bluuebunny/crossref_metadata_2025 huggingface dataset:
Blockade of recruitment of ovarian follicles by suppression of the secondary surge of follicle-stimulating hormone with porcine follicular field.
Authors: Hoak D C , Schwartz N B
Published: May 2006

The increased serum concentration of follicle-stimulating hormone (FSH; follitropin) between proestrus and estrus in the rat has been hypothesized to recruit the follicles destined to ovulate in the next cycle. Injection of porcine follicular fluid (PFF) late in proestrus suppresses the secondary FSH surge; injection early in proestrus suppresses the primary FSH surge without affecting the secondary FSH surge. Thus, it is possible to use PFF to test the FSH/follicular recruitment hypothesis and to distinguish between the contributions of the primary and secondary FSH surges to this recruitment. The normal recruitment of follicles occurs in the diameter range 350-499 microns between the day of proestrus and the day of estrus. When the secondary FSH surge was suppressed by injection of PFF late in proestrus, PFF, but not porcine serum (PS), blocked follicular recruitmenet into size groups of 350-499 microns on the morning of estrus. The number of ova ovulated did not differ between PFF- and PS-treated animals. When we suppressed only the primary FSH surge, by injecting PFF early in proestrus, there were no differences between PFF- and PS-treated animals in the number of ova ovulated, follicle size distribution, or hormones. In the last experiment, the secondary FSH surge was blocked with PFF but was replaced with exogenous ovine FSH which caused a dose-related increase in follicular recruitment, substantiating the interpretation that the follicular fluid suppressed recruitment by suppressing FSH secretion. Thus, in mammals with short reproductive cycles, the gonadotropin surges provide a "fail-safe" mechanism whereby luteinizing hormone triggers ovulation, thus ending one cycle, and the secondary increase in FSH levels recruits follicles for the next cycle.
Nucleotide sequence of influenza virus RNA segment 8 indicates that coding regions for NS1 and NS2 proteins overlap.
Authors: Porter A G , Smith J C , Emtage J S
Published: May 2006

The smallest RNA segment of influenza A viruses (vRNA segment 8) has recently been shown to code for two unrelated nonstructural proteins (NS1 and NS2) translated from separate mRNAs. Molecular weight considerations indicated that there might not be enough space on vRNA segment 8 for the two coding regions unless they overlap. We have recently cloned in bacterial plasmids several genes of an avian influenza A virus, fowl plague virus (EPV), and now present the complete nucleotide sequence of FPV RNA segment 8 largely determined from the cloned DNA. The DNA sequence predicts two open protein synthesis reading frames that can be translated into polypeptides of sizes similar to those of NS1 and NS2. The coding regions for these polypeptides overlap by the equivalent of 43-60 amino acids, the exact amount depending on which of several possible methionines initiates the synthesis of NS2.
Novel histone H2A-like protein of escherichia coli.
Authors: Hübscher U , Lutz H , Kornberg A
Published: May 2006

A histone-like protein (H) from Escherichia coli has been purified to more than 98% homogeneity by using its capacity to inhibit DNA functions. H protein behaves as a dimer of 28,000-dalton subunits. The histone H2A-like properties of H protein are: (i) binding to DNA at a stoichiometry of 1 H protein dimer per 75 bases; (ii) abundance of about 30,000 molecules per cell, sufficient to bind about 20% of the chromosome; (iii) limiting digestion of double-stranded DNA by micrococcal nuclease; (iv) reannealing of complementary single-stranded DNA; (v) amino acid composition resembling that of eukaryotic histone H2A; (vi) neutralization of H protein by antibody specific for H2A; (vii) heat stability; and (viii) acid solubility. The capacity of H protein to bind DNA prevents its template or substrate functions n several reactions in vitro: DNA synthesis by several polymerases; transcription by RNA polymerase; DNA topoisomerase activity; and DNA-dependent ATP hydrolysis by rep protein, dnaB protein, or protein n'. Together with other histone-like proteins of E. coli, H protein may organize the E. coli chromosome into nucleosomes, such as in eukaryotic chromatin.
Amino-terminal amino acid sequence of human leukocyte interferon.
Authors: Levy W P , Shively J , Rubinstein M , Del Valle U , Pestka S
Published: May 2006

We report the amino-terminal sequence of the first 22 amino acids of human leukocyte interferon. These and other results indicate that human leukocyte interferon consists of many individual species. We, therefore, postulate that diversity in this protein is routinely present and that the human leukocyte interferons represent a multigene family.
Role of arachidonic acid in stimulation of hexose transport by human polymorphonuclear leukocytes
Authors: Bass David A. , O'Flaherty Joseph T. , Szejda Pamela , DeChatelet Lawrence R. , McCall Charles E.
Published: May 2006

