bluuebunny/crossref_metadata_2025 huggingface dataset:
Neural cell adhesion molecules in rodent brains isolated by monoclonal antibodies with cross-species reactivity.
Authors: Chuong C M , McClain D A , Streit P , Edelman G M
Published: May 2006

Previous studies in this laboratory have led to the identification and purification of a chicken cell surface protein named "neural cell adhesion molecule" (N-CAM) that is involved in neural cell-cell and neurite-neurite interactions. In the present investigation, we have found that a similar molecule exists in the mouse and have confirmed that it is also present in rat neural tissue. A monoclonal antibody to chicken N-CAM that crossreacted with mouse and rat brains and an independently derived monoclonal antibody to mouse N-CAM were used to purify an antigen from perinatal mouse and rat brains. The purified neural antigen resembles chicken N-CAM in its ability to neutralize antibodies that inhibit neural cell aggregation and also in its biochemical properties including molecular weight, sialic acid content, amino acid composition, and autoconversion to a smaller polypeptide. Like chicken N-CAM, the murine molecule is found throughout the nervous system and over the entire neuronal cell surface. These results strongly suggest that the molecule is evolutionarily related to chicken N-CAM and prompt the hypothesis that cell adhesion involving N-CAM is a fundamental mechanism existing in nervous systems of different phylogenetic classes of animals.
alpha-Actinin interacts specifically with model membranes containing glycerides and fatty acids.
Authors: Meyer R K , Schindler H , Burger M M
Published: May 2006

A method was developed to identify specific protein-lipid interactions of complex lipid mixtures and to assess their effect upon the arrangement of such complexes in monolayers at an air-water interface. Its application to striated muscle alpha-actinin revealed that just two lipids selectively interact with alpha-actinin. One molecule of glyceride and one molecule of fatty acid were found to be associated in a constant stoichiometry with one molecule of the alpha-actinin dimer. In the presence of both glycerides and fatty acids unexpectedly rigid monolayer areas formed. This lipid specificity could be confirmed by brief protease digestion of alpha-actinin liposome mixtures followed by peptide analysis; the peptide patterns of alpha-actinin depended on the presence or absence of only these two lipids. Possible implications of these findings are discussed in the context of Z-line formation.
Tissue-specific expression of mRNAs for dipeptidyl carboxypeptidase isoenzymes.
Authors: El-Dorry H A , Pickett C B , MacGregor J S , Soffer R L
Published: May 2006

The molecular weight of newly synthesized dipeptidyl carboxypeptidase (angiotensin-converting enzyme; peptidyldipeptide hydrolase, EC 3.4.15.1) polypeptide primed in a reticulocyte lysate by poly(A)-containing RNA from mature rabbit testis was only about 65% that of the immunologically related species programmed by pulmonary RNA. Furthermore, in contrast to the pulmonary RNA-dependent product, the synthesis of this testicular protein was not directed by RNA from testes of immature animals. These findings indicate that a shorter polypeptide chain and pubertal expression--the structural and regulatory properties that distinguish the testicular dipeptidyl carboxypeptidase isozyme--are determined pretranslationally.
Control of transmembrane ion fluxes to select halorhodopsin-deficient and other energy-transduction mutants of Halobacterium halobium.
Authors: Spudich E N , Spudich J L
Published: May 2006

We describe a selection method for mutants altered in the generation and regulation of transmembrane ion flux in Halobacterium halobium. The method is based on experimental control of ion fluxes by a combination of light, ionophore, and external pH to generate an imbalance in the cells' proton circulation through their membranes. The steady-state proton circulation is increased by the introduction of a small inward proton leak with a protonophore. The cells are then illuminated to excite halorhodopsin, which hyperpolarizes the membrane and drives protons into the cells. As a result, wild-type cells suffer cytoplasmic acidification, which causes a dramatic loss of motility and suppresses their growth. These properties can be used to select for mutants that escape cytoplasmic acidification because either they lack halorhodopsin function or they have a greater capacity to eject protons during the illumination. In a popular selected by this method, 97% of the individual cells were demonstrably altered in ion flux properties. Cells were selected with alterations in the halobacterial rhodopsin, specifically with deficiencies in membrane potential generation by halorhodopsin and with increased cellular proton ejection by bacteriorhodopsin. We describe properties of one of the halorhodopsin-deficient strains, Flx37.
Electron microscopic visualization of the tetrodotoxin-binding protein from Electrophorus electricus.
Authors: Ellisman M H , Agnew W S , Miller J A , Levinson S R
Published: May 2006

Preparations of highly purified tetrodotoxin-binding protein (sodium channel) from the electric organ of the eel Electrophorus electricus were examined in negatively stained preparations. Structures observed in preparations exhibiting the highest tetrodotoxin binding tended to aggregate into ordered clusters with a unique ribbon-like conformation. The individual particles of these aggregates are elongated or rod-shaped, approximately 40 A wide and 170 A long. Stereoscopic imaging of the three-dimensional aspects of the structures revealed that the rod-like image is not an edge view of a flattened disc but represents a cylindrical structure. Individual rods in nonclustered forms were also observed but with greater frequency in preparations with lower specific activity. The dimensions of the particles suggest that they represent a protein core formed by perhaps one copy of the large glycopeptide previously identified as being part of the sodium channel. The structure of the sodium channel component visualized by negative staining is discussed in the context of the excitable properties it contributes to biological membranes.
Interleukin 1 can replace the requirement for I-A-positive cells in the proliferation of antigen-primed T cells.
Authors: Durum S K , Gershon R K
Published: May 2006

