascorbate had been considered to suggest an external-internal transition of protein-bound labels, coupled with ion translocation [Tonomura, Y. & Morales, M.F. (1974) Proc. Natl. Acad. Sci. USA 71, 3687-3691]. We show that these ascorbate studies do not lead to convincing conclusions. We demonstrate, on the contrary, that transition ions (nickel and ferricyanide) can be used as selective line-broadening agents for the signals arising from external labels. No significant difference in nickel- or ferricyanide-label interaction can be attributed to a different orientation of the label in any of the enzyme chemical states tested. Our results therefore contradict the current interpretation of ascorbate quenching experiments in terms of calcium ATPase rotatory motion; rather they are consistent with ion transport models involving only limited conformational rearrangements of the pump.
Structure of genes for virus-associated RNAI and RNAII of adenovirus type 2.
Authors: Akusjärvi G , Mathews M B , Andersson P , Vennström B , Pettersson U
Published: May 2006

A DNA sequence, 552 base pairs in length, encoding the two "virus-associated" (VA) RNAs of adenovirus type 2 is presented. Comparison of the oligonucleotide maps of VA RNAI and VA RNII with the established sequence permits identification of the genes for these RNAs. VA RNAI is 157-160 nucleotides long and VA RNAII 158-163 nucleotides long, depending on the exact length of their heterogeneous 3' end. The genes are separated by a spacer of about 98 nucleotides. The RNAs exhibit scattered regions of primary sequence homology and can adopt secondary structures which resemble each other closely in their configuration and stability. VA RNAII is also capable of assuming a different configuration that is energetically more favorable. The data suggest that the two RNA genes may have arisen by duplication of an ancestral gene and that the folding of the RNA chain may be of importance for the function of VA RNAs. Hypothetical RNA polymerase III recognition sequences and the coding potential of the region are discussed.
Stimulatory effect of low ATP pools on transport of purine nucleosides in cells of Escherichia coli.
Authors: Munch-Petersen A , Pihl N J
Published: May 2006

Cells of Escherichia coli contain two nucleoside-transport systems. Energy-starved cells of a strain containing only one of these systems and, in addition, carrying a mutation in the Ca2+- and Mg2+-dependent ATPase (ATP phosphohydrolase 3.6.1.3) are still able to transport nucleosides. The rate is only slightly lower than the rate measured in unstarved cells. Freshly harvested uncA cells transport purine nucleosides at a higher rate than cells from the isogenic strain containing a functional ATPase. If cells from the latter strain are treated with arsenate, transport rates increase to the same levels as found in uncA cells. The presence of an uncA mutation has no effect on the transport rates for cytidine, deoxycytidine, and uridine, nor has arsenate treatment. These findings indicate that ATP is not required as energy donor for nucleoside transport. The enhanced transport rate for purine nucleosides after treatment with arsenate seems to suggest a regulatory relationship between the transport of these nucleosides and the cellular levels of ATP or a closely related metabolite.
Transfer of mannose from GDP-mannose to lipid-linked oligosaccharide by soluble mannosyl transferase.
Authors: Spencer J P , Elbein A D
Published: May 2006

A solubilized mannosyl transferase(s) was obtained by treatment of the pig aorta particulate enzyme with the nonionic detergent Nonidet P-40 followed by centrifugation at 100,000 X g for 60 min. This enzyme preparation catalyzed the transfer of mannose from GDP-[14C]mannose (but not from [14C]mannosylphosphoryldolichol) to form a heptasaccharide-lipid. The synthesis of this heptasaccharide-lipid required the addition of an acceptor lipid that was isolated from pig liver. The oligosaccharide portion of the acceptor lipid appeared to be a mixture of trisaccharide and pentasaccharide. The formation of heptasaccharide-lipid did not require divalent cation, was not inhibited by EDTA, and was not inhibited by the antibiotic amphomycin. The heptasaccharide portion of the heptasaccharide-lipid had the same migration properties on paper chromatograms in two different solvent systems as a previously characterized Man5GlcNAc2 oligosaccharide [Li, E. & Kornfeld, S. (1979) J. Biol. Chem. 254, 2754-2758]. It also had the same migration properties as the oligosaccharide that accumulates in the presence of amphomycin. All three of these oligosaccharides emerged from a Bio-Gel P-4 column in the same position. Radioactive mannose was released from the heptasaccharide by digestion with alpha-mannosidase. These data demonstrate that at least some of the alpha-linked mannose residues in the heptasaccharide-lipid are donated directly from GDP-mannose.
Opiate regulation of estradiol-2-hydroxylase in brains of male rats: mechanism for control of pituitary hormone secretion.
Authors: Fishman J , Norton B I , Hahn E F
Published: May 2006