Whereas insulin does not stimulate hexose transport in polymorphonuclear leukocytes, we recently reported that C5a causes the leukocytes to take up 2-[
3
H]deoxyglucose. We now find that fMet-Leu-Phe, in a concentration-related manner with an EC
50
(concentration producing 50% of stimulatory activity) of 1.2 nM, causes a 5.5-fold stimulation of deoxyglucose uptake. Moreover, arachidonic acid (5,8,11,14-eicosatetraenoic acid) similarly stimulated deoxyglucose uptake with an EC
50
of 0.6 μM. Stimulation by arachidonic acid exhibited structural specificity; five structural analogues of arachidonic acid, including arachidonyl alcohol, 8,11,14-eicosatrienoic acid, 11,14,17-eicosatrienoic acid, 5,8,11,14-eicosatetraynoic acid, and arachidic acid, did not stimulate deoxyglucose uptake. Release and metabolism of arachidonic acid may also be involved in the stimulation of deoxyglucose uptake by fMet-Leu-Phe. Inhibitors of arachidonic acid metabolism (5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, indomethacin, aspirin, and benzylimidazole) caused parallel changes in the responses to both arachidonic acid and fMet-Leu-Phe. Stimulation of deoxyglucose uptake of polymorphonuclear leukocytes by chemotactic factors or arachidonic acid had the characteristics of carrier-facilitated hexose transport. The response was saturable with increasing concentrations of stimulant or substrate (deoxyglucose). It was stereospecific (inhibited by D-glucose but not by L-glucose) and was inhibited in resting and stimulated cells by 5 μg of cytochalasin B per ml. It was separable from the stimulation of oxidative metabolism; it occurred normally in polymorphonuclear leukocytes from a patient with chronic granulomatous disease (these are incapable of an oxidative metabolic response to membrane stimuli). Thus, stimulation of polymorphonuclear leukocytes is associated with enhanced hexose transport. Moreover, carrier-facilitated hexose transport and arachidonic acid metabolism may be linked, at least in these leukocytes: arachidonic acid mimies the stimulatory effects of chemotactic factors, and blockade of arachidonic acid metabolism inhibits the stimulation of hexose transport by these agents.
Origin and functional properties of the major gene product of the Snyder-Theilen strain of feline sarcoma virus.
Authors: Barbacid M , Beemon K , Devare S G
Published: May 2006

The only known product of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV) is a 85,000-dalton protein, designated ST P85, that contains feline leukemia virus gag gene encoded proteins (p15, p12, and a fragment of p30) and a sarcoma virus-specific polypeptide. Antibodies directed against the latter immunoprecipitated a 92,000-dalton phosphoprotein (NCP 92) expressed at low levels in normal feline embryo fibroblasts as well as in feline cells of epithelial or lymphoid origin. Normal cellular proteins crossreactive with ST P85 were also detected in cell lines from various other mammalian species. These results suggest that the ST-FeSV sequences encoding for the sarcoma virus-specific domain of ST P85 originated from an evolutionarily conserved cellular gene expressed in cells of independent differentiation lineage. Immunoprecipitates containing ST-FeSV P85 exhibited a protein kinase activity that specifically phosphorylated tyrosine residues. The physiological significance of this finding is illustrated by the finding that phosphotyrosine is an intrinsic component of ST P85. Furthermore, 5- to-fold higher levels of this unusual phosphorylated amino acid were present in ST-FeSV transformants than in uninfected control cells. Phosphorylation of tyrosine residues appears to be associated with cellular transformation caused by Rous sarcoma virus and Abelson murine leukemia virus. Thus, independent transforming virus isolates from birds, mice, and cats may utilize common pathways in exerting their oncogenic potential.
Novel bacteriophage lambda cloning vector.
Authors: Karn J , Brenner S , Barnett L , Cesareni G
Published: May 2006

A simple method for generating phage collections representing eukaryotic genomes has been developed by using a novel bacteriophage lambda vector, lambda 1059. The phage is a BamHI substitution vector that accommodates DNA fragments 6-24 kilobases long. Production of recombinants in lambda 1059 requires deletion of the lambda red and gamma genes. The recombinants are therefore spi- and may be separated from the spi+ vector phages by plating on strains lysogenic for bacteriophage P2. Random fragments suitable for insertion into lambda 1059 are obtained by partial digestion of high molecular weight eukaryotic DNA with Sau3a. This restriction enzyme cleaves at the sequence G-A-T-C and leaves a 5'-tetranucleotide "sticky end." Because G-A-T-C extensions are also produced by BamHI cleavage, these fragments may be annealed directly to BamHI-cleaved lambda 1059. By using these methods, a set of clones covering the entire Caenorhabditis elegans genome was constructed. DNA segments which include the unc-54 myosin heavy chain gene have been isolated from this collection.
Chloroplast phosphoproteins: regulation of excitation energy transfer by phosphorylation of thylakoid membrane polypeptides.
Authors: Bennett J , Steinback K E , Arntzen C J
Published: May 2006

Incubation of isolated chloroplast thylakoid membranes with [gamma-32P]ATP results in phosphorylation of surface-exposed segments of several membrane proteins. The incorporation of 32P is light dependent, is blocked by 3(3,4-dichlorophenyl)-1,1-dimethylurea (diuron, an inhibitor of electron transport), but is insensitive to uncouplers of photophosphorylation. Polypeptides of the light-harvesting chlorophyll a/b-protein complex are the major phosphorylated membrane proteins. Addition of ATP to isolated chloroplast thylakoid membranes at 20 degrees C results in a time-dependent reduction of chlorophyll fluorescence emission; this is blocked by diuron but not by nigericin. ADP could not substitute for ATP. Chlorophyll fluorescence induction transients showed a decrease in the variable component after incubation of the membranes with ATP. Chlorophyll fluorescence at 77 K of phosphorylated thylakoid membranes showed an increase in long-wavelength emission compared with dephosphorylated controls. We conclude that a membrane-bound protein kinase can phosphorylate surface-exposed segments of the light-harvesting pigment-protein complex, altering the properties of its interaction with the two photosystems such that the distribution of absorbed excitation energy increasingly favors photosystem I.
Molecular cloning and characterization of potato spindle tuber viroid cDNA sequences
Authors: Owens Robert A. , Cress Dean E.
Published: May 2006

