bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selective in vitro transcription of chloroplast genes
Authors: Gruissem Wilhelm , Narita Jonathan O. , Greenberg Bruce M. , Prescott David M. , Hallick Richard B. 
Published: December 2004

AbstractTranscription of Euglena gracilis chloroplast genes has been investigated by using in vitro transcription systems. A DNA‐dependent RNA polymerase responsible for the transcription of rRNA genes has been isolated as a nucleoprotein complex (transcriptionally active chromosome). The RNA polymerase remains tightly bound to the chloroplast DNA template and does not initiate transcription with cloned chloroplast genes. A transcriptionally active extract has been prepared from intact Euglena chloroplasts. The soluble RNA polymerase in this extract recognizes cloned chloroplast tRNA genes and tRNA‐sized products have been detected after transcription. The tRNA‐sized molecules specifically hybridize to the tRNA genes in the plasmid DNA. At least five tRNA‐sized products have been identified from transcription of a trnY1‐trnH1‐trnM1‐trnE1‐trnW1‐trnG1 cluster. Evidence is also presented that processing enzymes in the chloroplast‐extract can recognize a polycistronic tRNAVal‐tRNAAsn‐tRNAArg precursor and process it into tRNA‐sized molecules. Truncated templates have been used to demonstrate that the chloroplast tRNA genes are actively transcribed. From a comparison of 5′ flanking sequences in chloroplast tRNA genes, a consensus sequence which might function as a promoter, has been identified. The properties of the RNA polymerase involved in the transcription of chloroplast rRNA genes and tRNA genes have been investigated and compared.
Pyrazolopyrimidine metabolism in Leishmania and Trypanosomes: Significant differences between host and parasite
Authors: Marr J. Joseph 
Published: December 2004

AbstractThe pathogenic hemoflagellates of the genera Leishmania and Trypanosoma are major causes of human disease in the tropical and subtropical areas of the world. In general, the agents used to treat diseases caused by these organisms are toxic and not suitable for administration to the millions of people infected. Investigations over the past several years have shown that there are several major differences between man and these protozoans with respect to purine metabolism. The differences appear to offer promise for the development of effective chemotherapeutic compounds. These organisms do not synthesize purines de novo, as does man. They are able to concentrate pyrazolopyrimidines within the cell and metabolize them as purines through the salvage pathways, ultimately incorporating them into nucleic acids. This does not occur in mammals. The pyrazolopyrimidine base allopurinol, which has served as a prototype, is activated by a phosphoribosyltransferase to the ribonucleotide. The ribonucleotide is aminated to the 4‐amino‐pyrazolopyrimidine ribonucleotide and subsequently phosphorylated to the triphosphate form and incorporated into RNA. The pyrazolopyrimidine ribonucleosides formycin B and allopurinol ribonucleoside are activated through a nucleoside phosphotransferase. The resulting ribonucleotide is aminated and incorporated into RNA as described above. These metabolic peculiarities occur not only in the forms of these parasites which are found in the insect vectors but also in the intracellular forms which are pathogenic in man. The differences in the enzymology and metabolism of purines which exist in the genera Leishmania and Trypanosoma offer excellent opportunities for chemotherapeutic exploitation.
Expression and regulation of protein K, an Escherichia coli K1 porin, in Escherichia coli K‐12
Authors: Sutcliffe Joyce A. , Foulds John D. 
Published: December 2004

AbstractUsing a modified lambda phage as a vector and a procedure developed in Dr. C. Schnaitman's laboratory, we have cloned the structural gene for protein K from an Escherichia coli KI strain to an E coli K‐I2 strain. The cloned inserts consist of two HindIII fragments, 4 kb and 6.5 kb in size. The protein produced by the insert is nearly identical to “authentic” protein K when chymotryptic peptides of 125I‐labeled proteins are compared. Protein K was found to respond to changes in the osmolarity of the medium, being favored in trypticase soy broth (high osmolarity). This fluctuation was not dependent on a functional ompR gene. However, protein K was not expressed in strains carrying the envZ473 mutation. Thus, protein K appears to be within a class of exported proteins whose expression is regulated by the envZ gene independent of the ompR gene.
Alterations in the transport and processing of Rous sarcoma virus envelope glycoproteins mutated in the signal and anchor regions
Authors: Wills John W. , Hardwick J. Marie , Shaw Karen , Hunter Eric 
Published: December 2004

