Biomedical subjects
Publications and source records attributed to C Zwieb.
The SRPDB (signal recognition particle database) provides aligned SRP RNA and protein sequences, annotated and phylogenetically ordered. This release includes 82 SRP RNAs (including 22 bacterial and 9 archaeal homologs) and a total of 20 protein sequences representing SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72. The offerings also include representative RNA secondary structure diagrams.
The epithelial lining of the airways is subject to injury through several processes, including infections, bronchiolitis, and fume exposures. Because airway fibrin deposition influences the course of local injury, we examined how two inflammatory cytokines influenced fibrin formation and clearance in human tracheal epithelial cells (TEC). TEC were treated with transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha). TNF-alpha increased release of tissue factor (TF)-related procoagulant activity that, through generation of factor Xa, promotes assembly of the prothrombinase complex at the cell surface. Fibrinolytic activity was plasminogen dependent and due to both urokinase (uPA) and tissue plasminogen activator (tPA). The cells expressed plasminogen activator inhibitor 1 (PAI-1), but relatively little PAI-2. Depression of fibrinolysis by TGF-beta correlated with increased PAI-1. Conversely, TNF-alpha increased plasminogen activator (PA) activity due to increased uPA. Fibrinolytic activity was inhibited by actinomycin D and cyclohexamide, but changes in mRNAs for uPA, tPA, PAI-1, and TF by either cytokine were not appreciable. PAI-2 mRNA was not found. The data indicate that TGF-beta decreases the fibrinolytic capacity of TEC, suggesting that this cytokine promotes fibrin retention. TNF-alpha increases expression of both procoagulant and fibrinolytic activities; this differential regulation could favor both pericellular fibrin formation and dissolution.
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The SRPDB (signal recognition particle database) provides annotated SRP RNA sequences from Eucaryotes and Archaea, phylogenetically ordered and aligned with their bacterial equivalents. We also make available representative RNA secondary structure diagrams, where each base pair is proven by comparative sequence analysis. New to this release are 17 SRP RNA sequences (a total of 64 sequences) and alignments of proteins SRP19 and SRP54 with their RNA binding sites.
A group of RNA binding proteins, termed tetraloop binding proteins, includes ribosomal protein S15 and protein SRP19 of signal recognition particle. They are primary RNA binding proteins, recognize RNA tetranucleotide loops with a GNAR consensus motif, and require a helical region located adjacent to the tetraloop. Closely related RNA structures that fit these criteria appear in helix 6 of SRP RNA, in helices 22 and 23A of 16 S ribosomal RNA, and, as a pseudoknot, in the regulatory region of the rpsO gene.
The interaction of protein SRP19 with the RNA component of human signal recognition particle (SRP) was studied by site-directed mutagenesis of the SRP RNA. The effects of nucleotide changes in the tetranucleotide loop (tetraloop) of helix 6 showed that SRP19 recognizes a tetraloop in a sequence-specific manner. Adenosine 149 at the third position of the tetraloop was essential for binding. In contrast, changes of the base at the second position had no effect. Mutations that disrupt or compensate individual SRP RNA helices were generated to investigate the importance of base pairing and to identify other binding sites. Considerable base pairing was essential in helix 6. Another SRP19-binding site was located in the distal part of helix 8. The primary sequences of the tetraloop-binding protein SR19 and of bacterial ribosomal protein S15 are shown to be similar.
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Fibrin gels form within the alveolar and interstitial compartments of the injured lung, and fibroblasts invade and facilitate organization of these transitional gels. We studied the effects of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) on fibrinolytic and procoagulant activities of human lung fibroblasts (HLF) to determine their capacity to regulate pulmonary fibrin deposition. Fibrinolytic activity of cell lysates and media (n = 6 HLF cultures) were uniformly depressed by TGF-beta or TNF-alpha. In dose and time-course studies, HLF plasminogen activator inhibitor-1 (PAI-1) was increased by TGF-beta, whereas TNF-alpha induced release of PAI-1 into the media. HLF and media urokinase concentrations were depressed by TGF-beta, whereas urokinase was unchanged or increased by TNF-alpha. Tissue plasminogen activator was mainly cell associated and unchanged by TGF-beta or TNF-alpha. HLF antiplasmin activity was not detected. Plasma recalcification times of HLF media were decreased by TNF-alpha but unchanged by TGF-beta. These studies suggest that TGF-beta and TNF-alpha impair the ability of HLF to degrade fibrin by disturbing the balance of HLF plasminogen activators and PAI and that these cytokines concurrently leave unchanged or increase the capacity of HLF to initiate fibrin formation. Cytokines likely to occur in the injured lung induce abnormalities of fibrinolysis in HLF from adults; such abnormalities favor extravascular fibrin deposition, a characteristic feature of alveolitis.
