bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma prorenin response to human chorionic gonadotropin in ovarian-hyperstimulated women: correlation with the number of ovarian follicles and steroid hormone concentrations.
Authors: Itskovitz J , Sealey J E , Glorioso N , Rosenwaks Z
Published: May 2006

Plasma prorenin and active renin were measured before and after human chorionic gonadotropin (hCG) administration in two groups of patients undergoing ovarian stimulation for 4-6 days with follicle-stimulating hormone alone or in combination with luteinizing hormone, for in vitro fertilization. Baseline total plasma renin (prorenin plus active renin; n = 12) averaged 25 +/- 8 ng/ml per hr (mean +/- SD). Total renin did not change during ovarian stimulation but it increased to 46 +/- 16 ng/ml per hr (P less than 0.05) 1 or 2 days later, just before hCG administration. Thirty-six hours after hCG administration, just before laparoscopy and egg retrieval, total renin was 123 +/- 97 ng/ml per hr; a peak of 182 +/- 143 ng/ml per hr occurred 2-6 days later--i.e., during the luteal phase of the menstrual cycle. In eight of the patients who did not conceive, total renin returned to baseline 14 days after hCG administration. In four who conceived, a nadir was reached (57 +/- 13 ng/ml per hr) 8-12 days after hCG administration and then total renin increased again as the plasma beta hCG measurement began to rise. By day 16 it averaged 225 +/- 157 ng/ml per hr. In a second group of five patients active renin and prorenin were measured separately. Active renin comprised less than 20% of the total renin at all times. It was unchanged until day 4 after hCG administration and then increased significantly only when plasma progesterone was high. Thus, the initial response to hCG was entirely due to an increase in prorenin. A highly significant correlation was observed between the number of follicles and the total renin increases on the day of aspiration (r = 0.93, P less than 0.001) and at the peak (r = 0.89, P less than 0.001). After hCG administration, a temporal relationship was observed between the rise in total renin and plasma estradiol and progesterone levels. These results demonstrate that plasma prorenin increases markedly after administration of hCG and that the rise is directly related to the number of ovarian follicles and to plasma estrogen and progesterone levels. The findings suggest that prorenin is produced by the mature ovarian follicle and by the corpus luteum in response to gonadotropin stimulation.
Retinal horizontal cell gap junctional conductance is modulated by dopamine through a cyclic AMP-dependent protein kinase.
Authors: Lasater E M
Published: May 2006

The action of many neuromodulators is mediated by intracellular second messengers such as cyclic AMP. In the retina, exogenously applied dopamine alters the conductance of gap junctions between cultured horizontal cells and this effect is mediated by cyclic AMP. However, it is not known how cyclic AMP modulates horizontal cell gap junction function. Here I report that cyclic AMP works by way of a cyclic AMP-dependent protein kinase. Cyclic AMP-dependent protein kinase injected into coupled horizontal cells from white bass (Roccus chrysops) rapidly and reversibly uncoupled the cells, mimicking the actions of dopamine. The threshold for the effect was between 0.06 and 0.03 microM. Injection of Walsh inhibitor of protein kinase [Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, E. H. & Krebs, E. G. (1971) J. Biol. Chem. 246, 1977-1985] blocked the effect of dopamine. Thus, the action of dopamine is to raise intracellular levels of cyclic AMP, which then activates a cyclic AMP-dependent protein kinase. Although not tested, it is likely that the cyclic AMP-dependent protein kinase phosphorylates a protein, possibly a gap junction protein, to alter conductance.
Excitatory dyad synapse in rabbit retina.
Authors: Raviola E , Dacheux R F
Published: May 2006

In the inner plexiform layer of the rabbit retina, the synaptic endings of bipolar cells contact a pair of postsynaptic processes at an unusual type of specialized junction, the dyad synapse. One of the members of the postsynaptic dyad may return conventional feedback synapses onto the bipolar endings. Freeze-fracturing demonstrates that, opposite the presynaptic active zone, both postsynaptic membranes contain an aggregate of intramembrane particles that remain associated with the outer leaflet (E face) of the fractured plasmalemma; this is a feature typical of excitatory synapses in the central nervous system. Intracellular recordings followed by injection of horseradish peroxidase showed that at the dyad synapse the endings of rod bipolar cells are usually presynaptic to the dendrites of two amacrine cells, one narrow-field and bistratified (AII) and the other wide-field (A17). Only the A17 rod amacrine cell returns feedback synapses onto the bipolar endings. Both amacrine cells respond to illumination with transient-sustained depolarizations, dominated by rods; thus, the polarity of their light responses is the same as that of rod bipolar cells. We conclude that the dyad synapses established by rod bipolar cells with the two types of amacrine cells are excitatory.
Insulin stimulates choline acetyltransferase activity in cultured embryonic chicken retina neurons.
Authors: Kyriakis J M , Hausman R E , Peterson S W
Published: May 2006