AbstractThe env gene of Rous sarcoma virus codes for two glycoproteins which are located on the surface of infectious virions. Subcloning of these coding sequences in the place of the late region of SV40 DNA has allowed the expression of a normally glycosylated, functionally active glycoprotein complex on the surface of monkey cells. Through the use of site‐directed mutagenesis, the role of specific amino acids in the signal peptide, signal peptidase cleavage site, and membrane anchor region have been investigated. Amino‐terminal mutations have shown that deletion of the signal peptidase cleavage site along with one or two amino acids of the hydrophobic signal peptide results in the synthesis of an unglycosylated. uncleaved, and presumably cytoplasmically located precursor. Nevertheless, changing the signal peptidase cleavage site from ala/asp to ala/asn does not block the translocation of the glycoprotein across the membrane or the action of the peptidase. At the other end of the molecule, carboxy‐terminal mutations have shown that the deletion of the hydrophobic membrane anchor region is not sufficient for the secretion of the truncated glycoprotein. Interpretations of these results based on recent models for protein transport and secretion are discussed.
Pigment‐protein complexes of purple photosynthetic bacteria: An overview
Authors: Thornber J. Philip , Cogdell Richard J. , Pierson Beverly K. , Seftor Richard E. B. 
Published: December 2004

AbstractA minireview of antenna and reaction center pigmenl‐protein complexes of purple bacteria is presented. Advances in our knowledge of their structure and composition during the past 3 yr are emphasized and some new thoughts are introduced.
Genetic control of resistance to murine malaria
Authors: Stevenson Mary , Lemieux Suzanne , Skamene Emil 
Published: December 2004

AbstractStrain variation in the level of resistance to malaria was investigated in inbred mice after infection with Plasmodium chabaudi. Following intraperitoneal infection with the typing dose of parasitized erythrocytes, mice of 11 inbred strains could be separated using survival time as the criterium into resistant and susceptible groups. Genetic analysis of F1 hybrid and backcross progeny derived from one of the most resistant (B10.A) and from the most susceptible (A/J) strains as parents suggested that host resistance in this strain combination was genetically controlled by a dominant, non‐H‐2‐linked, autosomal gene or closely linked genes. Analysis of the mechanisms of resistance to P chabaudi showed (1) phenotypic expression of the resistance gene was apparent within 6 days of infection as a significant difference between resistant and susceptible mice in the level of parasitemia; (2) the level of host NK cell activity was not related to the level of host resistance to malaria; (3) compared with susceptible A/J mice, resistant B1O.A hosts had an augmented erythropoietic response during the course of malaria as well as during phenylhydrazine‐induced anemia and (4) treatment with BCG or P acnes resulted in an equal degree of protection, measured by parasitemia and survival, in both resistant and susceptible mice.
The effect of phenformin and other adenosine triphosphate (ATP)‐lowering agents on insulin binding to IM‐9 human cultured lymphocytes
Authors: Vigneri Riccardo , Maddux Betty , Goldfine Ira D. 
Published: December 2004

AbstractIn the present study, we investigated the mechanism by which the antidiabetic drug phenformin increases insulin binding to its receptors in IM‐9 human cultured lymphocytes. After a 24‐hr preincubation, phenformin induced a twofold increase in specific 125I‐insulin binding, and removal of phenformin was followed 6 hr later by a return in binding to control levels. This effect of phenformin on insulin binding was not a consequence of either inhibition of cell growth, changes in cellular cyclic adenosine monophosphate (AMP) levels, or changes in guanosine triphosphate (GTP) content. Since phenformin is known to inhibit various aspects of cellular energy metabolism, the relationship between 125I‐insulin binding and energy metabolism in IM‐9 cells was investigated. The phenformin‐induced increase in insulin binding to IM‐9 cells was related to a time‐ and dose‐dependent decrease in ATP levels. Other agents that lowered ATP levels, including antimycin, dinitrophenol, and 2‐deoxyglucose, also raised insulin binding. These studies indicated, therefore, that phenformin enhances insulin binding to receptors on IM‐9 cells and that this effect on insulin receptors may be related to alterations in metabolic functions that are reflected by a lowering of ATP levels.
Role of membrane potential in protein folding and domain formation during secretion in escherichia coli
Authors: Copeland Bruce R. , Landick Robert , Nazos Penelope M. , Oxender Dale L. 
Published: December 2004