Antigen-primed T cells have been shown to require I-region-compatible adherent cells, as well as the priming antigen, to proliferate in vitro. We postulated that the Ia-recognition event is required for the T cell to induce secretion of the monokine interleukin 1 (IL 1) from adherent cells; the conventionally held view is that Ia is directly required for T cell activation. Our hypothesis predicts that IL I could replace the requirement for Ia+ cells in T cell proliferation assays in vitro. To test this prediction, we depleted keyhole limpet hemocyanin (KLH)-primed C57BL/6 mouse lymph node cells of I-A+ cells by treating with monoclonal anti-I-Ab and complement. As expected, this treatment eliminated the ability of KLH to provoke a proliferative response by primed T cells. Proliferation was restored by providing exogenous IL 1, but only in conjunction with added KLH. The proliferative response of primed T cells could also be blocked by adding anti-I-Ab to culture, and this inhibition could similarly be reversed by providing IL 1 in the presence of the specific antigen KLH. On the basis of these findings we propose a model of T cell activation and discuss its implications.
Inactivation of interferons: halomethyl ketone derivatives of phenylalanine as affinity labels.
Authors: McCray J W , Weil R
Published: May 2006

Antiviral activity of rabbit and mouse fibroblast interferons was irreversibly destroyed by treatment with halomethyl ketone derivatives of phenylalanine but not by treatment with a halomethyl ketone derivative of lysine. The inactivation reaction was pH dependent, suggesting the involvement of an amino acid residue ionizing in the region of pH 7. Tryptophan and phenylalanine, known ligands of interferons, protected rabbit interferon substantially against inactivation by the chloromethyl ketone derivative of N-tosylphenylalanine. Mixed bovine brain gangliosides protected rabbit and mouse interferons against inactivation by this reagent. Although halomethyl ketone derivatives of phenylalanine were originally designed and used for affinity labeling of the active site of chymotrypsin and similar enzymes, no evidence was found for a chymotrypsin-like activity of interferons. It is proposed that halomethyl ketone derivatives of phenylalanine inactivate interferon by an affinity labeling mechanism, first binding to a hydrophobic binding site and then reacting irreversibly with a nearby nucleophilic amino acid residue, which appears to be a histidine. This conclusion implies that a hydrophobic site on interferons is necessary for their antiviral activity.
Activation of early adenovirus transcription by the herpesvirus immediate early gene: evidence for a common cellular control factor.
Authors: Feldman L T , Imperiale M J , Nevins J R
Published: May 2006

Adenovirus mutants carrying a defective E1A gene, such as dl312, are unable to express any of the early viral genes upon infection of HeLa cells. However, efficient expression of the other early adenovirus genes was obtained when dl312-infected HeLa cells were coinfected with pseudorabies virus, a herpesvirus. By employing a temperature-sensitive pseudorabies mutant (tsG1) it was demonstrated that the herpesvirus function responsible for the induction of adenovirus transcription was the immediate early gene, a gene required for the activation of herpesvirus early gene expression and the maintenance of early and late herpesvirus transcription. Specifically, HeLa cells coinfected with dl312 and tsG1, when shifted to the nonpermissive temperature, lost their capacity to express the early adenovirus genes. Furthermore, activation of early adenovirus gene expression in herpesvirus coinfection occurred earlier and at a higher level than in wild-type adenovirus infection. Therefore, the herpesvirus immediate early protein not only activates the early adenovirus transcription units but apparently does so more efficiently than the adenovirus E1A gene product. Because of this fact, we argue that the activation, either by the E1A protein or the herpesvirus immediately early protein, most likely occurs indirectly through interaction with a cellular protein rather than by a direct recognition of regulatory sequences at the adenovirus promoters.
Diffuse structural alterations in cell membranes of spontaneously hypertensive rats.
Authors: Devynck M A , Pernollet M G , Nunez A M , Aragon I , Montenay-Garestier T , Helene C , Meyer P
Published: May 2006

Plasma membranes from heart, nerve endings, and liver were compared in 3-week-old male spontaneously hypertensive rats from the Okamoto substrain (SHR) and normotensive Wistar/Kyoto control rats (WKY) [systolic blood pressure 105 +/- 4 and 95 +/- 4 mm Hg, respectively (1 mm Hg = 133 Pa)] according to two criteria: calcium binding at physiological intracellular concentrations and polarization of an embedded fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Whatever the tissue of origin, the density of high-affinity calcium binding sites was lower in SHR than in WKY plasma membranes, and the polarization of diphenylhexatriene fluorescence was constantly higher in SHR than in WKY membranes. These membrane abnormalities are similar to those previously described in the erythrocyte membrane from SHR. The presence of diffuse structural alterations in cellular membrane from young spontaneously hypertensive rats when blood pressure is still in the normotensive range suggests a genetic origin. Such inherited abnormalities may by themselves participate in the rise in blood pressure.
Expression of receptors for tetanus toxin and monoclonal antibody A2B5 by pancreatic islet cells.
Authors: Eisenbarth G S , Shimizu K , Bowring M A , Wells S
Published: May 2006

Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.
Platelet adhesion to damaged coronary arteries: Comparison in normal and von Willebrand disease swine.
Authors: Reddick R L , Griggs T R , Lamb M A , Brinkhous K M
Published: May 2006