Double-stranded cDNA has been synthesized from a polyadenylylated potato spindle tuber viroid (PSTV) template and inserted in the
Pst
I endonuclease site of plasmid pBR322 by using the oligo(dC)·oligo(dG)-tailing procedure. Tetracycline-resistant ampicillin-sensitive transformants contained sequences complementary to PSTV [
32
P]cDNA, and one recombinant clone (pDC-29) contains a 460-base-pair insert. This cloned double-stranded PSTV cDNA contains the cleavage sites for six restriction endonucleases predicted by the published primary sequence of PSTV as well as one additional site each for
Ava
I,
Hae
III,
Hpa
II, and
Sma
I. The additional
Ava
I,
Hpa
II, and
Sma
I sites are explained by the presence of a second C-C-C-G-G-G sequence in the cloned double-stranded cDNA. The largest fragment released by
Hae
III digestion contains approximately 360 base pairs. These results suggest that we have cloned almost the entire sequence of PSTV, but the sequence cloned differs slightly from that published. Hybridization probes derived from pDC-29 insert have allowed detection and preliminary characterization of RNA molecules having the same size as PSTV but the opposite polarity. This RNA is present during PSTV replication in infected tomato cells.
Four heat shock proteins of Drosophila melanogaster coded within a 12-kilobase region in chromosome subdivision 67B.
Authors: Corces V , Holmgren R , Freund R , Morimoto R , Meselson M
Published: May 2006

Unique coding sequences for four heat shock proteins of Drosophila melanogaster, hsp 28, hsp 26, hsp 23, and hsp 22, are clustered in a 12-kilobase interval at chromosome subdivision 67B. The four genes are not transcribed in the same direction and each gives rise to a separate messenger RNA, with no indication of intervening sequences. Including the present results, the genes for all seven major heat shock proteins of D. melanogaster are now cloned are found to exhibit a variety of patterns of organization at the five loci they occupy.
Lasting damage to bacterial ribosomes by reversibly bound virginiamycin M.
Authors: Parfait R , Cocito C
Published: May 2006

The M and S components of virginiamycin (VM and VS) inhibit protein synthesis in bacteria--reversibly when a single component is present and irreversibly when both are present. In cell-free systems, each factor binds to the large ribosomal subunit, and the affinity of ribosomes for VS is enhanced in the presence of VM. The present work shows that the action of VM (a 500-dalton modified depsipeptide) in vivo and in vitro persists upon its removal. The in vivo demonstration is based on the loss of viability of uninfected bacteria, and on the irreversible inactivation of virus-infected cells, that are caused by a sequential incubation with VM and VS (the inhibitory action of either component alone is reversible). In vitro, the binding of labeled VM to ribosomes, followed by its detachment, yields particles unable to perform poly(U)-directed polyphenylalanine synthesis. Also, the association constant for the binding of VS to these particles is equal to that of particles incubated with a mixture of VM and VS. Our findings indicate that VM action is catalytic rather than stoichiometric, and suggest the occurrence of two states of the large ribosomal subunit, a situation leading to a complex equilibrium with multiple transitional steps in the presence of virginiamycin.
Induction of pulmonary indoleamine 2,3-dioxygenase by interferon.
Authors: Yoshida R , Imanishi J , Oku T , Kishida T , Hayaishi O
Published: May 2006

Pulmonary indoleamine 2,3-dioxygenase [indoleamine: oxygen 2,3-oxidoreductase(decyclizing)] has been found to be induced (30- to 100-fold) in the mouse after a single intraperitoneal administration of bacterial endotoxin [Yoshida, R. & Hayaishi, O. (1978) Proc. Natl. Acad. Sci. USA 75, 3998-4000] or during in vivo virus infection [Yoshida, R., Urade, Y., Tokuda M. & Hayaishi, O. (1979) Proc. Natl. Acad. Sci. USA 76, 4084-4086]. In the present study, an in vitro system with mouse lung slices was developed in which bacterial endotoxin (5 micrograms/ml)produced an induction (approximately 10-fold) of indoleamine 2,3-dioxygenase. The endotoxin was substituted by interferon from mouse L cells or mouse brain. The pulmonary enzyme activity increased almost linearly for 48 hr after addition of mouse interferon (10(4) units/ml) to lung slices. Interferon from mouse L cells or mouse brain produced a 10- to 15-fold increase in the enzyme activity, whereas that from human leukocytes was all but ineffective. The effect also was observed using highly purified L-cell interferon, prepared by poly(U) affinity column chromatography. When interferon was treated either by heat, alpha-chymotrypsin, or anti-interferon serum, such increase in the enzyme activity was diminished essentially to the same extent as seen in the antiviral activity. The increase in the enzyme activity was blocked when actinomycin D or cycloheximide was added to the slices before interferon treatment. These results suggest that the enzyme induction was produced by interferon and not by possible contaminants in the interferon preparations.
Flow of information in the light-triggered cyclic nucleotide cascade of vision.
Authors: Fung B K , Hurley J B , Stryer L
Published: May 2006