AbstractThe synthesis and processing of the periplasmic components of the leucine transport system of E coli have been studied to determine the role played by transmembrane potential in protein secretion. Both the leucine‐isoleucine‐valine binding protein and the leucine‐specific binding protein are synthesized as precursors with 23 amino acid N‐terminal leader sequences. The processing of these precursors is sensitive to the transmembrane potential. Since the amino acid sequence and the crystal structure have been determined for the leucine‐isoleucine‐valine binding protein, it and the closely related leucine‐specific binding protein represent convenient models in which to examine the mechanism of protein secretion in E coli. A model for secretion has been proposed, suggesting a role for transmembrane potential. In this model, the N‐terminal amino acid sequence of the precursor is assumed to form a hairpin of two helices. The membrane potential may orient this structure to make it accessible to processing. In addition, the model suggests that a negatively charged, folded domain of the secretory protein may electrophorese toward the trans‐positive side of the membrane, thus providing an additional role for the transmembrane potential.
The role of membrane lectins in dictyostelium discoideum aggregation as ascertained by specific univalent antibodies against discoidin I
Authors: Cano Amparo , Pestaña Angel 
Published: December 2004

AbstractAntibodies against pure discoidin I have been used as a tool to ascertain the role of this lectin in aggregation of Dictyostelium discoideum. Discoidin I is widely expressed over the cell surface of aggregation‐competent AX‐2 cells, as ascertained by indirect immunofluorescence with specific (antidiscoidin I) antibodies. Univalent antidiscoidin I antibodies (Fab fragments) inhibit the aggregation‐specific intercellular adhesion of D discoideum AX‐2 cells in an in vitro assay. This inhibition depends on antibody concentration and cell density; a 50% inhibition of cell aggregation was obtained at antidiscoidin I Fab concentration of 4.5 mg/ml and 1 × 106 cells/ml. Aggregation and morphogenesis on solid support is also effectively inhibited when AX‐2 cells are starved in the presence of antidiscoidin I Fab fragments. The inhibition of morphogenesis is also dose dependent and more effective than in the in vitro assay. No inhibition of aggregation either in the in vitro assay or on morphogenesis on solid support was observed with preimmune Fab fragments at any of the concentrations tested (up to 9.6 mg/ml).
Role of membrane thermotropic properties on hypotonic hemolysis and hypertonic cryohemolysis of human red blood cells
Authors: Minetti Maurizio , Ceccarini Marina , Maria Anna , Di Stasi M. 
Published: December 2004

AbstractThe hypothesis of a correlation between the effects of temperature on red blood cells hypotonic hemolysis and hypertonic cryoheniolysis and two thermotropic structural transitions evidenced by EPR studies has been tested. Hypertonic cryoheniolysis of red blood cells shows critical temperatures at 7°C and 19°C. In hypotonic solution, the osmotic resistance increases near 10°C and levels off above 20°C. EPR studies of red blood cell membrane of a 16‐dinyloxyl stearic acid spin label show, in the 0–50°C range, the presence of three thermotropic transitions at 8, 20, and 40°C. Treatments of red blood cells with acidic or alkaline pH, glutaraldehyde, and chlorpromazine abolish hypertonic cryoheniolysis and reduce the effect of temperature on hypotonic hemolysis. 16‐Dinyloxyl stearic acid spectra of red blood cells treated with glutaraldehyde and chlorpromazine show the disappearance of the 8°C transition. Both the 8°C and the 20°C transitions were abolished by acidic pH treatment. The correlation between the temperature dependence of red blood cell lysis and thermotropic breaks might be indicative of the presence of structural transitions producing areas of mismatching between differently ordered membrane components where the osmotic resistance is decreased.
Calmodulin, a calmodulin acceptor protein, and calcimedins: Unique antibody localizations in hamster sperm
Authors: Moore Pamela B. , Dedman John R. 
Published: December 2004

AbstractA calmodulin acceptor protein has been identified in isolated hamster caudal sperm by immunofluoresence and Western transfer techniques. The protein shows a localization in sperm heads identical to calmodulin. Fluorescence of both calmodulin and the acceptor protein are lost by treatment with MgCl2, conditions which release the acrosome. These results are consistent with the proposed function of calmodulin in a sperm function.
Specific anion effects on ATPase activity, calmodulin sensitivity, and solubilization of dynein ATPases
Authors: Blum Jacob J. , Hayes Alvernon 
Published: December 2004