The early response to coronary artery injury was investigated in normal swine and in swine with von Willebrand disease (vWD). Thirty minutes after coronary endothelial denudation, a monolayer of platelets was adherent to areas of simple injury in both bleeder and normal swine. The number of adherent platelets was not significantly different in the two phenotypes. Injury involving the media of the vessel produced platelet-fibrin thrombi. Platelet activation, as judged by pseudopod formation and platelet spreading over areas of simple injury, was significantly less in bleeder animals than in normal animals. These studies suggest that chemotaxis and initial contact adhesion of platelets to injured arterial wall is independent of the von Willebrand factor. On the other hand, the spreading and activation of platelets on the subendothelium appear to be dependent on the presence of plasma von Willebrand factor. Through this mechanism von Willebrand factor may contribute to arterial thrombosis and atherogenesis.
cAMP stimulates transcription of the gene for cytosolic phosphoenolpyruvate carboxykinase in rat liver nuclei.
Authors: Lamers W H , Hanson R W , Meisner H M
Published: May 2006

The effects of starvation, glucose refeeding, dibutyryl cAMP, and dexamethasone on expression of the gene for phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from rat liver cytosol was studied by using a cloned cDNA probe. The rate of transcription of the gene for phosphoenolpyruvate carboxykinase in hepatic nuclei isolated from starved rats decreased rapidly after refeeding with glucose. Administration of dibutyryl cAMP to glucose-refed animals increased the rate of phosphoenolpyruvate carboxykinase gene transcription seven-fold within 20 min. Phosphoenolpyruvate carboxykinase mRNA in the cytosol is 2.8 kilobases long whereas liver nuclei contain four precursor RNA species that are up to 6.5 kilobases long. Feeding glucose to starved rats rapidly decreased the sequence abundance of enzyme mRNA in both nuclei and cytosol. However, the decrease in cytosolic phosphoenolpyruvate carboxykinase mRNA was preceded by a transient increase in enzyme mRNA over the first 20 min after glucose refeeding. Administration of dibutyryl cAMP to glucose-refed starved animals increased the concentration of the nuclear RNA precursors of phosphoenolpyruvate carboxykinase five- to eight-fold within 30 min and induced the mRNA for the cytosolic enzyme over a period of 60 min. We conclude that cAMP induces phosphoenolpyruvate carboxykinase mRNA by increasing the rate of gene transcription.
Regulated expression of human interferon beta 1 gene after transduction into cultured mouse and rabbit cells.
Authors: Canaani D , Berg P
Published: May 2006

The human interferon beta 1 gene has been inserted into simian virus 40 hybrid plasmid vectors carrying the bacterial phosphotransferase gene (neo), and introduced into cultured mammalian cells by DNA transfection. A majority of the transformants resistant to the antibiotic G418 were capable of synthesizing and secreting biologically active human interferon. The neo/interferon transformants contain several copies of the transfecting DNA integrated into cellular DNA sequences. In most transformants the production of human interferon and its mRNA is induced by the addition of poly(rI) X poly(rC); by contrast, the level of neo mRNA is not increased under the same conditions. The 5' end of the human interferon mRNA produced after induction was indistinguishable from the interferon mRNA induced in human fibroblasts. This indicates that information enabling human beta 1 interferon gene to be induced by poly(rI) X poly(rC) is localized to sequences within, or 5'-proximal to, the coding sequence.
Calcium/phospholipid regulates phosphorylation of a Mr "87k" substrate protein in brain synaptosomes.
Authors: Wu W C , Walaas S I , Nairn A C , Greengard P
Published: May 2006

Depolarization-induced calcium influx into rat cerebral cortex synaptosomes increased the phosphorylation of several synaptosomal proteins as examined by 32Pi incorporation. A phosphopeptide mapping technique involving NaDodSO4/polyacrylamide gels has been used to show that phosphorylation of a Mr 87,000 substrate protein is stimulated by depolarization-induced calcium influx. Phosphorylation of this Mr 87,000 substrate occurred in synaptosomal cytosol and was markedly stimulated by calcium/phosphatidylserine. Calmodulin inhibited this phosphorylation reaction. This substrate for calcium/phospholipid-dependent protein kinase is enriched in and appears to be specific to neurons.
Native phytochrome: Inhibition of proteolysis yields a homogeneous monomer of 124 kilodaltons from
Avena
Authors: Vierstra Richard D. , Quail Peter H.
Published: May 2006