Treatment of male rats with a single high dose of morphine (10 mg/kg, subcutaneously) results in a dramatic suppression of brain estradiol-2-hydroxylase activity. The suppression is blocked by naloxone and is decreased upon the development of tolerance. The injection of naloxone (0.4 mg/kg) alone produces a significant increase in brain estradiol-2-hydroxylase activity over control levels. The effects of the opiate agonists and antagonists on the activity of this brain enzyme coincide in degree and direction with their effects on plasma lutropin (luteinizing hormone) concentrations. Because the 2-hydroxyestrogens were shown to induce pituitary lutropin release, the present results indicate that the action of opiates, endogenous or exogenous, on pituitary gonadotropin release can be mediated by brain catechol estrogens.
Structure of a mycobacterial polysaccharide-fatty acyl-CoA complex: nuclear magnetic resonance studies.
Authors: Maggio J E
Published: May 2006

MMP, a linear alpha 1 leads to 4 linked polymer of 3-O-methylmannose, regulates the fatty acid synthetase from Mycobacterium smegmatis by forming stoichiometric complexes with the long-chain acyl-CoA synthetase products. In agreement with previous proposals [Bloch, K. (1977) in Advances in Enzymology and Related Areas of Molecular Biology, ed. Meister, A. (Wiley, New York), Vol. 45, pp. 1-84], nuclear magnetic resonance studies show that the polysaccharide, a random coil in its free form, undergoes a major conformational transition upon enclosing long-chain acyl-CoA. The polysaccharide, probably in helical conformation in the complexed form, interacts with both the paraffinic chain and the CoA moieties of the included fatty acyl thioester.
Immunological identification of high molecular weight forms common to bovine neurophysin and vasopressin
Authors: Nicolas Pierre , Camier Maryse , Lauber Marc , Masse Marie-J. O. , Möhring Jan , Cohen Paul
Published: May 2006

Extracts of bovine neurohypophysis made in acid/ethanol solution containing protease inhibitors were fractionated by two successive filtrations on Sephadex G-75 columns equilibrated in the presence and then in the absence of 4 M urea. Analysis of the pattern of neurophysin-like immunoreactivity in the eluate, with two different antibodies, indicated the presence of high
M
r
forms of neurophysin (apparent sizes, [unk]70,000 and 20,000-25,000, respectively) besides the
M
r
10,000 neurophysin. [8-Arginine]vasopressin-like immunoreactivity was also detected, coeluting with the neurophysin-like species, in the material recovered in the exclusion and
M
r
20,000-25,000 elution volumes of the same molecular sieve fractionation of neurohypophyseal extracts. Upon subsequent Sephadex G-150 filtration, the immunoreactive material recovered in the exclusion volume of the Sephadex G-75 filtration showed an apparent
M
r
of approximately 140,000. Both neurophysin-like and vasopressin-like immunoreactivities coeluted in the same volume. The elution profile of this
M
r
140,000 material was unmodified when reanalyzed by the same molecular sieve filtration after exposure to 8 M urea. When these
M
r
140,000 immunoreactive forms of vasopressin and neurophysin were submitted to affinity chromatography on anti-neurophysin antibodies immobilized on Sepharose, both immunoreactivities were selectively coadsorbed to the immunoadsorbent. Similarly, the neurophysin and vasopressin immunoreactivities associated with
M
r
≈25,000 were retained together on the same anti-neurophysin immunoadsorbent. The
M
r
140,000 and
M
r
25,000 species having both neurophysin and [8-arginine]vasopressin antigenic determinants generated the two neurosecretory components when exposed to proteolytic activities. This
in vitro
processing was inhibited in acid medium, at low temperature, and in the presence of a mixture of protease inhibitors. It is concluded that these two large forms of proteins containing both neurophysin and vasopressin may represent common biosynthetic precursors of these two neurohypophyseal components.
Autogenous control of Escherichia coli ribosomal protein L10 synthesis in vitro.
Authors: Brot N , Caldwell P , Weissbach H
Published: May 2006

The DNA-dependent in vitro synthesis of Escherichia coli ribosomal protein L10 was inhibited when L10 was added to the protein-synthesizing incubations. Addition of L10 had little or no effect on the synthesis of ribosomal protein L12, elongation factor Tu (tufB), or the beta and beta' subunits of RNA polymerase. In addition, ribosomal protein L12 did not inhibit its own synthesis or the synthesis of L10. Experiments using a mRNA-directed system showed that the inhibition of the synthesis of L10 by itself is at the level of translation of protein synthesis. The mechanism of inhibition does not appear to be due to increased degradation of L10 mRNA.
DNA sequence encoding the NH2-terminal peptide involved in transport of lambda receptor, an Escherichia coli secretory protein.
Authors: Hedgpeth J , Clement J M , Marchal C , Perrin D , Hofnung M
Published: May 2006

lamB encodes the lambda receptor of Escherichia coli, an outer membrane protein. We have identified the beginning of the lamB gene by correlating DNA nucleotide sequence with a partial sequence of the primary translation product of lamB. We show that lambda receptor is synthesized as a precursor containing an extra 25 amino acids at its NH2 terminus. These amino acids are predominately hydrophobic and probably comprise a structure required for initiation of transport of lambda receptor from the cytoplasm to the outer membrane.