Penn Dental Medicine
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PublicationIn Vitro Replication of Cyanobacterial Plasmids from Synechocystis PCC 6803(1994-09-01) Yang, Xiaoyu; Daniell, Henry; McFadden, BruceLittle knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column. DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation. DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain. The in vitro replication products consist mostly of the supercoiled form of the plasmids. Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide. These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis. Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication. PublicationEvaluating Two Methods for Fingerprinting Genomes of Actinobacillus Actinomycetemcomitans(1993-12-01) Slots, J.; Liu, Y. B.; DiRienzo, J. M.; Chen, C.The arbitrary primer polymerase chain reaction (AP-PCR) and Southern blot restriction fragment length polymorphism (RFLP) were used to genotype the periodontal pathogen A. actinomycetemcomitans. Total genomic DNA from 73 strains was extracted by conventional methods. Three random-sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 1% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI, separated on a 0.8% agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterized 5.2 kilobases (kb) DNA fragment cloned from A. actinomycetemcomitans strain Y4. The probe was labeled with digoxigenin, and hybridized fragments were detected with anti-digoxigenin antibody. AP-PCR produced 4–10 DNA bands in the 0.5–5 kb regions and distinguished 9, 13 or 17 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 12 hybridization patterns consisting of 1 or 2 DNA fragments (2–23 kb). The addition of the Southern blot analysis to the AP-PCR analysis gave rise to a total of 30 DNA profiles among the 73 A. actinomycetemcomitans study strains. The results indicate that both AP-PCR and Southern blot analysis are useful in clonal analysis of A. actinomycetemcomitans. PublicationChemoattractant Receptor-Induced Phosphorylation of L-Selectin(1997-05-23) Haribabu, Bodduluri; Steeber, Douglas A.; Ali, Hydar; Richardson, Ricardo M.; Snyderman, Ralph; Tedder, Thomas F.The selectin adhesion molecules and chemoattractant receptors synergistically regulate leukocyte migration into lymphoid tissues and sites of inflammation, but little is known about how these families of receptors modulate each other's function. In this study, L-selectin was found to be phosphorylated in lymphoblastoid cell lines, and phosphorylation was enhanced by phorbol ester (phorbol 12-myristate 13-acetate (PMA)) treatment. Interactions between L-selectin and chemoattractant receptors were therefore examined using transfected rat basophilic leukemia cell lines (RBL-2H3) that expressed human L-selectin along with human leukocyte chemoattractant receptors. L-selectin was rapidly phosphorylated in cells treated with chemoattractants, thrombin, IgE receptor agonists, or PMA. Pertussis toxin or the protein kinase C inhibitor, staurosporine, completely blocked chemoattractant receptor-induced phosphorylation of L-selectin. PMA-induced phosphorylation was on serine residues within the cytoplasmic tail of L- selectin that have been well conserved during recent evolution. Although L- selectin phosphorylation was not essential for basal levels of adhesion through L-selectin in transformed cell lines, the rapid increase in ligand binding activity of L-selectin that occurs following leukocyte activation was blocked by staurosporine. These results demonstrate that L-selectin can be phosphorylated following engagement of chemoattractant receptors and suggest that this may be a physiologically relevant mechanism for the synergistic regulation of these receptors during leukocyte migration. PublicationRole of Phospholipase Cβ3 Phosphorylation in the Desensitization of Cellular Responses to Platelet-Activating Factor(1997-05-02) Ali, Hydar; Fisher, Ian; Haribabu, Bodduluri; Richardson, Ricardo M.; Snyderman, RalphPlatelet-activating factor (PAF) stimulates a diverse array of cellular responses through receptors coupled to G proteins that activate phospholipase C (PLC). Truncation of the cytoplasmic tail of the receptor to remove phosphorylation sites (mutant PAF receptor, mPAFR) results in enhancement of PAF-stimulated responses. Here we demonstrate that PAF or phorbol 12- myristate 13-acetate (PMA) pretreatment inhibited wild type PAFR-induced PLC- mediated responses by ~90%, whereas these responses to the phosphorylation- deficient mPAFR were inhibited by ~50%, despite normal G protein coupling, suggesting a distal inhibitory locus. PAF and PMA, as well as a membrane permeable cyclic AMP analog, stimulated phosphorylation of PLCβ3. A protein kinase C (PKC) inhibitor blocked phosphorylation of PLCβ3 stimulated by