Phytochrome purified from
Avena
as the red-absorbing form, Pr, by an established immunoaffinity column procedure is heterogeneous. Two major polypeptides and one minor polypeptide with apparent molecular masses of 118, 114, and 112 kilodaltons (kDal), respectively, are observed on NaDodSO
4
/polyacrylamide gel electrophoresis. In contrast, only a single band of 124 kDal is obtained when phytochrome is rapidly immunoprecipitated after extraction either (
i
) as the far-red absorbing form, Pfr, in detergent-free buffer or (
ii
) in either spectral form in a 100°C NaDodSO
4
-containing buffer. On two-dimensional gel electrophoresis the three column-purified species have pIs of 5.8, 6.0, and 6.0, whereas 124-kDal phytochrome is a single spot with a pI of 5.9. Incubation as Pr in extracts causes progressive conversion of the 124-kDal polypeptide to the 118- and 114-kDal species. This process is inhibited by phenylmethylsulfonyl fluoride, suggesting that Pr is susceptible and Pfr resistant to limited proteolysis during extraction. These data, and the fact that the cell-free translation product of phytochrome mRNA is also 124 kDal [Bolton, G. W. & Quail, P. H. (1982)
Planta
, in press], indicate that the native monomer from
Avena
is a single species of 124 kDal. Thus the heterogeneous preparations of slightly lower molecular weight (“large” or “120-kilodalton” phytochrome) previously extensively characterized appear to have consisted of a mixture of partially degraded molecules that have undergone limited proteolysis during purification as Pr, as is established practice. A reexamination of the molecular properties of phytochrome appears necessary.
Evidence for DNA modification in the maintenance of X-chromosome inactivation of adult mouse tissues.
Authors: Chapman V M , Kratzer P G , Siracusa L D , Quarantillo B A , Evans R , Liskay R M
Published: May 2006

The role of DNA modification in the maintenance of mammalian X-chromosome inactivation was investigated by using the technique of DNA transformation in mammalian cells. The ability of inactive X-chromosome DNA from adult mouse tissues to act in transformation for the X-linked hypoxanthine phosphoribosyltransferase gene (Hprt) could be ascertained by utilizing a recently discovered electrophoretic variant form of the hypoxanthine phosphoribosyltransferase enzyme and a previously available X:autosome translocation. Our findings indicate that inactive X-chromosome DNA from several tissues of adult female mice is strikingly inefficient, in comparison to active X-chromosome DNA, in eliciting genetic transformation for hypoxanthine phosphoribosyltransferase. These results provide in vivo evidence that is consistent with DNA modification playing an important role in the maintenance of X-chromosome inactivation.
Immunological castration of male mice by a totally synthetic vaccine administered in saline.
Authors: Carelli C , Audibert F , Gaillard J , Chedid L
Published: May 2006

It had been reported that immunological neutralization of the hypothalamic luteinizing hormone-releasing hormone (LH-RH) can be achieved by injection of the synthetic hormone, a decapeptide, linked to a carrier and administered with Freund's complete adjuvant. Data presented here demonstrate that the resulting immunological castration can be obtained by administering in an aqueous medium the synthetic decapeptide directly conjugated to the synthetic immunomodulator glycopeptide N-acetylmuramyl-L-alanyl-D-isoglutamine. In the group receiving the conjugate, a higher titer of anti-LH-RH antibodies was obtained than in the group receiving LH-RH in Freund's emulsion. Moreover, histological examination showed that seminiferous tubules were atrophied and that no spermatozoides could be observed. This procedure could lead to applications in the veterinary field and also serve as a useful model for other antiviral or antibacterial synthetic vaccines.
A small hypervariable segment in the variable domain of an immunoglobulin light chain stimulates formation of anti-idiotypic suppressor T cells.
Authors: Sakato N , Semma M , Eisen H N , Azuma T
Published: May 2006

The induction in BALB/c mice of suppressor T cells that block a delayed-type hypersensitivity (DTH) response to the idiotype of M315, a myeloma protein of BALB/c origin, was examined with a variety of immunoglobulin chains and fragments whose amino acid sequences are known. Normal BALB/c mice receiving either the light chain of M315 (L315, lambda 2 isotype) or the variable (V) domain of this chain prior to sensitization with M315 showed marked suppression of DTH to the M315 idiotype. In contrast, neither the heavy chain nor the variable domain of the heavy chain of M315 affected the DTH response. Two other lambda 2 chains were tested and they also failed to suppress DTH to M315. Comparison of amino acid sequences in the three lambda 2 chains indicates that in L315 at most four V region amino acid substitutions (each resulting from a somatic mutation in the V lambda 2 germ-line gene) determine the specificity of the T-cell suppressor pathway. One of the four is in the framework and probably of negligible importance; the other three, however, are all clustered in the third hypervariable loop of the L315 V domain. The tertiary structure of L315 may also be essential, because disruption of intrachain disulfide bonds abolished the ability of the chain to induce suppression.
High-level expression of an interferon alpha 2 gene cloned in phage M13mp7 and subsequent purification with a monoclonal antibody.
Authors: Slocombe P , Easton A , Boseley P , Burke D C
Published: May 2006

A cloned interferon alpha 2 (IFN-alpha 2) gene was partially digestd with Pvu II to give a fragment that was inserted into the HincII site of the lacZ gene of bacteriophage M13mp7. Two recombinant phages containing the IFN-alpha 2 sequences in the correct orientation for expression from the lac promoter were characterized in detail. DNA sequence analysis showed that the inserted IFN-alpha 2 gene was in phase with the initiation codon of the lacZ gene. The polypeptide product has an additional 19 amino amino acids at the amino terminus of the mature IFN-alpha 2. The first 11 amino acids originate from the amino terminus of beta-galactosidase, and the remaining 8 amino acids are part of the signal sequence of pre-IFN-alpha 2. Infection of Escherichia coli with these phage followed by induction of the lac promoter with isopropyl thiogalactoside gives high yields (up to 10(9) units/liter with an average of 1.5 X 10(8) units/liter) of the modified IFN-alpha 2. This was purified to homogeneity in a single step by immunochromatography using the monoclonal antibody NK2. The nonreduced product had an apparent molecular weight of 20,500 and was shown by immunoradiometric assay to have the same specific activity as IFN made in Namalwa cells. It exhibited the characteristic cross-species antiviral activity of IFN-alpha 2.
Stability of carbohydrate-modified vesicles in vivo: comparative effects of ceramide and cholesterol glycoconjugates.
Authors: Wu P S , Wu H M , Tin G W , Schuh J R , Croasmun W R , Baldeschwieler J D , Shen T Y , Ponpipom M M
Published: May 2006