The effect of insulin on the appearance of the enzyme choline acetyltransferase (ChoAcT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) in embryonic chicken retina neurons cultured in defined medium was studied. In the presence of a minimal level of insulin (1 ng/ml), ChoAcT activity increased with time in culture. A correspondence between the insulin concentration in the defined medium (1-100 ng/ml) and both the rate of increase and maximum attained level of ChoAcT activity was observed. Maximal ChoAcT activity was 2- to 3-fold greater in cells cultured in the presence of 100 ng of insulin per ml than in cells cultured in the presence of 1 ng of insulin per ml. To elicit maximum ChoAcT activity, insulin at 100 ng/ml was required in the medium for only the first 4 days of the culture period, at which time insulin could be reduced to maintenance levels (10 ng/ml) without affecting ChoAcT activity. Insulin binding assays performed during a 7-day culture period revealed that irrespective of the insulin concentration in the medium during culture, cell-surface insulin receptors decreased by approximately 90% between 4 and 7 days in culture. This decrease in insulin binding corresponded to the observed decrease in the sensitivity of ChoAcT activity to insulin. Our findings suggest that insulin plays a role in mediating cholinergic differentiation in the embryonic chicken retina.
Phosphatidylinositol kinase is activated in membranes derived from cells treated with epidermal growth factor.
Authors: Walker D H , Pike L J
Published: May 2006

The ability of epidermal growth factor (EGF) to stimulate phosphatidylinositol (PtdIns) kinase activity in A431 cells was examined. The incorporation of 32P from [gamma-32P]ATP into PtdIns by A431 membranes was increased in membranes prepared from cells that had been pretreated with EGF. Demonstration of a stimulation of the PtdIns kinase activity by EGF required the use of subconfluent cultures and was dependent on the inclusion of protease inhibitors in the buffers used to prepare the membranes. Stimulation of the PtdIns kinase activity was rapid. The activation peaked 2 min after the addition of EGF and declined slowly thereafter. Half-maximal stimulation of the PtdIns kinase occurred at 7 nM EGF. Kinetic analyses of the reaction indicated that treatment of the cells with EGF resulted in a decrease in the Km for PtdIns with no change in the Vmax. The kinetic parameters for the utilization of ATP were unchanged in the EGF-treated membranes compared to the control membranes. Pretreatment of the cells with the phorbol ester phorbol 12-myristate 13-acetate blocked the ability of EGF to stimulate PtdIns kinase activity. These findings demonstrate that a PtdIns kinase activity in A431 cells is regulated by EGF and provide a good system for examining the mechanism by which EGF stimulates the activity of this intracellular enzyme.
Activators of protein kinase C enhance acetylcholine receptor desensitization in sympathetic ganglion neurons.
Authors: Downing J E , Role L W
Published: May 2006

Recent studies suggest that phosphorylation may regulate the rate of desensitization of nicotinic acetylcholine (AcCho) receptors (AcChoR) in vertebrate muscle and Torpedo. It is not known if phosphorylation is involved in regulation of the neuronal AcChoR, however. In this study we examine the possibility that protein kinase C might regulate nicotinic AcChoR function in neurons. Several activators of protein kinase C (1-oleoyl-2-acetylglycerol, phorbol 12,13-diacetate, and phorbol 12,13-dibutyrate) were tested for their ability to modulate AcChoR function in embryonic chicken sympathetic ganglion neurons. Neurons were voltage-clamped at the resting potential, and the response to AcCho was tested before and after treatment with activators of protein kinase C. We find that all of these agents enhance the rate of decay of AcCho-induced current without affecting peak current amplitude or cellular input resistance. The drugs were ineffective if applied concurrently with AcCho: significant effects could be detected after 60 sec of pretreatment. A phorbol that does not increase protein kinase C activity (4 beta-phorbol) was ineffective in enhancing the decay of AcCho-induced current. Thus, the effects of these agents on AcChoR function are likely to be mediated by their interaction with C kinase, rather than by direct interaction with the AcChoR channel. Our data suggest that kinase C may regulate agonist-induced desensitization of the neuronal AcChoR channel.
Proton nuclear magnetic resonance study on the solution conformation of human epidermal growth factor.
Authors: Makino K , Morimoto M , Nishi M , Sakamoto S , Tamura A , Inooka H , Akasaka K
Published: May 2006

This paper describes elucidation of the conformation of the human epidermal growth factor (hEGF) in an aqueous environment, using one- and two-dimensional proton nuclear magnetic resonance methods. The noted structural information obtained is that a rigid core structure is formed by the interplay of the three disulfide bridges and an antiparallel beta-sheet consisting of Val-19 to Glu-24 and Asp-27 to Asn-32. Furthermore, the hydrophobic amino acid residues of the long C-terminal segment fold back to interact locally with residues in the beta-sheet. It is suggested that the C-terminal residues play an inevitable role in the formation of the receptor-binding site.
Aminoacyl-tRNA synthetases catalyze AMP----ADP----ATP exchange reactions, indicating labile covalent enzyme-amino-acid intermediates.
Authors: Rapaport E , Remy P , Kleinkauf H , Vater J , Zamecnik P C
Published: May 2006