The mesothelium contains both procoagulant and fibrinolytic activities. An imbalance between these activities could account for the abnormal fibrin turnover and pleural fibrin deposition that is characteristic of pleural inflammation. Procoagulant activity of human pleural mesothelial cells (HPMC) is in part due to tissue factor, and the prothrombinase complex can also assemble at the HPMC surface. HPMC express tissue plasminogen activator (tPA) but no detectable fibrinolytic activity in a fibrin plate assay. Inhibition of HPMC fibrinolytic activity is due, in part, to elaboration of plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) as well as antiplasmins. Synthesis of PAI-1 and PAI-2 is inhibited by actinomycin D and cyclohexamide. HPMC PAI-1 is increased by transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha), as is tPA release, while PAI-1 mRNA is unchanged and tPA mRNA is increased. PAI-2 release is induced by TNF-alpha and TGF-beta. Because they are a rich source of PAI-1 and PAI-2, HPMC may contribute to the high levels of these inhibitors in pleural exudates. Stimulation of HPMC by TNF-alpha or TGF-beta in vitro did not alter HPMC procoagulant activity nor the balance of elevated PAI and antiplasmins relative to PA, changes that collectively favor formation and persistence of pericellular fibrin.
Derivatives of human SRP-RNA were constructed by site-directed mutagenesis and tested for their ability to interact with protein SRP19. An RNA missing helix 6 barely interacts with SRP19, while the helix 8-deletion mutant retains much binding capability. A mutant RNA consisting just of helix 6 also binds the protein, but not as well as the unaltered molecule. SRP19 interacts to a full extent with the fourth mutant RNA composed of helices 6, 7, 8 and a portion of helix 5. It is concluded that helix 6- and not helix 8- is the major SRP19 binding site. Helices 7, 8 and portions of helix 5 contribute to the formation of a functional site. These results agree with data suggesting a proximity of helix 6 and the conserved part of SRP-RNA.
The secondary structures of the RNAs from the signal recognition particle, termed SRP-RNA, were derived buy comparative analyses of an alignment of 39 sequences. The models are minimal in that only base pairs are included for which there is comparative evidence. The structures represent refinements of earlier versions and include a new short helix.
The Galactosidase repressor (GalR) of Escherichia coli modulates the expression of the gal operon by binding to two DNA operators, OE and O1. The OE and O1 elements are 16 bp pallindromic DNA sequences, differing in four of the base pairs. OE and O1 DNA fragments, both free and complexed with repressor, were analyzed by "quantitative gel electrophoresis". By the criteria of that method, applied to the linear Ferguson plots of both DNA fragments and the linear ranges of those of the DNA-GalR complexes, it was shown that the apparent size of DNA increases upon repressor binding. Moreover, this size increase is greater for the complex with the O1 operator than for the complex with the OE operator in the case that GalR is located in the center of a 155 bp DNA fragment. This is not the case when GalR is located in a peripheral position. By contrast with their size differences, the centrally located GalR-O1 and GalR-OE complexes appear to possess indistinguishable net surface charge densities as judged from the intercepts with the mobility axis. The larger size of the complex with centrally located O1 fragment, as compared with that bearing the OE fragment, is interpreted as being due to bending of the DNA-protein complex, since an authentically bent fragment of a plasmid with bent upstream activator sequence also exhibits a larger slope of the Ferguson plot, and thus the larger size, than predicted on the basis of its DNA chain length (bp).(ABSTRACT TRUNCATED AT 250 WORDS)
To identify some of the determinants in the 19-kilodalton protein of signal recognition particle (SRP19) for binding to signal recognition particle RNA, two mutant derivatives of the SRP19 were constructed, lacking 14 and 24 C-terminal amino acids. Polypeptides were transcribed and translated in vitro and tested for their ability to bind to signal recognition particle RNA by retention of protein-RNA complexes on DEAE-Sepharose. Both mutant polypeptides form complexes with the RNA, demonstrating that the 24 C-terminal amino acids, which include a lysine-rich sequence at positions 136-144, are dispensable. A third mutant polypeptide, in which eight additional amino acids were removed by oligonucleotide-directed digestion of the mRNA, was unable to bind. The amino acids in the sequence PKLKTRTQ correspond to positions 113-120; they are suggested to be involved in interaction with signal recognition particle RNA.