AbstractThe basal ATPase activity of 30S dynein, whether obtained by extraction of ciliary axonemes with a high (0.5 M NaCl) or low (1 mM Tris‐0.1 mM EDTA) ionic strength buffer is increased by NaCl, NaNO3, and Na acetate, with NaNO3 causing the largest increase. The calmodulin‐activated ATPase activity of 30S dynein is also increased by addition of NaCl, NaNO3, or Na acetate, but the effects are less pronounced than on basal activity, so that the calmodulin activation ratio (CAR) decreases to 1.0 as salt concentration increases to 0.2 M. These salts also reduce the CAR of 14S dynein ATPase to 1.0 but by strongly inhibiting the calmodulin‐activated ATPase activity and only slightly inhibiting the basal activity. Sodium fluoride differs both quantitatively and qualitatively from the other three salts studied. It inhibits the ATPase activity of both 14S and 30S dyneins at concentrations below 5 mM and, by a stronger inhibition of the calmodulin‐activated ATPase activities, reduces the CAR to 1.0. Na acetate does not inhibit axonemal ATPase, nor does it interfere with the drop in turbidity caused by ATP and extracts very little protein from the axonemes. NaCl and, especially, NaNO3, cause a slow decrease in A350 of an axonemal suspension and an inhibition of the turbidity response to ATP. NaF, at concentrations comparable to those that inhibit the ATPase activities of the solubilized dyneins, also inhibits axonemal ATPase activity and the turbidity response. Pretreatment of demembranated axonemes with a buffer containing 0.25 M sodium acetate for 5 min followed by extraction for 5 min with a buffer containing 0.5 M NaCl and resolution of the extracted dynein on a sucrose density gradient generally yields a 30S dynein that is activated by calmodulin in a heterogeneous manner, ie, the “light” 30S dynein ATPase fractions are more activated than the “heavy” 30S dynein fractions. These results demonstrate specific anion effects on the basal and calmodulin‐activated dynein ATPase activities, on the extractability of proteins from the axoneme, and on the turbidity response of demembranated axonemes to ATP. They also provide a method that frequently yields 30S dynein fractions with ATPase activities that are activated over twofold by added calmodulin.
Solubilization, reconstitution, and attempted affinity chromatography of the sugar transporter of the erythrocyte membrane
Authors: Weber Jakob , Warden Dean Allan , Semenza Giorgio , Diedrich Donald F. 
Published: December 2004

AbstractReconstitution of the sugar transport system of human erythrocytes into artificial liposomes was achieved by freezing, thawing, and sonicating preformed phospholipid vesicles in the presence of intact ghosts, protein‐depleted ghosts, or detergent‐treated ghosts. D‐glucose equilibrium exchange activities and affinity constants in the range of the reported erythrocyte values were reached in the best experiments. Whereas the extraction of peripheral membrane proteins did not depress the transport function crucially after reconstituting these protein‐depicted ghosts, the selective Solubilization of integral membrane proteins by a variety of nonionic detergents resulted in an uncontrollable, continuously increasing inactivation of the carrier. However, Emulphogene BC‐720 extracts could be prepared in which the glucose transporter retained activity for days at 4°C. These extracts were applied to affinity chromatography matrices of phloretin‐Agarose, prepared by coupling phloretinyl‐3′‐benzylamine (PBA) to CH‐Sepharose 4B and to Affigel 202. Although the solubilized sugar transporter appeared to be selectively adsorbed to both PBA matrices, it could not be eluted by specific counter ligands or gentle eluants in a biologically active form. However, chaotropic agents could be used to elute intrinsic proteins, including bands 3 and 4. 5, from the Affigel affinity medium.
Membrane‐cytoskeleton interactions and the regulation of chemotactic peptide‐induced activation of human granulocytes: The effects of dihydrocytochalasin B
Authors: Jesaitis Algirdas J. , Tolley James O. , Painter Richard G. , Sklar Larry A. , Cochrane Charles G. 
Published: December 2004