Aminoacyl-tRNA synthetases (amino acid-tRNA ligases, EC 6.1.1.-) catalyze the aminoacylation of specific amino acids onto their cognate tRNAs with extraordinary accuracy. Recent reports, however, indicate that this class of enzymes may play other roles in cellular metabolism. Several aminoacyl-tRNA synthetases are herein shown to catalyze the AMP----ADP and ADP----ATP exchange reactions (in the absence of tRNAs) by utilizing a transfer of the gamma-phosphate of ATP to reactive AMP and ADP intermediates that are probably the mixed anhydrides of the nucleotide and the corresponding amino acid. AMP and ADP produce active intermediates with amino acids by entering the back-reaction of amino acid activation, reacting with labile covalent amino acid-enzyme intermediates. Gramicidin synthetases 1 and 2, which are known to activate certain amino acids through the formation of intermediate thiol-esters of the amino acids and the enzymes, catalyze the same set of reactions with similar characteristics. Several lines of evidence suggest that these activities are an inherent part of the enzymatic reactions catalyzed by the aminoacyl-tRNA synthetases and gramicidin synthetases and are not due to impurities of adenylate kinase, NDP kinase, or low levels of tRNAs bound to the enzymes. The covalent amino acid-enzyme adducts are likely intermediates in the aminoacylation of their cognate tRNAs. The use of gramicidin synthetases has thus helped to illuminate mechanistic details of amino acid activation catalyzed by the aminoacyl-tRNA synthetases.
Ligands internalized through coated or noncoated invaginations follow a common intracellular pathway.
Authors: Tran D , Carpentier J L , Sawano F , Gorden P , Orci L
Published: May 2006

Cholera toxin (CT) represents a class of ligands that binds preferentially to noncoated pits on the cell surface. In the present study, we have investigated the mechanism of endocytosis of this class of ligand and compared it to the classic coated pit mechanism. When either CT coupled to colloidal gold particles (CT-gold) or 125I-labeled CT were incubated with 3T3 L1 fibroblasts at 4 degrees C, both ligands bound in a preferential fashion to small noncoated pits on the cell surface. CT-gold surface-labeled cells were then incubated at 22 degrees C. The labeled ligand progressively moved into noncoated vesicles and a tubulovesicular compartment composed of a network of tubules and vesicles closely associated with multivesicular bodies but distinct from the Golgi complexes. The ligand next passed into multivesicular bodies. By contrast, alpha 2-macroglobulin (alpha 2m)-gold initially localized preferentially to coated pits and subsequently to coated vesicles and tubulovesicular structures before associating with multivesicular bodies. To directly compare the intracellular pathway followed by CT-gold to that followed by alpha 2m-gold, CT-gold (7 nm) was coincubated with alpha 2m-gold (15 nm). By 10 min of incubation at 22 degrees C, up to 66% of tubulovesicular units contained both ligands when analyzed in serial sections. Subsequently, both ligands were colocalized in multivesicular bodies. We conclude that CT-gold endocytosed via noncoated vesicles and alpha 2m-gold endocytosed through coated vesicles subsequently associate with the same tubulovesicular units, multivesicular bodies, and lysosomes.
The regulated expression of erythropoietin by two human hepatoma cell lines.
Authors: Goldberg M A , Glass G A , Cunningham J M , Bunn H F
Published: May 2006

The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. We have screened multiple renal and hepatic cell lines (including MDCK, LLC-PK1, BHK, WRL 68, CLCL, A704, CRFK, A498, ACHN, TCMK-1, LLC-MK2, CaKi-2, HepG2, and Hep3B) for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10(6) cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities less than 3.3 X 10(5) cells per cm2, there was little constitutive release of Epo in the medium (less than 30 milliunits per 10(6) cells in 24 hr). With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 microM cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.
Three-dimensional structure of an antibody-antigen complex.
Authors: Sheriff S , Silverton E W , Padlan E A , Cohen G H , Smith-Gill S J , Finzel B C , Davies D R
Published: May 2006

We have determined the three-dimensional structure of two crystal forms of an antilysozyme Fab-lysozyme complex by x-ray crystallography. The epitope on lysozyme consists of three sequentially separated subsites, including one long, nearly continuous, site from Gln-41 through Tyr-53 and one from Gly-67 through Pro-70. Antibody residues interacting with lysozyme occur in each of the six complementarity-determining regions and also include one framework residue. Arg-45 and Arg-68 form a ridge on the surface of lysozyme, which binds in a groove on the antibody surface. Otherwise the surface of interaction between the two proteins is relatively flat, although it curls at the edges. The surface of interaction is approximately 26 X 19 A. No water molecules are found in the interface. The positive charge on the two arginines is complemented by the negative charge of Glu-35 and Glu-50 from the heavy chain of the antibody. The backbone structure of the antigen, lysozyme, is mostly unperturbed, although there are some changes in the epitope region, most notably Pro-70. One side chain not in the epitope, Trp-63, undergoes a rotation of approximately 180 degrees about the C beta--C gamma bond. The Fab elbow bends in the two crystal forms differ by 7 degrees.
Evidence for a nonprolactin, non-growth-hormone mammary mitogen in the human pituitary gland.
Authors: Newman C B , Cosby H , Friesen H G , Feldman M , Cooper P , De Crescito V , Pilon M , Kleinberg D L
Published: May 2006