The stability and tissue distribution of lipid vesicles modified at the surface by the incorporation of either a galactosyl ceramide (GalCer) or a galactosyl cholesterol (GalChol) glycoconjugate have been studied in mice by measuring the release of vesicle-entrapped 111In. Although the tissue distributions of both vesicle types were similar, the GalCer-containing vesicles were markedly less stable than those prepared with GalChol, whether administered orally or by intraperitoneal injection. Physical characterization of the vesicles in vitro suggests that the increased disruption rate for GalCer vesicles in vivo is related to structural instabilities induced by the cerebroside, which can then result in either an increased rate of vesicle uptake by tissues or a greater susceptibility to lysis. These studies demonstrate the importance of the nonpolar anchoring groups in determining the fate of surface-modified vesicles in vivo.
Abelson murine leukemia virus: structural requirements for transforming gene function.
Authors: Srinivasan A , Dunn C Y , Yuasa Y , Devare S G , Reddy E P , Aaronson S A
Published: May 2006

The integrated Abelson murine leukemia virus (A-MuLV) genome cloned in bacteriophage lambda gtWES.lambda B was used to localize viral genetic sequences required for transformation. Comparison of the biological activity of cloned A-MuLV genomic and subgenomic fragments showed that subgenomic clones that lacked the 5' long terminal repeat and adjoining sequences (300 base pairs downstream of the repeat) were not biologically active. In contrast, subgenomic clones that lacked the 3' long terminal repeat and as much as 1.3 kilobase pairs of the A-MuLV cell-derived abl gene were as efficient as wild-type viral DNA in transformation. The A-MuLV-encoded polyprotein P120 and its associated protein kinase activity were detected in transformants obtained by transfection with Cla I, BamHI, and HindIII subgenomic clones. In contrast, individual transformants obtained with subgenomic Sal I clones expressed A-MuLV proteins ranging in size from 82,000 to 95,000 daltons. Each demonstrated an associated protein kinase activity. These results provide direct genetic evidence that only the proximal 40% of abl with its associated 5' helper viral sequences is required for fibroblast transformation.
DNA methyltransferase-dependent transcription of the phage Mu mom gene.
Authors: Hattman S
Published: May 2006

The phage Mu mom gene controls an unusual DNA modification. Expression of the mom function requires an active host (dam+) DNA adenine methylase [S-adenosyl-L-methionine:DNA (6-aminopurine)-methyltransferase]; in dam- hosts, Mu development is normal except that the viral DNA does not undergo the mom modification. The present communication compares transcription of the mom gene in dam+ versus dam- cells. 32P-labeled probes were prepared by nick-translation of a purified mom gene-containing restriction fragment and of virion DNA, respectively. These probes were hybridized with various RNAs blotted onto nitrocellulose filters (after fractionation by agarose gel electrophoresis). The salient findings are: (i) mom-specific RNA was readily detected in dam+ lysogenic cells, but only after induction of the Mu prophage; (ii) the level of mom RNA was decreased at least to 1/20th in induced dam- Mu lysogens; and (iii) little difference, if any, was observed between dam+ and dam- cells with respect to total Mu transcripts produced after prophage induction. These results are in accord with the known pattern of mom gene expression and Mu development. They show that the host (dam+) DNA adenine methylase activity is required for transcription of the mom gene. This represents a unique example where a DNA methylase exerts a positive regulatory role in mRNA transcription; alternative mechanisms for this process will be discussed.
Movement of yeast transposable elements by gene conversion.
Authors: Roeder G S , Fink G R
Published: May 2006

We have constructed yeast strains in which Ty (transposon yeast) elements at the HIS4 locus are genetically marked with the yeast URA3 gene. By isolating and analyzing Ura- derivatives of these strains, we have detected a variety of Ty-mediated recombination events. In this paper, we describe events in which the DNA sequence of the Ty element at the HIS4 locus is replaced by the DNA sequence of a different Ty element. These replacements occur without alterations in the flanking DNA sequence and without chromosomal aberrations. We believe that these events result from gene conversion between the Ty element at HIS4 and a Ty element at a different site in the yeast genome. Gene conversion can occur between Ty elements that differ by large insertion and substitution mutations. These recombination events result not only in the movement of Ty sequences but also in alterations in expression of the adjacent HIS4 gene. Different Ty elements at the same site in the HIS4 regulatory region can result in His-, His+, and cold-sensitive His+ phenotypes. Several Ty elements render expression of the HIS4 gene subject to control by genes at the mating type locus.
Restriction map variation in the Adh region of Drosophila.
Authors: Langley C H , Montgomery E , Quattlebaum W F
Published: May 2006