Photolyzed rhodopsin catalyzes the exchange of GTP for FDP bound to a protein in retinal rod outer segments. We previously proposed that the GTP complex of this protein regulates the cyclic GMP phosphodiesterase and that it may be the first amplified intermediate in visual excitation [Fung, B. K.-K. & Stryer, L. (1980) Proc. Natl. Acad. Sci. USA 77, 2500-2504]. We report here the identification and characterization of transducin, a regulatory protein consisting of three kinds of polypeptide chains: T alpha (39 kilodaltons), T beta (36 kilodaltons), and T gamma (approximately 10 kilodaltons). Reconstituted membranes containing transducin and rhodopsin but no phosphodiesterase exhibit GTPase activity and amplified binding of guanosine 5'[beta, gamma-imido]triphosphate (p[NH]ppG), a nonhydrolyzable analog of GTP, on illumination. A single photolyzed rhodopsin molecule led to the uptake of p[NH]ppG by 71 molecules of transducin. High-pressure liquid chromatography showed that the binding site for GTP is on the alpha subunit of transducin. The isolation of the complex of ;[NH]ppG with T alpha enabled us to determine whether this species is the activator of the phosphodiesterase. We found that phosphodiesterase on unilluminated disc membranes can indeed be fully activated by addition of T alpha containing bound p[NH]ppG. These findings strongly suggest that transducin is the first amplified information-carrying intermediate in the cyclic nucleotide cascade of vision.
Shadow-cast electron microscopy of fibrinogen with antibody fragments bound to specific regions.
Authors: Price T M , Strong D D , Rudee M L , Doolittle R F
Published: May 2006

Specimens of human fibrinogen mixed with Fab fragments of antibodies that were specific for various portions of the fibrinogen molecule were tungsten shadow-cast and examined by electron microscopy. Typical trinodular fibrinogen molecules were observed when Fab fragments were omitted or when fragments from nonimmune sera were used. In the experimental fibrinogen-Fab preparations, a significant number of molecules were found with an extra nodule. In the case of Fab fragments from antibodies directed to fragment E, the additional nodule was attached to the central sphere of the fibrinogen molecule. Similarly, anti-fragment D preparations yielded molecules that were derivatized on the terminal spheres. Fragments from antibodies raised against a cyanogen bromide fragment of fibrinogen alpha chains (residues 241-476) also led to exclusive derivatization of the terminal domains, although in these cases the additional material was often separated discretely from the terminal sphere by a gap. These experiments confirm longstanding notions that the central domain of a trinodular fibrinogen molecule corresponds to the plasmin-derived fragment E and that the terminal spheres correspond to fragments D. Moreover, the carboxy-terminal two-thirds of alpha chains protrude from the extremities of the molecule, as had been inferred on the basis of indirect biochemical data.
Mode of action of the heme-controlled translational inhibitor: relationship of eukaryotic initiation factor 2-stimulating protein to translation restoring factor.
Authors: Siekierka J , Mitsui K I , Ochoa S
Published: May 2006

We have purified the translation restoring factor (RF) and the eukaryotic initiation factor 2 (eIF-2) stimulating protein (ESP) to near homogeneity from the postribosomal supernatant and the ribosomal salt wash, respectively, of rabbit reticulocyte lysate. They were isolated in the form of eIF-2 complexes, apparently in a 1:1 ratio. Their virtually identical NaDodSO4/polyacrylamide gel electrophoretic patterns show, in addition to the eIF-2 alpha (38,000), beta (52,000), and gamma (54,000) bands, peptide bands at approximately 80, 65, 57, 40, and 32 kilodaltons. The apparent Mr of either complex is about 450,000, whereas that of free translation restoring factor (RF) is approximately 25,000. At 0.5 mM Mg2+, both ESP and RF stimulate ternary complex (eIF-2.GTP.Met-tRNAi) formation catalytically with unphosphorylated eIF-2. Phosphorylation of the eIF-2 alpha subunit by preincubation with eIF-2 alpha kinase and ATP, which virtually blocks eIF-2-ESP interaction, results in only partial blocking of the interaction with RF. This may explain the translation restoring activity of RF.
Nucleotide sequence of cloned rat serum albumin messenger RNA.
Authors: Sargent T D , Yang M , Bonner J
Published: May 2006

The nucleotide sequences of the recombinant DNA inserts of three bacterial plasmid clones containing nearly all of the rat serum albumin mRNA have been determined. A statistical analysis of the nucleotide sequence reveals a pattern of repeated internal homology that confirms the "intragenic triplication" model of albumin evolution.
Chemical potential of bound ligand, an important parameter for free energy transduction
Authors: Tanford Charles
Published: May 2006

The chemical potential (μ
L,b
) of a ligand L bound to a protein or enzyme can be rigorously defined, and this paper describes some of its properties in relation to other thermodynamic parameters, with emphasis on thermodynamic parameters that may be used in the elucidation of the mechanism of biological free energy transduction. Free energy transduction involves the transfer of free energy from one molecule to another, and the actual transfer may often occur while both molecules are bound to the transducer enzyme, which means that μ
L,b
for one bound ligand increases at the expense of μ
L,b
for the other. The free energy change for the overall reaction may be very small, and it is not possible to express the phenomenon of transfer, in thermodynamic terms, without the explicit use of μ
L,b
as a parameter.
Mutant defective in processing of an enzyme located in the lysosome-like vacuole of Saccharomyces cerevisiae.
Authors: Hemmings B A , Zubenko G S , Hasilik A , Jones E W
Published: May 2006

Carboxypeptidase Y, a vacuolar enzyme in Saccharomyces cerevisiae, is synthesized as a larger precursor whose apparent molecular mass is approximately 67,000 daltons. We have characterized a recessive mutation, pep4-3, that prevents maturation of this precursor. The accumulated precursor does not possess enzymatic activity. We have shown that the precursor accumulating in the pep4-3 mutant is not produced in a doubly mutant strain that also bears a mutation in the carboxypeptidase Y structural gene that eliminates production of carboxypeptidase Y. We have also shown that a nonsense fragment of carboxypeptidase Y is processed. Although there is evidence that proteinase B can catalyze the conversion of the precursor to a mature form in vitro, nonsense mutations in the structural gene for proteinase B, PRB1, do not affect the levels of carboxypeptidase Y activity, and strains bearing these mutations produce a carboxypeptidase Y of apparently normal size. Hence, proteinase B is not essential for the maturation of carboxypeptidase Y precursor in vivo. The pep4-3 mutation affects at least five vacuolar enzymes. This suggests that there is a processing event common to all of these enzymes.
Genetic control of major histocompatibility complex-linked immune responses to synthetic polypeptides in man.
Authors: Hsu S H , Chan M M , Bias W B
Published: May 2006