To determine whether the human pituitary contains a previously unidentified, nonprolactin (non-hPRL), non-growth-hormone (non-hGH) factor capable of stimulating mammary development, we tested the effects of whole human pituitary extract (hPE) and pituitary extracts depleted of hPRL and hGH ("stripped hPE") in hypophysectomized, castrated estradiol (E2)-treated male rats and rhesus monkeys. Both whole and stripped hPE significantly stimulated rat mammary development (mean scores = 3.3 and 2.0, respectively, on a scale ranging from 0 to 4) in comparison with controls (mean score = 1.0). Mammary development was not due to minute concentrations of hGH or hPRL remaining in stripped hPE because 30- to 100-fold higher concentrations of hGH (Genentech) and 1000-fold higher concentrations of hPRL were required to stimulate significant mammary development. Non-pituitary extracts of human ovary, muscle, and serum, and bovine serum albumin did not stimulate rat mammary gland growth. Trypsin destroyed the mammary mitogenic activity of whole hPE, indicating that the unidentified factor is likely a protein. Mammary growth and development were also stimulated in hypophysectomized, E2-treated monkeys by stripped hPE (mean histological score = 3.25 vs. 1.35 in control animals). Monkeys receiving stripped hPE had undetectable levels of hGH and hPRL in serum sampled over a 24-hr period. These findings suggest that the human pituitary contains a non-hPRL, non-hGH factor that stimulates mammary growth and may be important in normal mammary growth and development and perhaps in breast cancer.
Genomic RNAs of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle.
Authors: Hsu M T , Parvin J D , Gupta S , Krystal M , Palese P
Published: May 2006

The viral RNA segments in influenza virions were shown to be circular in conformation by using psoralen crosslinking methods. Electron microscopy of purified RNA following treatment of virus with the psoralen reagent 4'-aminomethyltrioxsalen (AMT) revealed circles with lengths corresponding to the individual segments. RNA blot analysis using polyacrylamide gels demonstrated that RNA from AMT-treated virus had a slowed migration, consistent with it being a single-stranded circle. Furthermore, nuclease S1 protection assays indicated that the termini of the RNA segments form an approximately 15-base-pair-long panhandle. This structure is consistent with the partial sequence complementarity that has been observed for the termini of all influenza virus RNAs. By RNA blot analysis, circular structures of viral sense RNA were also found in influenza virus-infected cells at early and late time points. The circular RNA was the predominant species at the time when the major transcription product is message RNA. This finding and the observation that the termination signal for mRNA synthesis directly abuts the panhandle suggest that a panhandle in the template viral RNA is a cis regulatory signal promoting the synthesis of mRNA instead of plus-sense template. Also, since the panhandle is present in high concentration in virions, we suggest that it is required for packaging and that the input RNA after infection is in the proper conformation for synthesis of primary transcripts.
Adenovirus E1A gene autorepression: revertants of an E1A promoter mutation encode altered E1A proteins.
Authors: Larsen P L , Tibbetts C
Published: May 2006

Revertants have been isolated from Ad3hr15, a mutant of human adenovirus type 3 that carries a defective E1A promoter. Transcription of these revertant E1A genes is restored--from nil for Ad3hr15 mutant to levels exceeding that of the wild-type virus. The mutant Ad3hr15 virus and the revertants all have an aberrant E1A promoter that contains two short tandem duplications of viral DNA sequence. The E1A gene-coding region of the mutant is the same as that for wild-type adenovirus type 3, whereas the revertants are characterized by short in-frame deletions within the 5' exon region of their E1A genes. Location of these reverting, second-site deletions is discussed in relation to E1A gene autoregulation and the evolved diversity of E1A-related oncogenic potential among different human adenoviruses.
Segment-specific inverted repeats found adjacent to conserved terminal sequences in wound tumor virus genome and defective interfering RNAs.
Authors: Anzola J V , Xu Z K , Asamizu T , Nuss D L
Published: May 2006

Defective interfering (DI) RNAs are often associated with transmission-defective isolates of wound tumor virus (WTV), a plant virus member of the Reoviridae. We report here the cloning and characterization of WTV genome segment S5 [2613 base pairs (bp)] and three related DI RNAs (587-776 bp). Each DI RNA was generated by a simple internal deletion event that resulted in no sequence rearrangement at the deletion boundaries. Remarkably, although several DI RNAs have been in continuous passage for more than 20 years, their nucleotide sequences are identical to that of corresponding portions of segment S5 present in infrequently passaged, standard, transmission-competent virus. The positions of the deletion breakpoints indicate that the minimal sequence information required for replication and packaging of segment S5 resides within 319 bp from the 5' end of the (+)-strand and 205 bp from the 3' end of the (+)-strand. The terminal portions of segment S5 were found to contain a 9-bp inverted repeat immediately adjacent to the conserved terminal 5'-hexanucleotide and 3'-tetranucleotide sequences shared by all 12 WTV genome segments. The presence of a 6- to 9-nucleotide segment-specific inverted repeat immediately adjacent to the conserved terminal sequences was found to be a feature common to all WTV genome segments. These results reveal several basic principles that govern the replication and packaging of a segmented double-stranded RNA genome.
Angiogenin abolishes cell-free protein synthesis by specific ribonucleolytic inactivation of ribosomes.
Authors: St Clair D K , Rybak S M , Riordan J F , Vallee B L
Published: May 2006