Restriction maps of a 12,000-nucleotide region containing the alcohol dehydrogenase (Adh) locus of the second chromosome of Drosophila were determined for (i) 18 independent second chromosome stocks of D. melanogaster established from several natural populations and (ii) five closely related Drosophila species. All of the detected map differences appear to lie outside the Adh coding block. Four polymorphic restriction sites and four insertion/deletion variants were identified among D. melanogaster chromosomes. The estimated heterozygosity per nucleotide site was 0.006. The relative distributions of restriction sites and insertion/deletion variations among subpopulations and species suggest that the insertion/deletion variation may be mildly deleterious.
Monoclonal antibodies identify the protective antigens of sporozoites of Plasmodium knowlesi.
Authors: Cochrane A H , Santoro F , Nussenzweig V , Gwadz R W , Nussenzweig R S
Published: May 2006

Nine monoclonal antibodies against surface antigens of sporozoites of the simian malaria parasite Plasmodium knowlesi were produced by fusion of plasmacytoma cells with spleen cells of a mouse immunized with the parasites. Immunoprecipitation of extracts of [35S]methionine-labeled sporozoites with seven of the monoclonals identified the same three polypeptides with apparent molecular weights of 52,000 (Pk52), 50,000 (Pk50) and 42,000 (Pk42). These antigens also were recognized by serum of a rhesus monkey immunized with and protected against P. knowlesi sporozoites. Pulse--chase experiments indicated that the higher molecular weight proteins are precursors of Pk42. As shown by trypsin treatment of viable sporozoites, Pk42 is a surface antigen whereas Pk52 and Pk50 appear to be intracellular. Three of the monoclonal antibodies also reacted with a membrane antigen of sporozoites of another simian malaria, P. cynomolgi, and one monoclonal antibody reacted with sporozoites of human malaria, P. falciparum. When assayed for sporozoite neutralizing activity, most of the antibodies and their Fab fragments, which recognize Pk52, Pk50, and Pk42, abolished parasite infectivity.
An amber suppressor tRNA gene derived by site-specific mutagenesis: cloning and function in mammalian cells.
Authors: Laski F A , Belagaje R , RajBhandary U L , Sharp P A
Published: May 2006

We describe the synthesis, cloning, expression, and in vivo function of a suppressor tRNA gene in mammalian cells. By using "primer-directed mutagenesis" on a Xenopus laevis tyrosine tRNA gene cloned into the recombinant single-strand phage M13mp5, we have generated an amber suppressor tRNA gene that has a nucleotide change--GTA leads to CTA--in the anticodon sequence. The suppressor (Su) tRNA gene was introduced into monkey kidney cells (CV-1) by using simian virus 40 (SV40) DNA as vector (SV40-tRNATyrSu+). CV-1 cells infected with virus containing the mutant, but not the wild-type, tRNA gene produce a functional amber suppressor tRNA as indicated by suppression of amber mutations in co-infecting adenovirus serotype 2-SV40 hybrids. Further evidence that suppression of these amber mutations is tRNA mediated was derived by isolation of total tRNA from CV-1 cells infected with the SV40-tRNATyr (Su+) recombinant and its use in demonstration of read through of an amber codon during in vitro translation of tobacco mosaic virus RNA in reticulocyte extracts. Interestingly, the amplification of an amber suppressor gene in CV-1 cells does not interfere with SV40 production, suggesting that suppression of amber codons may not be very deleterious to mammalian cell metabolism.
Transcription initiation by RNA polymerase II is inhibited by S-adenosylhomocysteine.
Authors: Jove R , Manley J L
Published: May 2006

Most eukaryotic mRNAs are blocked at their 5' termini by guanylylation and methylation. These "cap structures" have been shown to play important roles in increasing the stability and translatability of mRNAs. Previous in vitro and in vivo data suggest that these modifications occur extremely early in the synthesis of RNA transcripts by RNA polymerase II. Here we show that S-adenosylhomocysteine (AdoHcy), both a product and an inhibitor of transmethylation reactions, inhibits transcription initiation by RNA polymerase II, but not by RNA polymerase III, in a HeLa whole-cell lysate. AdoHcy must be present during initiation to inhibit transcription and does not affect elongation by RNA polymerase II or the stability of the resultant transcript. Furthermore, AdoHcy does not inhibit transcription by purified HeLa RNA polymerase II. These results suggest that formation of the 5'-cap structure is coupled to initiation of transcription and is consistent with a close association between the capping enzymes and RNA polymerase II at the time of initiation.
DNA motions in the nucleosome core particle.
Authors: Wang J , Hogan M , Austin R H
Published: May 2006

We have used time-resolved triplet state anisotropy decay techniques to measure the conformational flexibility of DNA in the nucleosome. From these measurements we conclude that, in a nucleosome, the DNA helix experiences substantial internal flexibility, which occurs with a time constant near 30 nsec. We find that our data can be fit well by a modified version of the Barkley-Zimm model for DNA motion, allowing only DNA twisting motions and the overall tumbling of the nucleosome. That fit yields a calculated torsional rigidity equal to 1.8 X 10(-19) erg X cm, a value equal to that measured for uncomplexed DNA. We conclude from such similarity that large, fast twisting motions of the DNA helix persist, nearly unaltered, when DNA is wrapped to form a nucleosome.
Ricin inhibition of
in vitro
protein synthesis by plant ribosomes
Authors: Harley Suzanne M. , Beevers Harry
Published: May 2006