Vigorous lymphocyte proliferative response to synthetic polypeptides was observed in cells from 50 normal volunteers. Results indicated that 64% responded to poly(LHis, LGlu)-poly(DLAla)--poly(LLys) [(H, G)-A--L] and 54% to poly(LTyr, LGlu)-poly(DLAla)--poly(LLys) [(T, G)-A--L]. Subjects could be classified into high-, intermediate-, and non-responder phenotypes according to their stimulation indices. Family studies indicated that high responses to these antigens are inherited as histocompatibility antigen gene (HLA)-linked dominant traits. Two matings suggested gene complementation in response to (T, G)-A--L and (H, G)-A--L. One, with an intra-HLA recombinant offspring, provided evidence localizing the immune response gene(s) controlling lymphocyte proliferation to (T, G)-A--L and (H, G)-A--L, presumably the homologue to Ir-1 of mouse, closer to the HLA-B than to the HLA-D region.
Estimation of evolutionary distances between homologous nucleotide sequences.
Authors: Kimura M
Published: May 2006

By using two models of evolutionary base substitutions--"three-substitution-type" and "two-frequency-class" models--some formulae are derived which permit a simple estimation of the evolutionary distances (and also the evolutionary rates when the divergence times are known) through comparative studies of DNA (and RNA) sequences. These formulae are applied to estimate the base substitution rates at the first, second, and third positions of codons in genes for presomatotropins, preproinsulins, and alpha- and beta-globins (using comparisons involving mammals). Also, formulae for estimating the synonymous component (at the third codon position) and the standard errors are obtained. It is pointed out that the rates of synonymous base substitutions not only are very high but also are roughly equal to each other between genes even when amino acid-altering substitution rates are quite different and that this is consistent with the neutral mutation-random drift hypothesis of molecular evolution.
13C NMR study of gluconeogenesis from labeled alanine in hepatocytes from euthyroid and hyperthyroid rats.
Authors: Cohen S M , Glynn P , Shulman R G
Published: May 2006

Metabolism of [3-13C]alanine in the presence and absence of beta-hydroxybutyrate or ethanol has been followed at 25 degrees C by 13C NMR at 90.5 MHz in primary hepatocytes from untreated rats and rats treated with triiodothyronine and not allowed to eat for 24 hr. The phosphoenolpyruvate/pyruvate futile cycle was followed in situ by comparing the concentration of 13C at the scrambled alanine C2 position with that at glucose C5. In the absence of ethanol, the flux through pyruvate kinase was 60% of the gluconeogenic flux in hepatocytes from hyperthyroid rats, compared with 25% in the controls. Incubation with ethanol reduced the pyruvate kinase flux in the hyperthyroid state to that measured in the controls. Under all conditions, the relative concentration of label at the aspartate C2 and C3 sites was 1:2, whereas at the corresponding carbons in glutamate, randomization was almost complete. These observations, which require flux of unscrambled label into aspartate, are consistent with intramitochondrial synthesis of aspartate only if there is incomplete mixing of the intramitochondrial oxaloacetate pool. The 13C enrichment measured in the ketone bodies is increased by the presence of exogenous beta-hydroxybutyrate. The greater labeling that we observe at C2 of beta-hydroxybutyrate compared with C4 under this condition is explained by the flow through 3-hydroxy-3-methylglutaryl-coenzyme A synthase.
Purification of the opiate receptor from rat brain.
Authors: Bidlack J M , Abood L G , Osei-Gyimah P , Archer S
Published: May 2006

The opiate receptor was purified from a Triton-solubilized preparation of rat neural membranes by the use of affinity chromatography. The affinity gel was prepared by coupling 14-beta-bromoacetamidomorphine, a newly synthesized ligand, to omega-aminohexyl-Sepharose. After elution of the nonspecific proteins with 50 mM Tris (pH 7.5), the receptor proteins were eluted with 1 microM levorphanol or etorphine. NaDodSO4/polyacrylamide gel electrophoresis revealed three major proteins associated with the opiate receptor, having molecular weights of 43,000, 35,000, and 23,000. The purified receptor binds 10(-11) mol of dihydromorphine/per mg of protein, with a Kd of 3.8 X 10(-9) M. Other opiates, naloxone, and methionine-enkephalin, inhibit [3H]dihydromorphine binding in a manner similar to that observed with intact and solubilized neural membranes.
Interaction of sickle cell hemoglobin with erythrocyte membranes.
Authors: Shaklai N , Sharma V S , Ranney H M
Published: May 2006