Angiogenin is a potent inhibitor of cell-free protein synthesis. When incubated with rabbit reticulocyte lysate at a concentration of 40-60 nM, it completely abolishes the capacity of the lysate to support protein synthesis. The inhibition appears to be due to its ribonucleolytic activity since it (i) generates limited cleavage products from reticulocyte RNA and (ii) is prevented from both cleaving RNA and inhibiting protein synthesis by placental RNase inhibitor. The ribonucleolytic activity of angiogenin toward the reticulocyte RNA system is highly specific. Thus, under conditions where angiogenin totally abolishes protein synthesis, an equivalent concentration of pancreatic RNase A inhibits it only partially. In contrast, RNase A is a much more effective enzyme than angiogenin using isolated RNA as substrate. Angiogenin inhibits protein synthesis by cleaving rRNA, thereby inactivating the protein synthesis machinery. Addition of isolated reticulocyte ribosomes to an angiogenin-treated lysate restores the capacity for protein synthesis, whereas addition of tRNA or mRNA does not. This potent effect on protein synthesis suggests a possible physiological function of angiogenin whose overall relevance and implications should become evident as the mechanisms of neovascularization are deciphered. The use of angiogenin may also further elucidate ribosome structure and its role in protein synthesis.
High-frequency deletion between homologous sequences during retrotransposition of Ty elements in Saccharomyces cerevisiae.
Authors: Xu H , Boeke J D
Published: May 2006

By following the fates of genetically marked Ty elements, we observed a very high frequency (80-90%) of deletion between directly repeated marker sequences during transposition. From blot hybridization analyses of Ty RNA and DNA species found in the Ty virus-like particles, we determined that the deletion events occurred during or immediately after reverse transcription of Ty RNA but before integration of Ty DNA. The results suggest that the Ty reverse-transcription machinery can recognize homologous sequences in the template. This capacity may be utilized in the replication and recombination processes of retrotransposons and retroviruses.
Three-dimensional model of the active site of the self-splicing rRNA precursor of Tetrahymena.
Authors: Kim S H , Cech T R
Published: May 2006

The rRNA intervening sequence of Tetrahymena is a catalytic RNA molecule, or "ribozyme." A tertiary-structure model of the active site of this ribozyme has been constructed based on comparative sequence analysis of related group I intervening sequences, data on the accessibility of each nucleotide to chemical and enzymatic probes, and principles of RNA folding derived from a consideration of the structure of tRNA determined by x-ray crystallography. In the model, the catalytic center has a two-helix structural framework composed of the base-paired segments of the group I conserved sequence elements. The structural framework supports and orients the conserved nucleotides that are adjacent to the base-paired sequence elements; these conserved nucleotides are proposed to form the active site and to bind the 5' splice-site duplex and the guanine nucleotide substrate. Tests of the model are proposed.
A mutational analysis of the insulin gene transcription control region: expression in beta cells is dependent on two related sequences within the enhancer.
Authors: Karlsson O , Edlund T , Moss J B , Rutter W J , Walker M D
Published: May 2006

Cell-specific expression of the insulin gene is controlled by cis-acting DNA sequences located within approximately equal to 350 base pairs of the 5' flanking DNA immediately upstream from the transcription start site. Using synthetic oligonucleotides, we have constructed a systematic series of block replacement mutants spanning this region. No single sequence appears to be absolutely required for expression. However, three of the mutants exhibit 5-10 times less activity and several others show 2-3 times less. Simultaneous mutation of two of the most mutationally sensitive regions leads to virtual abolition of activity. These two elements are structurally related and presumably represent key components of the machinery determining the cell-specific expression of the insulin gene.
Activation of casein kinase II in response to insulin and to epidermal growth factor.
Authors: Sommercorn J , Mulligan J A , Lozeman F J , Krebs E G
Published: May 2006

Insulin treatment enhances casein kinase II (CKII) activity in 3T3-L1 mouse adipocytes and H4-IIE rat hepatoma cells, the magnitude of the activation varying from 30% to 150%. Activation of CKII was apparent after 5 min of exposure of 3T3-L1 cells to insulin, was maximal by 10 min, and persisted through 90 min. The insulin-stimulated activity was inhibited by low concentrations of heparin and was stimulated by spermine. Activation of CKII was effected by physiological concentrations of insulin (EC50 = 0.15 nM), suggesting that the effect is a true insulin response and not one mediated through insulin-like growth factor receptors. Epidermal growth factor (100 ng/ml for 10 min) also activated CKII in A431 human carcinoma cells, which is consistent with other observations that insulin and epidermal growth factor may have some common effects. Insulin stimulation of CKII activity was due to an increase in the maximal velocity of the kinase; the apparent Km for peptide substrate was not altered. Enhanced activity did not appear to result from increased synthesis of CKII protein, because cycloheximide did not block the effect and because an immunoblot developed with antiserum to CKII showed no effect of insulin on the cytosolic concentration of CKII. Because insulin-stimulated CKII activity was maintained after chromatography of cell extracts on Sephadex G-25, it is unlikely that the effect is mediated by a low-molecular-weight activator of the kinase. Rather, the results are consistent with the possibility that insulin activates CKII by promoting a covalent modification of the kinase.
Interleukin 6 enhancement of interleukin 3-dependent proliferation of multipotential hemopoietic progenitors.
Authors: Ikebuchi K , Wong G G , Clark S C , Ihle J N , Hirai Y , Ogawa M
Published: May 2006