PAF and PMA but not by cAMP. Activation of protein kinase A (PKA) by cAMP did not result in inhibition of Ca2+ mobilization stimulated by PAF. In contrast, cAMP did inhibit the response to formylpeptide chemoattractant receptor. These data suggest that homologous desensitization of PAF-mediated responses is regulated via phosphorylation at two levels in the signaling pathway, one at the receptor and the other at PLCβ3 mediated by PKC but not by PKA. Phosphorylation of PLCβ3 by PKA could explain the inhibition of formylpeptide chemoattractant receptor signaling by cAMP. As PAF and formylpeptide chemoattractant receptors activate PLC via different G proteins, phosphorylation of PLCβ3 by PKC and PKA could provide distinct regulatory control for classes of G protein-coupled receptors. PublicationThrombin Primes Responsiveness of Selective Chemoattractant Receptors at a Site Distal to G Protein Activation(1996-02-09) Ali, Hydar; Tomhave, Eric D.; Richardson, Ricardo M.; Haribabu, BodduluriTo define the molecular basis of human chemoattractant receptor regulation, rat basophilic leukemia RBL-2H3 cells, which are thrombin- responsive, were transfected to stably express epitope-tagged receptors for C5a, interleukin-8 (IL-8), formylpeptides (e.g. N-formylmethionyl-leucyl- phenylalanine (fMLP)), and platelet-activating factor (PAF). Here we demonstrate that both thrombin and a synthetic peptide ligand for the thrombin receptor (sequence SFLLRN) caused phosphorylation and heterologous desensitization of the receptors for C5a, IL-8, and PAF but not that for formylpeptides as measured by agonist-stimulated [35S]guanosine 5'-3-O- (thio)triphosphate binding to membranes. Consistent with the PAF receptor phosphorylation, both thrombin and thrombin receptor peptide inhibited phosphoinositide hydrolysis, Ca2+ mobilization, and degranulation stimulated by PAF. Unexpectedly, despite heterologous desensitization at the level of receptor/G protein activation, there was enhancement ('priming') by thrombin of subsequent activities stimulated by C5a and IL-8 as well as fMLP. The priming effect of thrombin was blocked by its inhibitor, hirudin. However, two other activators of the thrombin receptor, the peptide SFLLRN and trypsin, stimulated Ca2+ mobilization in RBL-2H3 cells but did not cause priming. In addition, SFLLRN and the thrombin receptor antagonist peptide FLLRN both inhibited thrombin-induced Ca2+ mobilization but not priming. Furthermore, the proteolytically active γ-thrombin, which does not stimulate the tethered ligand thrombin receptor and caused little or no Ca2+ mobilization in RBL-2H3 cells, effectively primed the response to fMLP. These data demonstrate that heterologous receptor phosphorylation and attenuation of G protein activation are not, by themselves, sufficient for the inhibition of biological responses mediated by C5a and IL-8. Moreover, thrombin appears to utilize mechanism(s) independent of its tethered ligand receptor to selectively prime phospholipase C-mediated biological responses of the C5a, IL-8, and formylpeptide receptors but not PAF. Because C5a, IL- 8, and formylpeptide activate phospholipase Cβ2, whereas PAF stimulates a different phospholipase C, the striking selectivity of thrombin's priming may be mediated via its ability to enhance receptor-mediated activation of phospholipase Cβ2. PublicationNeonatal Exposure to Thymotropic Gross Murine Leukemia Virus Induces Virus-Specific Immunologic Nonresponsiveness(1990-12-01) Korostoff, Johnathan M; Nakada, Marian T; Faas, Susan J; Blank, Kenneth J; Gaulton, Glen NNeonatal exposure to Gross murine leukemia virus results in a profound inhibition of the virus-specific T and B cell responses of adult animals. Animals exposed to virus as neonates exhibit a marked depression in virus-specific T cell function as measured by the virtual absence of in vivo delayed type hypersensitivity responses and in vitro proliferative responses to virally infected stimulator cells. Further, serum obtained from neonatally treated mice failed to either immunoprecipitate viral proteins or neutralize virus in an in vitro plaque assay, suggesting the concurrent induction of a state of B cell hyporesponsiveness. The specificity of this effect at the levels of both T and B cells was demonstrated by the ability of neonatally treated mice to respond normally after adult challenge with either irrelevant reovirus or influenza virus. The replication of Gross virus within both stromal and lymphocytic compartments of the neonatal thymus suggests that thymic education plays a key role in the induction of immunologic nonresponsiveness to viruses. PublicationOverexpression of the Bacillus Thuringiensis (Bt) Cry2Aa2 Protein in Chloroplasts Confers Resistance to Plants Against Susceptible and Bt-resistant Insects(1999-03-02) Kota, Madhuri; Daniell, Henry; Varma, Sam; Garczynski, Stephen F.; Gould, Fred; Moar, William J.Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000–10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field. PublicationDifferential Cross-Regulation of the Human Chemokine Receptors CXCR1 and CXCR2. Evidence for Time-Dependent Signal Generation(1998-09-11) Richardson, Ricardo M.; Pridgen, Bryan C.; Haribabu, Bodduluri; Ali, Hydar; Snyderman, RalphNeutrophils and transfected RBL-2H3 cells were used to investigate the mechanism of cross-regulation of the human interleukin-8 (IL-8) receptors CXCR1 and CXCR2 by chemoattractants. In neutrophils, Ca2+ mobilization by the CXCR2-specific chemokine, growth-related oncogene α (Groα), was desensitized by prior exposure to the chemoattractants N-formylated peptides (fMLP) or a complement cleavage product (C5a). In contrast, growth-related oncogene α did not desensitize the latter receptors. To investigate this phenomenon, CXCR2 was stably expressed in RBL-2H3 cells and mediated phosphoinositide hydrolysis, Ca2+ mobilization, chemotaxis, and secretion. In cells co-expressing CXCR2 and receptors for either C5a (C5aR) or fMLP (FR), CXCR2 was cross-phosphorylated and cross-desensitized by C5a and fMLP. However, neither C5aR nor FR was cross-phosphorylated or cross-desensitized by CXCR2 activation, although CXCR1 did mediate this process. Receptor internalization induced by IL-8 was more rapid and occurred at lower doses with CXCR2 than CXCR1, although both receptors mediated equipotent chemotaxis and exocytosis in RBL. Truncation of the cytoplasmic tail of CXCR2 (331T) prolonged its signaling relative to CXCR2, increased its resistance to internalization, and induced phospholipase D activation. 331T was resistant to homologous phosphorylation and cross-phosphorylation but not cross- desensitization of its Ca2+ mobilization by fMLP or C5a, indicating an inhibitory site distal to receptor/G protein coupling. In contrast to CXCR2, stimulation of 331T cross-desensitized Ca2+ mobilization by both FR and C5aR. CXCR2 and the mutant 331T induced phospholipase C β3 phosphorylation to an extent equivalent to that of CXCR1. Taken together, these results suggest that CXCR1 and CXCR2 bind IL-8 to produce a group of equipotent responses, but their ability to generate other signals, including receptor internalization, cross-desensitization, and phospholipase D activation, are very different. The latter phenomena apparently require prolonged receptor activation, which in the case of CXCR2 is precluded by rapid receptor phosphorylation and internalization. Thus, receptors coupling to identical G proteins may trigger different cellular responses dependent on the length of their signaling time, which can be regulated by receptor phosphorylation. PublicationInsertional Inactivation of Binding Determinants of Streptococcus Crista CC5A Using Tn916(1995-08-01) Correia, F. F.; DiRienzo, J. M.; Lamont, R. J.; Anderman, C.Intermicrobial binding plays an important role in the ecology of the oral cavity because itrepresents one mechanism by which specific bacteria colonize dental plaque. The formation of“corncobs”, a morphologically distinct microbial unit composed of Streptococcus crista andFusobacterium nucleatum, is a highly specific binding interaction that depends on the presence ofpolar tufts of fimbriae on the streptococci. We have used a genetic approach to examine the role ofstreptococcal cell surface components involved in the binding of S. crista to F. nucleatum. Suchbinding may be an important component of corncob formation. A method for the genetictransformation of S. crista was used to transfer the broad host range transposon, Tn916, into thebacteria. Cells were grown to early log phase in brain heart infusion broth containing 10% fetalcalf serum. The competent cells were mixed with purified DNA from pDL916, a plasmidconstruct consisting of Tn916 and the streptococcal/Escherichia coli shuttle vector pDL278. Over300 transformants were screened for a reduction in binding to F. nucleatum. Five of thetransformants showed a change in binding ranging from 59% to 29% of the positive controlvalues. Southern blots revealed that the binding-deficient transformants contained the Tn916element integrated into one of 4 different sites in the chromosome. The transposon, integrated into4 different sites, appeared to be stable in the absence of selective pressure. Based on thesefindings, it appears that some strains of S. crista are naturally competent and that insertionalinactivation methods can be used to facilitate the study of binding receptors in this group of oralstreptococci. PublicationCholesterol Dynamics in Membranes(1990-03-01) Yeagle, P. L.; Albert, A. D.; Boesze-Battaglia, Kathleen; Young, J.; Frye, J.Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.