The interactions of hemoglobin S with the erythrocyte membrane were compared with the corresponding interactions of hemoglobin A by measuring in both steady-state and kinetic experiments the quenching of the fluorescence of a probe embedded in erythrocyte membranes. Whereas hemoglobin A could be dissociated from membranes, a fraction of hemoglobin S was irreversibly bound even in the oxy state. Deoxyhemoglobin S interacted much more strongly with erythrocyte membranes than did deoxyhemoglobin A: a portion of the deoxyhemoglobin S was irreversibly bound, and the reversibly bound fraction of hemoglobin S dissociated more slowly than did deoxyhemoglobin A. It is suggested that the binding of deoxyhemoglobin S is a two-step reaction in which the first step involves electrostatic interaction with band III erythrocyte membrane protein and the second step involves a hydrophobic interaction with membrane lipids. The latter reaction reflects the greater hydrophobicity of hemoglobin S. The unique interaction of hemoglobin S with erythrocyte membranes may be important in the formation of irreversibly sickled cells.
Radioimmunoreactivity and receptor-binding activity of the recombined molecule obtained by complementation of two fibrinolysin fragments of ovine prolactin.
Authors: Wong T M , Cheng C H , Li C H
Published: May 2006

The recombined molecule obtained by complementation of two fibrinolysin fragments of ovine prolactin (oPRL) has been characterized by radioimmunoassay and radioreceptor assay. The recombinant alone exhibits very low radioimmunoreactivity and radioreceptor activity. However, in the presence of excess fragment oPRL-(1-53), which does not compete with oPRL in either assay, the recombinant has 101% of the radioimmunoreactivity and only 13% of the radioreceptor activity. The result shows that the low activity of the recombinant when assayed alone is due to dissociation of the molecule. When the molecule is completely in the recombined form in the presence of excess oPRL-(1-53), it retains full immunoreactivity but only part of the radioreceptor activity.
5'-O-Monophosphoryladenylyl(2' goes to 5')adenylyl(2' goes to 5')adenosine is an antagonist of the action of 5'-O-triphosphoryladenylyl-(2' goes to 5')adenylyl(2' goes to 5')adenosine and double-stranded RNA.
Authors: Torrence P F , Imai J , Johnston M I
Published: May 2006

5'-O-Monophosphoryladenylyl(2' goes to 5')adenylyl-(2' goes to 5')adenosine [(2' goes to 5')(pA)3] antagonizes the protein synthesis inhibitory effects of 5'-O-triphosphoryladenylyl-(2' goes to 5')adenylyl(2' goes to 5')adenosine by preventing activation of the (2' goes to 5')oligo(a)-activated ribonuclease which degrades mRNA. The oligoribonucleotide (2' goes to 5')(pA)3 also antagonizes the translational inhibitory capacity of poly(I).poly(C) in extracts of interferon-treated L cells, suggesting that (2' goes to 5')oligo(A) is the primary mediator of the protein synthesis inhibitory effects of poly(I).poly(C) in this cell-free system.
tif-1 mutation alters polynucleotide recognition by the recA protein of Escherichia coli.
Authors: McEntee K , Weinstock G M
Published: May 2006

The requirements for polynucleotide-dependent hydrolysis of ATP and for proteolytic cleavage of phage lambda repressor have been examined for both the wild-type (recA+ protein) and the tif-1 mutant form [tif(recA) protein] of the recA gene product. The recA+ and tif(recA) proteins catalyze both reactions in the presence of long single-stranded DNAs or certain deoxyhomopolymers. However, short oligonucleotides [(dT)12, (dA)14] stimulate neither the protease nor the ATPase activities of the recA+ protein. In contrast, these short oligonucleotides activate tif(recA) protein to cleave lambda repressor without stimulating its ATPase activity. Moreover, both the ATPase and protease activities of the tif(recA) protein are stimulated by poly(rU) and poly(rC) whereas the recA+ protein does not respond to these ribopolymers. We have purified the recA protein from a strain in which the tif mutation is intragenically suppressed. This mutant protein (recA629) is inactive in the presence of (dT)12, (dA)14, poly(rU), and poly(rC) for lambda repressor cleavage and ATP hydrolysis. These results argue that the tif-1 mutation (or mutations) alters the DNA binding site of the recA protein. We suggest that in vivo the tif(recA) protein is activated for cleaving repressors of SOS genes by complex formation with short single-stranded regions or gaps that normally occur near the growing fork of replicating chromosomes and are too short for activating the recA+ enzyme. This mechanism can account for the expression of SOS functions in the absence of DNA damage in tif mutant strains.
Kallikrein-induced uterine contraction independent of kinin formation.
Authors: Chao J , Buse J , Shimamoto K , Margolius H S
Published: May 2006

Responses of smooth muscle to kallikreins (EC 3.4.21.8) are generally considered to result from kinin formation. This premise was reexamined with the isolated rat uterus. Rat urinary kallikrein or bradykinin produced dose-dependent contractions of rat uterus but kallikrein was 5-fold more potent than bradykinin. Kallikrein caused an immediate series of rhythmic contractions which could be increased gradually with subsequent addition of kininogen substrate. Kallikrein-induced contractions were unaffected by carboxypeptidase B or a bradykinin antiserum whereas bradykinin-induced contractions were attenuated or abolished. Other serine proteinases, including trypsin, either did not induce contraction in the absence of added kininogen or did so minimally. Although small amounts of kininogen-like substrate were found in uterine tissue, detectable kinin levels (greater than 4 pg) could not be found in bathing media during maximal kallikrein-induced contractions or after uterine tissue was incubated with high concentrations of the enzyme in the presence of SQ 20881, a kininase II inhibitor. The data suggest that uterine contraction produced by a homologous kallikrein does not involve kinin formation but results from an action of this serine proteinase upon other accessible systems coupled to the contractile response.
Amino acid sequence of a human leukocyte interferon.
Authors: Levy W P , Rubinstein M , Shively J , Del Valle U , Lai C Y , Moschera J , Brink L , Gerber L , Stein S , Pestka S
Published: May 2006