AbstractWhen N‐formyl chemotaclic peptides bind to granulocyte receptors at 37°C they rapidly form a high‐affinity ligand‐receptor complex whose coisolation with cyto‐skeletal residues of Triton X‐100–extracted cells is under cellular control [Jesaitis et al: J Cell Biol 98:1378, 1984]. Experiments were performed to investigate the significance of this coisolation. When the granulocytes were preincubated with dihydrocytochalasin B (dhCB) for 10 min at 37°C and then stimulated with 50 nM N‐formyl‐Met‐Leu‐[3H]Phe, the rate of uptake of the radioligand by the cells was inhibited. Colocalization of the retained peptide with the Triton X‐100 fraction of these cells was also reduced relative to this fraction of the untreated cells. This inhibition was apparent before the onset of FMLP endocytosis. The inhibition was 50% effective at 0.25 μg dhCB/ml. Maximal inhibition (80–90%) occurred at doses of dhCB > 1 μg/ml. The 90% retention of two plasma membrane markers by the cytoskeleton was marginally affected. These results support the hypothesis that coisolation of the high‐affinity receptor‐peptide complexes with granulocyte cytoskeletons occurs because of specific association of the complexes with the cytoskeleton at the cell surface. In addition, since these events precede internalization, they suggest that formation of the association between the ligand‐receptor complex and cytoskeleton may be necessary for ligand‐receptor endocytosis. Experiments were also performed to evaluate other functional consequences of cytoskeletal disruption on chemotactic peptide‐stimulated functions. f‐Met‐Leu‐Phe stimulation of O production was potentiated due to prolongation of and increase in the rate of O production. This potentiation has the same dose dependency as the inhibition of receptor modulation. The possible relationship of these various functions is discussed.
Activators of protein kinase C and 5‐azacytidine induce IL‐2 receptor expression on human T lymphocytes
Authors: Depper Joel M. , Leonard Warren J. , Drogula Cynthia L. , Krönke Martin , Waldmann Thomas A. , Greene Warner C. 
Published: December 2004

AbstractResting human T lymphocytes do not express receptors for interleukin‐2, but expression is rapidly induced by exposure to PHA. After maximal expression 2–3 days after stimulation, a progressive decline in receptor number is observed. Receptor expression can be augmented by reexposure to PHA. In this study we show that activators of protein kinase C including phorbol diester, phospholipase C, and the diacylglycerol congener diC8 also increase IL‐2 receptor expression. Moreover, 5‐azacytidinc, which inhibits cytosine methyltransferase, and hydroxy‐urea, which inhibits ribonucleotide reductase, also increased receptor number. These studies demonstrate that IL‐2 receptor expression can be altered in vitro, and that IL‐2 receptor number, in combination with IL‐2 secretion, may contribute to the regulation of IL‐2–dependent immune responses.
Transferrin receptor induction is required for human B‐lymphocyte activation but not for immunoglobulin secretion
Authors: Neckers L. M. , Yenokida G. , Trepel J. B. , Lipford E. , James S. 
Published: December 2004

AbstractTransferrin receptors are expressed on proliferating cells and are required for their growth. Transferrin receptors can be detected after, but not before, mitogenic stimulation of normal peripheral blood T and B cells. In the experiments reported here we have examined the regulation of transferrin receptor expression on activated human B cells and whether or not these receptors are necessary for activation to occur. Activation was assessed by studying both proliferation and immunoglobulin secretion. We have determined that transferrin receptor expression on B cells is regulated by a factor contained in supernatants of mitogenstimulated T cells (probably B‐cell growth factor). This expression is required for proliferation to occur, since antibody to transferrin receptor (42/6) blocks B‐cell proliferation. Induction of immunoglobulin secretion, however, although dependent on PHA‐treated T‐cell supernatant, is not dependent on transferrin receptor expression and can occur in mitogen‐stimulated cells whose proliferation has been blocked by antitransferrin receptor antibody. In addition, we have demonstrated that IgM messenger RNA induction following mitogen stimulation is unaffected by antitransferrin receptor antibody. These findings support a model for B‐cell activation in which mitogen (or antigen) delivers two concurrent but distinct signals to B cells: one, dependent on B‐cell growth factor and transferrin receptor expression, for proliferation, and a second, dependent on T cell‐derived factors and not requiring transferrin receptors, which leads to immunoglobulin secretion.
Interaction of epidermal growth factor with initiators and complete carcinogens in the C3H10T1/2 cell culture System
Authors: Herschman Harvey R. , Brankow David 
Published: December 2004

AbstractUnlike 12‐O‐tetradecanoylphorbol‐l3‐acctate, epidermal growth factor (EGF) could not promote the appearance of type III foci from initiated C3H10T1/2 cells. At appropriate concentrations, EGF induced the formation of type II colonies in the absence of any initiator. At higher concentrations, EGF suppressed the induction of both type II and type III colonies elicited by methylcholanthrene.