Interleukin-6 (IL-6, also known as B-cell stimulatory factor 2/interferon beta 2) was previously shown to support the proliferation of granulocyte/macrophage progenitors and indirectly support the formation of multilineage and blast cell colonies in cultures of spleen cells from normal mice. We report here that IL-3 and IL-6 act synergistically in support of the proliferation of murine multipotential progenitors in culture. The time course of total colony formation by spleen cells isolated from mice 4 days after injection of 5-fluorouracil (150 mg/kg) was significantly shortened in cultures containing both lymphokines relative to cultures supported by either of the two factors. Serial observations (mapping) of individual blast cell colonies in culture revealed that blast cell colonies emerged after random time intervals in the presence of IL-3. The average time of appearance in IL-6 alone was somewhat delayed, and in cultures containing both factors the appearance of multilineage blast cell colonies was significantly hastened relative to cultures grown in the presence of the individual lymphokines. In cultures of day-2 post-5-fluorouracil bone marrow cells, IL-6 failed to support colony formation; IL-3 alone supported the formation of a few granulocyte/macrophage colonies, but the combination of factors acted synergistically to yield multilineage and a variety of other types of colonies. In this system, IL-1 alpha also acted synergistically with IL-3, but the effect was smaller, and no multilineage colonies were seen. Together these results indicate that IL-3 and IL-6 act synergistically to support the proliferation of hemopoietic progenitors and that at least part of the effect results from a decrease in the G0 period of the individual stem cells.
Identification of base pairs in the outside end of insertion sequence IS50 that are needed for IS50 and Tn5 transposition.
Authors: Phadnis S H , Berg D E
Published: May 2006

Short DNA sequences at ends of transposable elements are needed as sites for transposition. Previous deletion mapping showed that, in Tn5 and its component IS50 elements, these essential sites are about 19 base pairs long. To determine which positions are important in transposition, we made one or more sequence changes at each position in the IS50 outside (O) end and assayed the effects of these changes on transposition. Our results indicate that the specific base pairs at 18 of the 19 positions are important in transposition. A 9-base-pair segment in the O end corresponds to a binding site for the Escherichia coli DnaA protein. Comparisons of effects of mutations at different positions in this site, and also measurements of Tn5 transposition in dnaA- cells, indicate that DnaA protein participates in O-end-mediated transposition.
Close linkage of the mouse and human CD3 gamma- and delta-chain genes suggests that their transcription is controlled by common regulatory elements.
Authors: Saito H , Koyama T , Georgopoulos K , Clevers H , Haser W G , LeBien T , Tonegawa S , Terhorst C
Published: May 2006

Antigen receptors on the T-cell surface are noncovalently associated with at least four invariant polypeptide chains, CD3-gamma, -delta, -epsilon, and -zeta. The mouse CD3-gamma gene, consisting of seven exons, was found to be highly homologous to the CD3-delta gene described earlier. Both the high level of sequence homology and the exon/intron organization indicate that the CD3-gamma and -delta genes arose by gene duplication. Surprisingly, murine and human genomic DNA clones could be isolated that contained elements of both the CD3-gamma and CD3-delta genes. In fact, the putative transcription start site of the mouse CD3-gamma gene is less than 1.4 kilobases from the transcription initiation site of the mouse CD3-delta gene. Common elements that regulate the divergent transcription of the two genes are therefore proposed to be located in the intervening 1.4-kilobase DNA segment. This might contribute to the coordinate expression of the CD3-gamma and -delta genes during intrathymic maturation of T lymphocytes.
A model of high-affinity antibody binding to type III group B Streptococcus capsular polysaccharide.
Authors: Wessels M R , Muñoz A , Kasper D L
Published: May 2006

We recently reported that the single repeating-unit pentasaccharide of type III group B Streptococcus (GBS) capsular polysaccharide is only weakly reactive with type III GBS antiserum. To further elucidate the relationship between antigen-chain length and antigenicity, tritiated oligosaccharides derived from type III capsular polysaccharide were used to generate detailed saturation binding curves with a fixed concentration of rabbit antiserum in a radioactive antigen-binding assay. A graded increase in affinity of antigen-antibody binding was seen as oligosaccharide size increased from 2.6 repeating units to 92 repeating units. These differences in affinity of antibody binding to oligosaccharides of different molecular size were confirmed by immunoprecipitation and competitive ELISA, two independent assays of antigen-antibody binding. Analysis of the saturation binding experiment indicated a difference of 300-fold in antibody-binding affinity for the largest versus the smallest tested oligosaccharides. Unexpectedly, the saturation binding values approached by the individual curves were inversely related to oligosaccharide chain length on a molar basis but equivalent on a weight basis. This observation is compatible with a model in which binding of an immunoglobulin molecule to an antigenic site on the polysaccharide facilitates subsequent binding of antibody to that antigen.
Improving the
in vitro
protein digestibility of sorghum with reducing agents
Authors: Hamaker B. R. , Kirleis A. W. , Butler L. G. , Axtell J. D. , Mertz E. T.
Published: May 2006