The primary structures of three major species of human leukocyte interferon differ from the structure predicted from the DNA sequence of recombinants containing leukocyte interferon-coding regions. Compared to the recombinant interferon produced in bacteria, three of the purified natural proteins isolated from leukocytes lack the 10 COOH-terminal amino acids suggested by the DNA sequence.
Configuration of the four iron atoms in dissolved human hemoglobin as studied by anomalous dispersion.
Authors: Stuhrmann H B , Notbohm H
Published: May 2006

The anomalous dispersion of iron at its K-absorption edge in small-angle scattering of an aqueous solution of hemoglobin has been used to establish the geometrical arrangement of the four iron atoms in this protein. Although the anomalous contributions are 0.001-0.01 of the total scattering, experiments with synchrotron radiation from the storage ring DORIS have shown that these effects can be measured with an average precision of approximately 10% at each of the 50 points of the scattering curve. The anomalous scattering represents the convolution of the whole structure with the configuration of the four iron atoms of hemoglobin. Analysis in terms of multipoles suggests that tetrahedral symmetry of both the subunit arrangement and the iron structure is a dominant feature. The mean distance between the iron atoms of 26 A derived from this experiment compares well with that derived from crystallographic data.
Membrane differentiation in human myeloid cells: expression of unique profiles of cell surface glycoproteins in myeloid leukemic cell lines blocked at different stages of differentiation and maturation.
Authors: Fukuda M , Koeffler H P , Minowada J
Published: May 2006

Cell surface glycoprotein structures of human leukemic cell lines blocked at various stages of myeloid (granulocyte--monocyte) differentiation and maturation were characterized. Cell lines representing early stages of differentiation and maturation such as KG1a, KG1 and those representing later stages such as ML-1-3 and HL-60 were found to express discretely different sets of cell surface glycoproteins. The proteins (such as Gp130) present on mature granulocytes appear only in cell lines representing later stages of maturation. The quantity and structure of the carbohydrate chains susceptible to endo-beta-galactosidase ("lactosaminoglycan") are also significantly different among cell lines. A small quantity of essentially unbranched lactosaminoglycan is present on KG1a and KG1 cells, whereas a significant quantity of branched lactosaminoglycan is present on the more mature ML-1-3 and HL-60 cells. The process of myeloid maturation from promyeloblast to granulocyte is accompanied by the disappearance of the 105- and 95-kilodalton glycoproteins, the appearance of Gp130, and a great increase in the quantity and branching structure of lactosaminoglycan. These results are discussed together with our previous findings on erythroid differentiation and maturation.
Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera.
Authors: Hinuma Y , Nagata K , Hanaoka M , Nakai M , Matsumoto T , Kinoshita K I , Shirakawa S , Miyoshi I
Published: May 2006

Indirect immunofluorescence of certain human sera demonstrated an antigen(s) in the cytoplasm of 1--5% of the cells of a T-cell line, MT-1, from a patient with adult T-cell leukemia (ATL), which is endemic in southwestern Japan. The antigen was not detected in other human lymphoid cell lines, including six T-cell lines, seven B-cell lines, and four non-T non-B cell lines. The antigen did not show cross antigenicity with that of herpesviruses, including Epstein--Barr virus, herpes simplex virus, cytomegalovirus, varicella-zoster virus, herpesvirus saimiri, and Marek disease virus. The proportion of antigen-bearing cells was increased by a factor of approximately 5 on culture in the presence of 5-iodo-2'-deoxyuridine. Antibodies against the antigen in MT-1 cells were found in all 44 patients with ATL examined and in 32 of 40 patients with malignant T-cell lymphomas (most of them had diseases similar to ATL except that leukemic cells were not found in the peripheral blood). The antibodies were also detected in 26% of the healthy adults examined from ATL-endemic areas but in only a few of those examined from ATL-non-endemic areas. On electron microscopy, extracellular type C virus particles were detected in pelleted MT-1 cells cultured in the presence of 5-iodo-2'-deoxyuridine.
Atherosclerosis and hypertension induction by lead and cadmium ions: an effect prevented by calcium ion.
Authors: Revis N W , Zinsmeister A R , Bull R
Published: May 2006

In epidemiological studies, both positive and negative correlations have been found between cardiovascular disease and mortality and the presence of several inorganic ions in the drinking water. In an attempt to resolve this apparent disagreement, we exposed White Carneau pigeons to drinking water containing calcium (100 ppm), magnesium (30 ppm), lead (0.8 ppm), or cadmium (0.6 ppm) and used a 2(4)-factorial design to measure the effects of these elements in atherosclerosis and hypertension. The results indicate that (i) lead and cadmium induced aortic atherosclerosis and hypertension, and (ii) calcium protects against the cardiovascular effects of cadmium. Furthermore, the effects of lead and cadmium were promoted by magnesium, and there were indications that magnesium antagonized the atherosclerotic protective effect of calcium. We suggest that, if these results with the pigeon can be applied to humans, the incidence of aortic atherosclerosis and hypertension should be significantly higher in areas where the drinking water contains magnesium, lead, and cadmium with a relatively low calcium concentration. Furthermore, if hard and soft water produce similar levels of lead and cadmium uptakes, the level of magnesium may be an additional factor in aortic atherosclerosis.
The dicyclohexylcarbodiimide-binding protein c of ATP synthase from Escherichia coli is not sufficient to express an efficient H+ conduction.
Authors: Friedl P , Bienhaus G , Hoppe J , Schairer H U
Published: May 2006