In vitro
translation systems were prepared with supernatant factors from wheat germ and 80S ribosomes from wheat germ, barley embryos, watermelon cotyledons, pea cotyledons, and castor bean endosperm. Ricin A-chain, which strongly inhibits protein synthesis by mammalian ribosomes, inhibited all of the plant ribosomal systems by 50% when present at 25-45 μg/ml—≈23,000 times the concentration needed to inhibit mammalian systems.
Ricinus communis
agglutinin A-chain, a protein similar to ricin A-chain, inhibited translation by the plant systems 50% at concentrations 5-10 times those of the ricin A-chain. Ribosomes from castor bean endosperm, the source of ricin and the agglutinin, were just as susceptible to the inhibitors as were ribosomes from the other four plants. Compartmentation of the inhibitors within vacuoles derived from protein bodies of the endosperm appears to be responsible for protecting cytoplasmic protein synthesis during germination of castor beans.
Clustering of spore-specific genes in Aspergillus nidulans.
Authors: Orr W C , Timberlake W E
Published: May 2006

We have investigated the chromosomal organization of genes that are expressed specifically in the asexual spores (conidia) of the Ascomycete fungus Aspergillus nidulans, using two experimental approaches. In the first, 30 different recombinant clones, containing long nuclear DNA inserts and at least one spore-specific gene, were selected randomly. The total number of spore-specific genes present in each clone was then determined by RNA blot analysis. In the second approach, several chromosomal recombinant DNA libraries, having average insert lengths ranging from 1 to 15 kilobases, were constructed. The fraction of clones in each library having one or more spore-specific poly(A)+RNA-coding regions was then determined by colony or plaque filter hybridization with radiolabeled, spore-specific, complementary DNA. The results from these experiments were compared to statistical predictions based on the assumption that the spore-specific genes are randomly distributed in the Aspergillus genome. In both cases, the experimental values deviated significantly from the predicted values, demonstrating that the spore-specific genes are nonrandomly arranged in the genome. Rather, they appear frequently to occur in tightly linked clusters.
Variation in the polyadenylylation site of bovine prolactin mRNA.
Authors: Sasavage N L , Smith M , Gillam S , Woychik R P , Rottman F M
Published: May 2006

The poly(A) site of bovine prolactin (bPRL) mRNA was examined by phased priming of cDNA synthesis with oligodeoxynucleotides of the general sequence d(pT8-N-N'). The existence of multiple poly(A)-adjacent sequences in bPRL mRNA was indicated by the production of specific chain-termination fragments with at least three d(pT8-N-N') sequences. Comparison of the sequence bands produced by initiation of cDNA synthesis on the bPRL mRNA template with d(pT8-A-G), d(pT8-G-A), and d(pT8-C-G) revealed a shifted pattern of identical fragments. The shift in position of related sequence bands on the gel suggested that the difference in length of the three major bPRL mRNA species occurred within a span of 12 nucleotides. Sequence analysis conducted with the three d(pT8-N-N') primers gave identical nucleotide sequences for the 3' noncoding region of the bPRL mRNA species and suggested that the mRNA molecules were heterogeneous in length. The existence of multiple poly(A) sites was confirmed by determination of the nucleotide sequence of bPRL cDNA clones containing two of the major poly(A)-adjacent sequences predicted by the oligodeoxynucleotide primers. The mRNA molecules containing these multiple poly(A)-addition sites were shown to be present in the bPRL mRNA obtained from a single pituitary gland. The variation in the poly(A) junction of bPRL mRNA may be a reflection of the processing events at the 3' terminus of mRNAs.
Affinity labeling of muscarinic receptors in rat cerebral cortex with a photolabile antagonist.
Authors: Amitai G , Avissar S , Balderman D , Sokolovsky M
Published: May 2006

Highly potent photoaffinity probes for muscarinic binding sites were prepared by the incorporation of an azido group into the benzilic acid moiety in two compound, 3-quinuclidinyl benzilate (3QNB) and N-methyl-4-piperidyl benzilate (4NMPB). Inactivation of muscarinic sites in rat cortex depends on the formation of a reversible complex with the azides prior to their photolytic conversion to the highly reactive nitrenes. During photolysis, radiolabeled azido-4NMPB interacted specifically and with high affinity (Kd = 1.06 nM) with the muscarinic receptors, and the ligand could be covalently incorporated into a macromolecule of about 86,000 Mr, presumably the muscarinic receptor. The incorporation was almost stoichiometric when compared to determination of receptor density by reversible ligands. Atropine (10 microM) afforded specific protection (greater than 83%) of the receptor against inactivation by azido-[3H]4NMPB. This compound and the other ligands described here (i.e., amino-4NMPB, amino-3QNB, and azido-3QNB) represent powerful potential probes for the biochemical isolation and characterization of muscarinic receptors.
Induction of tyrosine aminotransferase mRNA by glucocorticoids and cAMP in fetal rat liver.
Authors: Ruiz-Bravo N , Ernest M J
Published: May 2006

Tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) enzyme and mRNA activity were not detectable in day 20 fetal rat liver. Precocious induction of catalytic activity by in utero injection of dibutyryl cAMP was a direct consequence of the de novo appearance of translatable tyrosine aminotransferase mRNA. In contrast, in utero injection of hydrocortisone acetate failed to elicit fetal liver enzyme activity. This failure was due to the inability of the steroid hormone to induce the appearance of tyrosine aminotransferase mRNA activity. In fetal rat liver explants, either compound was capable of stimulating the synthesis of adult levels of enzyme and mRNA. However, catalytic and mRNA activity comparable with that seen in vivo 24 hr after birth required the concerted action of both inducers.
Characterization of leucine side-chain reorientation in collagen-fibrils by solid-state 2H NMR.
Authors: Batchelder L S , Sullivan C E , Jelinski L W , Torchia D A
Published: May 2006