We have shown in previous reports that cooked sorghum protein is less digestible than other cooked cereal proteins. The pepsin-indigestible proteins in sorghum were found to be mainly prolamin proteins. Cooking sorghum in the presence of 2-mercaptoethanol increased protein digestibility (
in vitro
with pepsin or trypsin/chymotrypsin) to a level comparable with other cereals. At a concentration of 100 mM, other reducing agents (dithiothreitol, sodium bisulfite, and L-cysteine) were equally effective in improving sorghum digestibility. When maize was cooked in the presence of 2-mercaptoethanol, protein digestibility increased 5% compared to 25% for sorghum. Cooking barley, rice, and wheat with 2-mercaptoethanol had no significant effect on protein digestibility. The addition of reducing agents appears to prevent the formation of protein polymers linked by disulfide bonds.
Nucleotide sequence determining the first cleavage site in the processing of mouse precursor rRNA.
Authors: Craig N , Kass S , Sollner-Webb B
Published: May 2006

The first step in the processing of 47S precursor rRNA in mouse cells is reproduced in vitro in an S-100 transcription reaction and consists of an endonucleolytic cleavage at residue +650 of the primary transcript followed by rapid degradation of the fragment upstream from residue +650. An analogous processing occurs in human rRNA. The mouse and human rRNA sequences are approximately equal to 80% conserved for 200 nucleotides on the 3' side of these processing sites, suggesting that this conserved region may be important in specifying the processing. To test this hypothesis, we constructed a systematic series of deletion mutants approaching the mouse rDNA processing region from both the 5' and 3' directions and analyzed the processing of their transcripts in vitro. The 5' boundary of the region required for processing is quite sharp and corresponds to the rRNA cleavage site at the 5' end of the conserved sequence region. The 3' boundary is more complex: The 3' deletions extending to between 250 and 130 nucleotides beyond the processing site cause about a 50% decrease in the amount of the processed RNA. A 3' deletion that extends to 109 nucleotides beyond the processing site greatly reduces the processing efficiency. Deletions to or beyond 91 nucleotides on the 3' side of the processing site virtually eliminate processing. Under altered ionic conditions, transcripts of 3' deletions extending to only 41 nucleotides beyond the processing site can still direct a low level of accurate processing. These results demonstrate that the mouse/human conserved sequence just on the 3' side of the primary rRNA processing site consists of several domains that direct and/or augment both the initial endonucleolytic cleavage and the closely coupled selective degradation of the upstream fragment that together constitute the primary rRNA processing event.
Cloning, sequencing, and expression of cDNA for human beta-glucuronidase.
Authors: Oshima A , Kyle J W , Miller R D , Hoffmann J W , Powell P P , Grubb J H , Sly W S , Tropak M , Guise K S , Gravel R A
Published: May 2006

We report here the cDNA sequence for human placental beta-glucuronidase (beta-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. We also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH2-terminal amino acid sequence determined for human spleen beta-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human beta-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human beta-glucuronidase, demonstrate the existence of two populations of mRNA for beta-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.
Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and receptor kinase activity.
Authors: Ebina Y , Araki E , Taira M , Shimada F , Mori M , Craik C S , Siddle K , Pierce S B , Roth R A , Rutter W J
Published: May 2006

To test whether the tyrosine kinase activity of the insulin receptor is crucial for insulin action, we have constructed mutations of the human insulin receptor at Lys-1030, which is in the presumed ATP-binding region. By using oligonucleotide-directed mutagenesis, this lysine residue was replaced with either methionine, arginine, or alanine. Chinese hamster ovary cells were transfected by mutant cDNAs and the expressed insulin receptors were characterized. We show here that none of these mutants exhibited insulin-activated autophosphorylation and kinase activity in vitro. They also do not mediate insulin- and antibody-stimulated uptake of 2-deoxyglucose. The tyrosine kinase activity is thus required for a key physiological response of insulin.
Unusual structure of geranium chloroplast DNA: A triple-sized inverted repeat, extensive gene duplications, multiple inversions, and two repeat families
Authors: Palmer Jeffrey D. , Nugent Jacqueline M. , Herbon Laura A.
Published: May 2006

Physical and gene mapping studies reveal that chloroplast DNA from geranium (
Pelargonium hortorum
) has sustained a number of extensive duplications and inversions, resulting in a genome arrangement radically unlike that of other plants. At 217 kilobases in size, the circular chromosome is about 50% larger than the typical land plant chloroplast genome and is by far the largest described to date, to our knowledge. Most of this extra size can be accounted for by a 76-kilobase inverted duplication, three times larger than the normal chloroplast DNA inverted repeat. This tripling has occurred primarily by spreading of the inverted repeat into regions that are single copy in all other chloroplast genomes. Consequently, 10 protein genes that are present only once in all other land plants are duplicated in geranium. At least six inversions, occurring in both the inverted repeat and large single-copy region, must be postulated to account for all of the gene order differences that distinguish the geranium genome from other chloroplast genomes. We report the existence in geranium of two families of short dispersed repeats and hypothesize that recombination between repeats may be the major cause of inversions in geranium chloroplast DNA.
Molecular mapping of point mutations in the period gene that stop or speed up biological clocks in Drosophila melanogaster.
Authors: Yu Q , Jacquier A C , Citri Y , Hamblen M , Hall J C , Rosbash M
Published: May 2006