Bacteriophage Mu was inserted into the unc genes of Escherichia coli. The resulting mutation AS12 had a polar effect on the unc operon: membranes of the mutant AS12 contained the dicyclohexylcarbodiimide-binding protein c and the protein a as sole subunits of the ATP synthase. It was shown by peptide mapping and amino acid analysis of the fragments that protein c from mutant AS12 was identical with the wild-type protein c. The absence of subunit b in mutant AS12 drastically lowered the H+ conduction dependent on the membrane-integrated moiety (F0) of the ATP synthase. This suggests that both subunits b and c are necessary for an efficient expression of H+ conduction.
Arrangement of the subunits in the ribosome of Escherichia coli: demonstration by immunoelectron microscopy.
Authors: Kastner B , Stöffler-Meilicke M , Stöffler G
Published: May 2006

The three-dimensional locations of Escherichia coli ribosomal proteins S13, L1, and L7/L12 on the surface of ribosomal subunits and 70S monomeric ribosomes were determined by electron microscopy of antibody-labeled ribosomal particles. A new approach to orient the subunits within 70S ribosomes was developed that used 30S.70S.50S triples that were prepared by simultaneous combination with one antibody directed against a 30S protein and another directed against a 50S protein. Electron microscope studies of triples obtained with the antibody combinations anti-S13/anti-L1 and anti-S13/anti-L7/L12 showed that, in 70S monomeric ribosomes, the head of the 30S subunit is proximate to protein L1 and the peptidyl transferase center but far from the rod-like appendage containing proteins L7 and L12.
Characterization of brevin, a serum protein that shortens actin filaments
Authors: Harris David A. , Schwartz James H.
Published: May 2006

We have purified a protein from rabbit serum with a molecular weight of 90,000 that inhibits the polymerization of actin measured viscometrically and that we have named “brevin” (from the Latin
breviare
, to shorten). From the extent of purification, we estimate that this inhibitor constitutes 0.3% of the total protein in plasma and serum. Brevin is also present in sera from humans and rats. Almost all of the activity in blood is extracellular; only 1% is present in platelets or other cellular elements. Several lines of evidence indicate that brevin is the same protein as the factor described by Fagraeus and Norberg [Fagraeus, A. & Norberg, R. (1978)
Curr. Top. Microbiol. Immunol.
82, 1-13] as an actin-depolymerizing factor (ADF). If ADF and brevin are identical, then “ADF” is an inappropriate name because we find that the protein shortens actin filaments without depolymerizing them. Thus, brevin causes little change in the critical concentration of monomeric actin, even though the inhibitor binds to monomeric actin complexed to DNase I-agarose. Binding of brevin to filaments was demonstrated by sedimenting the inhibitor with F-actin. From the amounts of actin and brevin sedimented, and from the lengths of filaments measured by electron microscopy, we calculated that the stoichiometry of binding is one brevin molecule per filament over a wide range of inhibitor concentrations. This stoichiometry suggests that brevin inhibits polymerization by binding at the end of elongating actin filaments, a mechanism similar to that proposed for several intracellular actin-binding proteins and for the cytochalasins. Its abundance suggests that brevin plays an important physiological role in serum, but one not directly concerned with intracellular motility. Therefore its relationship to cytoplasmic actin-binding proteins remains to be determined.
Detergent extraction of a presumptive gating component from the voltage-dependent sodium channel.
Authors: Culp W J , McKenzie D T
Published: May 2006

A physiologically characterized radiolabeled neurotoxin complex obtained from venom of the scorpion Leiurus quinquestriatus has been used to identify detergent-solubilized presumptive sodium channel components in sucrose gradients. This toxin-binding component is found in extracts prepared from three sources of excitable membrane but appears to be absent from similar extracts prepared from nonexcitable membrane or from Torpedo californica membrane. Procedures that destroy the physiological activity of the Leiurus neurotoxin lead to a corresponding loss of toxin binding to the putative sodium channel component. The major component recognized by the Leiurus toxin sediments at 6.5 S. Scatchard analysis of quantitative binding experiments carried out in sucrose gradients shows approximately linear plots and indicates that the toxin recognizes a relatively small number of sites with a dissociation constant near 10 nM. Once formed, the channel element--toxin complex is quite stable. Experiments show diphasic dissociation kinetics with half-times near 70 hr and greater than 200 hr.
Ketone-body metabolism in glioma and neuroblastoma cells.
Authors: Patel M S , Russell J J , Gershman H
Published: May 2006

We have examined the metabolism of ketone bodies in neuroblastoma C1300 and glioma C6 cells, two established lines of neural origin. The three ketone body-metabolizing enzymes are present in cells of both lines in the relative proportions normally found in brain (D-3-hydroxybutyrate dehydrogenase less than acetoacetyl-CoA thiolase less than 3-ketoacid CoA-transferase), the activities of the first two are higher in glioma cells than in neuroblastoma, and that of the third is 2-fold higher in neuroblastoma cells than in glioma cells. The specific activity of 3-ketoacid CoA-transferase (EC 2.8.3.5) in both cell lines increased as the cultures achieved confluence, then decreased. Ketone bodies and especially acetoacetate are preferred substrates for synthesis of neural lipids in cells of both lines. The incorporation of glucose carbon into lipids is significantly reduced in cells of both lines in the presence of ketone bodies. Addition of acetoacetate but not DL-3-hydroxybutyrate to the culture medium resulted in a significant increase in the activity of 3-ketoacid CoA-transferase and also in the rate of acetoacetate oxidation in neuroblastoma cells but not glioma cells. These findings indicate that specific differences exist in the capacity of these two cell lines to metabolize ketone bodies and also that substrate-level regulation of the ketone body-metabolizing pathway exists. These two lines therefore provide a potentially useful system in which the mechanisms of regulation of these enzymes may be examined.