The extent and location of DNA-bending induced in the Xenopus laevis transcription factor IIIA-oocyte 5S RNA gene complex was determined by the gel retardation method. The electrophoretic mobilities of TFIIIA complexed with restriction fragments of 160, 177, 282 and 300 bp that contain the sequence of the major oocyte 5S RNA gene were compared. In these fragments the 120-bp gene is positioned either in the middle or at the end. Minor differences in the mobility of the complexes indicate that the degree of DNA bending is only slight. To determine the bending angle more precisely, a bending vector system, pBend3, was used to examine the complex of TFIIIA with the internal control region (ICR) of the 5S RNA gene. A 61-bp synthetic duplex corresponding to the ICR sequence was cloned into pBend3. Duplicated circular permuted restriction sites allow several 186-bp fragments to be generated in which the position of the ICR can be varied. Gel retardation of TFIIIA-DNA complexes with the ICR sequence contained in pBend3 indicates a bending angle of only 30 degrees and shows that interaction in the ICR could account for all of the bending found in the complete oocyte 5S RNA gene.
The binding of a protein to its specific sequence, borne on a DNA fragment, retards the mobility of the fragment in a characteristic way during gel electrophoresis. If the protein induces bending in the DNA, the contortion can also be monitored by gel electrophoresis, because the amount of retardation of the mobility of the DNA-protein complex is dependent upon the position and the degree of the bend induced in the DNA fragment [Wu and Crothers, Nature 308 (1984) 509-513]. We have constructed a plasmid, pBend2, which can generate a large number of DNA fragments of identical length in which the protein-binding nucleotide sequence is located in circular permutations. The vector contains two identical DNA segments containing 17 restriction sites in a direct repeat spanning a central region containing cloning sites. The protein-binding sequence is inserted at one of these cloning sites. To investigate the functional significance of bending, we have compared, using pBend2, the cAMP.cAMP-receptor protein (CPR)-induced bending of CRP-binding sites found in five different genes of Escherichia coli. We have also shown that the bacteriophage lambda 0R1 operator DNA is bent when complexed with the CI or Cro repressor of the phage.
An 11 S ribonucleoprotein particle was isolated from maize endosperm and shown to be functionally and structurally equivalent to the mammalian signal recognition particle. However, unlike animal cells which apparently contain a single 7 SL RNA species, maize endosperm contains a heterogeneous population of 7 SL RNA. To investigate this diversity, we have cloned and sequenced a number of the maize endosperm 7 SL RNAs isolated from functionally active SRP preparations. Some maize 7 SL RNAs are strikingly similar, differing by single base changes or short deletions; surprisingly, others share less than 70 percent sequence homology. Despite differences in primary sequence, nearly identical secondary structures can be suggested for all maize 7 SL RNAs, consistent with a proposed functional role in protein translocation for each of these RNAs. The amount of new available sequence data enabled us to define two conserved regions of presumed functional importance: A conserved sequence -G-N-A-R- in the center of a variable region which forms a well defined stem-loop and possibly is involved in an interaction with the 19 kDa protein of the SRP. Secondly, three short nucleotide stretches located in the central domain of 7 SL RNA may form part of a dynamic RNA-switch structure.
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A procedure was developed for casting thin-layer multistrip polyacrylamide gels and using them for the simultaneous gel electrophoresis at several gel (Ferguson plot analysis) at the sub-microgram load level, using silver staining, autoradiography and, potentially, blotting for detection. The lower viscosity of polymerization mixtures, compared to agarose gelation mixtures, required the redesign of the multistrip cassette with separation of channels by rubber gaskets and the application of a cassette press. The lowered viscosity also required addition of 35% sucrose and an increased rate of polymerization in application to multistrip gels formed on a common NetFix backing. The present design allows one to obtain Ferguson plots exemplified by those of 32P-labeled DNA followed by autoradiographic detection.