The pero1 and the pers mutations in Drosophila melanogaster, which seem to eliminate or speed up, respectively, the clocks underlying biological rhythmicity, were mapped to single nucleotides. Chimeric DNA fragments consisting of well-defined wild-type plus mutant DNA subsegments were constructed, introduced into flies by germ-line transformation, and assayed for biological activity. These experiments localized both pero1 and pers to a 1.7-kilobase DNA fragment that is mostly coding DNA. Sequencing of this subsegment from each mutant showed that pero1 is completely accounted for by a nonsense mutation in the third coding exon of a 4.5-kilobase RNA transcribed from this locus. The pers mutation is also a single nucleotide substitution, in the fourth coding exon, which results in a serine-to-asparagine substitution in the per gene protein product. The functional significance of these changes is discussed with reference to the phenotypes of the two mutations.
Recovery from anti-VIII:C (antihemophilic factor) autoimmune disease is dependent on generation of antiidiotypes against anti-VIII:C autoantibodies.
Authors: Sultan Y , Rossi F , Kazatchkine M D
Published: May 2006

Plasma samples obtained from a patient 6 wk, 6 months, and 4 yr after recovery from anti-VIII:C (anti-hemophilic factor, where VIII:C = factor VIII procoagulant activity) autoimmune disease were found to contain antibodies that inhibited anti-VIII:C activity in the patient's prerecovery plasma and in the plasma of two other patients with anti-VIII:C autoantibodies. F(ab')2 fragments from postrecovery IgG suppressed anti-VIII:C activity in F(ab')2 fragments from prerecovery IgG within a narrow range of molar ratios. Anti-VIII:C activity in F(ab')2 autoantibodies was also inhibited by F(ab')2 fragments from polyspecific therapeutic immunoglobulins prepared from a large pool of normal donors (IVIg). IgG from prerecovery plasma bound to F(ab')2 from postrecovery IgG and to F(ab')2 from IVIg, as assessed by ELISA. Affinity chromatography experiments demonstrated that F(ab')2 from postrecovery IgG preferentially bound anti-VIII:C antibodies among F(ab')2 fragments from prerecovery plasma containing anti-VIII:C autoantibodies. F(ab')2 from prerecovery plasma bound in higher amounts to postrecovery F(ab')2 than to IVIg. Insolubilized F(ab')2 fragments from postrecovery plasma also bound F(ab')2 fragments prepared from the plasma of another patient with anti-VIII:C autoimmune disease, although in lesser amounts than the patient's own prerecovery anti-VIII:C F(ab')2 antibodies. These observations suggest that human anti-VIII:C autoantibodies share idiotypic determinants and that spontaneous recovery from anti-VIII:C autoimmune disease occurs through idiotypic suppression of autoantibodies. In patients who recover from autoimmune disease and in patients in whom autoantibodies have been suppressed by infusions of IVIg, antiidiotypic antibodies, possibly by providing internal images of the antigen, may have shifted the immune system toward the steady-state equilibrium that prevents autoimmunity in normal individuals.
Insulin-like growth factor II in human adrenal pheochromocytomas and Wilms tumors: expression at the mRNA and protein level.
Authors: Haselbacher G K , Irminger J C , Zapf J , Ziegler W H , Humbel R E
Published: May 2006

Two forms of insulin-like growth factor (IGF) II with molecular masses of 10 and 7.5 kDa, respectively, were found in tumor tissue from human adrenal pheochromocytomas. The tumors contained 5.3-7.1 micrograms of immunoreactive IGF-II per g of tissue, which is about 20 times more than in adrenal medulla. The total bioactive IGF in the pheochromocytomas exceeded that in normal liver or kidney, which contained only the 7.5-kDa IGF-II species, by a factor of approximately equal to 100. By contrast, the amount of IGF-I was just measurable and did not vary significantly between tumor and normal tissue. The high amounts of IGF-II in the pheochromocytomas were not reflected, however, by a corresponding increase of mRNA. The opposite situation was found in Wilms tumors, where IGF-II content was in the same range as in nontumor tissues despite increased expression of IGF-II mRNA.
Gonadotropin-releasing peptide from human follicular fluid: isolation, characterization, and chemical synthesis.
Authors: Li C H , Ramasharma K , Yamashiro D , Chung D
Published: May 2006

A gonadotropin-releasing peptide has been isolated from human follicular fluid. Its amino acid composition and sequence are completely different from the hypothalamic lutropin-releasing hormone. It is designated human follicular gonadotropin-releasing peptide and abbreviated as hF-GRP. The primary structure of this peptide (H-Thr-Asp-Thr-Ser-His-His-Asp-Gln-Asp-His-Pro-Thr-Phe-Asn-OH) has been confirmed by chemical synthesis. In the mouse pituitary incubation assay, the ED50 value for follitropin or lutropin release is estimated to be 1.2-1.6 nM.