We have used 2H quadrupole-echo NMR spectroscopy to study the molecular dynamics of the leucine side chain in collagen fibrils labeled with [2H10]leucine. X-ray crystallographic studies of leucine and small leucyl-containing peptides and proteins [Benedetti, C. (1977) in Proceedings of the Fifth American Peptides Symposium, eds, Goodman, M. & Meienhofer, J. (Wiley, New York), pp. 257--274; Janin, J., Wodak, S., Levitt, M. & Maigret, B. (1978) J. Mol. Biol. 125, 357--386] show that the amino acid side chain exists predominantly in only two of the nine possible conformations. 2H NMR spectra of polycrystalline D,L [2H10]leucine obtained from -45 degrees C to +100 degrees C showed that interconversion of the two conformations did not take place on the 2H NMR timescale in this temperature range. In contrast, experimental lineshapes observed for [2H10]leucine-labeled collagen fibrils from -85 degrees C to +30 degrees C were simulated by using a model in which the side chain hops at various rates between the two predominant conformations found by the x-ray studies. A small difference between calculated and observed linewidths above the freezing point of water can be accounted for by backbone reorientation or by the presence of a small percentage of other side-chain conformations. Thus, these results provide strong evidence that the two predominant x-ray conformations not only exist in the fibrils as the preferred orientations but interconvert at rates that are proportional to temperature over the range - 85 degrees C to +30 degrees C. These observations concur with previous NNR studies of collagen fibrils that demonstrated a mobile contact region between collagen molecules.
Location of monovalent cation binding sites in the gramicidin channel.
Authors: Urry D W , Prasad K U , Trapane T L
Published: May 2006

Six syntheses of gramicidin A have been carried out, each with 90% 13 C enrichment of a single carbonyl carbon these being the formyl, Val-1, Trp-9, Trp-11, Trp-13, and Trp-15 carbonyl carbons. Each gramicidin A was incorporated as the channel state into phospholipid structures, and the chemical shift of the carbonyl carbon resonance was monitored by 13C NMR as a function of ion concentration. Plots of Na+- and Tl+-induced chemical shifts as a function of carbonyl location in the channel indicate two symmetrically related binding sites centered at the tryptophan carbonyls and separated by 23 A. The absence of ion-induced chemical shifts for the formyl and Val-1 carbonyl carbon resonances indicates that there is no binding site midway through the channel but rather a central free-energy barrier for ion transit through the channel. Ion induced chemical shifts of the tryptophan carbonyl carbon resonances at 100 mM Na+ verify that the tight binding constant (Kbt congruent to 70 M-1), observed with 23Na NMR, results from binding within the channel. This observation and the lateral, triangular distribution of the coordinating Trp-9, -11, and -13 carbonyls combine to provide an experimental demonstration that the carbonyls of the walls of the channel directly coordinate the ion, successfully competing with the polar solvent. With the binding sites verified and localized, it is possible to conclude that the transport mechanism for Na+ is well represented by the case of the two-site model [D. W. Urry, Venkatachalam, C. M., Spisni, A., Läuger, P. & Khaled, M. A. (1980) Proc. Natl. Acad. Sci. USA 77, 2028--2032].
Picosecond photochemistry of a cofacial diporphyrin containing iron(III) and zinc(II): Mimicking electron transfer between cytochrome
c
and the primary electron donor in reaction centers of photosynthetic bacteria
Authors: Fujita I. , Netzel T. L. , Chang C. K. , Wang C.-B.
Published: May 2006

Comparison of picosecond kinetic and spectroscopic data for Zn octaethylporphine and Fe(III)Cl octaethylporphine with that for Zn—Fe(III)Cl, a cofacial diporphyrin composed of a Zn porphyrin covalently bound to an Fe(III)Cl porphyrin with two chains of five atoms each, supports the assignment of a light-driven electron transfer (
k
> 10
11
s
-1
) within Zn—Fe(III)Cl to form [Zn
+
·—Fe(II)]Cl. The kinetics (
k
≈ 10
10
s
-1
) and thermodynamics of the reverse electron transfer are compared to those of a similar electron transfer in bacterial photosynthesis, the reduction of an oxidized bacteriochlorophyll dimer, (BChl)
2
+
·, by Fe(II) cytochrome
c
.
Early auxin-regulated polyadenylylated mRNA sequences in pea stem tissue
Authors: Theologis Athanasios , Ray Peter M.
Published: May 2006

Polyadenylylated mRNA from etiolated pea stem segments treated with or without 20 μM indoleacetic acid (IAA) for various periods of time was assayed by translating it in a wheat germ extract containing [
35
S
]methionine and separating the translation products by two-dimensional gel electrophoresis. Within 2 hr IAA causes at least five mRNA sequences to increase in translational activity, relative to initial levels and to simultaneous controls; three of these rise significantly within 20 min after exposure of tissue to IAA but are apparently not elevated at 10 min, whereas the others begin to increase at successive times later than 30 min, and still others begin to change only later than 2 hr. These observations indicate an early, highly selective IAA regulation of mRNA amounts or activities, becoming progressively more extensive with time. The earliest detected enhancement seems close to the primary action of IAA but appears not to be rapid enough to be responsible for auxin induction of cell enlargement.