Biomedical subjects

C C Kao

Publications and source records attributed to C C Kao.

At least 55 records · Page 3Linked to original sources

PubMed1999

Use of DNA, RNA, and chimeric templates by a viral RNA-dependent RNA polymerase: evolutionary implications for the transition from the RNA to the DNA world.

All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position -11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes.

Bromovirus↗

PubMed1998

Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure.

RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of the viral replicase required for the replication of all positive strand RNA viruses. The vast majority of RdRps have been identified solely on the basis of sequence similarity. Structural studies of RdRps have lagged behind those of the DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, and reverse transcriptases until the recent report of the partial crystal structure of the poliovirus RdRp, 3Dpol [Hansen, J. L., et al. (1997). Structure 5, 1109-1122]. We seek to address whether all RdRps will have structures similar to those found in the poliovirus polymerase structure. Therefore, the PHD method of Rost and Sander [Rost, B., and Sander, C. (1993a). J. Mol. Biol. 232, 584-599; Rost, B., and Sander, C. (1994). Protein 19, 55-77] was used to predict the secondary structure of the RdRps from six different viral families: bromoviruses, tobamoviruses, tombusvirus, leviviruses, hepatitis C-like viruses, and picornaviruses. These predictions were compared with the known crystal structure of the poliovirus polymerase. The PHD method was also used to predict picornavirus structures in places in which the poliovirus crystal structure was disordered. All five families and the picornaviruses share a similar order of secondary structure elements present in their polymerase proteins. All except the leviviruses have the unique region observed in the poliovirus 3Dpol that is suggested to be involved in polymerase oligomerization. These structural predictions are used to explain the phenotypes of a collection of mutations that exist in several RNA polymerases. This analysis will help to guide further characterization of RdRps.

Amino Acid Sequence↗

PubMed1998

Sequences 5' of the conserved tRNA-like promoter modulate the initiation of minus-strand synthesis by the brome mosaic virus RNA-dependent RNA polymerase.

Each of the brome mosaic virus (BMV) genomic RNAs contains a conserved tRNA-like structure that is sufficient to direct minus-strand RNA synthesis in vitro. The tRNA-like promoters, tB1 and tB3, direct approximately equal amounts of synthesis in vitro. However, 5' sequences were found to affect the amount of minus-strand synthesis, suggesting that sequences beyond the tRNA-like structure are important in moderating minus-strand synthesis. Consistent with this, sequences upstream the tRNA-like structure are able to partially suppress mutations at or near the initiation site. This activity is observed in the 5' sequences of both BMV and CMV (cucumber mosaic virus) templates. However, a chimeric RNA containing the CMV tRNA-like promoter fused to the 5' sequences of BMV was not able to suppress mutations at the initiation site, suggesting that homologous 5' and 3' sequences are required to affect initiation. The ability to suppress mutations at the initiation site was correlated with a slight increase in the ability of the BMV RNA-dependent RNA polymerase to interact with the RNA.

Base Sequence↗

PubMed1998

Subgenomic RNA promoters dictate the mode of recognition by bromoviral RNA-dependent RNA polymerases.

Both the brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) RNA-dependent RNA polymerases (RdRps) were found to recognize the BMV core subgenomic promoter in the same manner, requiring specific functional groups at positions -17, -14, -13, and -11 relative to the subgenomic initiation site (+1). For CCMV subgenomic RNA synthesis, both RdRps required the same nucleotides and four additional nucleotides at positions -20, -16, -15, and -10. The -20 nucleotide is partially responsible for the differential mode of recognition of the two promoters. These data provide evidence that the RNA can induce RdRps to alter the mode of promoter recognition.

Bromovirus↗

PubMed1998

Moieties in an RNA promoter specifically recognized by a viral RNA-dependent RNA polymerase.

RNAs 33 nucleotides in length can direct accurate initiation of subgenomic RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase (RdRp), provided that the native sequences are maintained at five positions: -17, -14, -13, -11, and the +1 initiation site. The functional groups in the bases of these essential nucleotides required to interact with RdRp were examined by using chemically synthesized RNAs containing base analogs at each of the five positions. Analysis using a template competition assay revealed that the mode of recognition for the initiation nucleotide (+1) is distinct from that of the other essential nucleotides in the promoter. Competition experiments also determined that three template nucleotides are sufficient for stable interaction with RdRp. These results identify base moieties in the brome mosaic virus subgenomic promoter required for efficient RNA synthesis and support the hypothesis that the recognition of a RNA promoter by a viral RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.

Base Sequence↗

PubMed1998

ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. Blockade of an autocrine/paracrine pathway to define receptor preference of an agonist.

Extracellular nucleotides regulate function in many cell types via activation of multiple P2-purinergic receptor subtypes. However, it has been difficult to define which individual subtypes mediate responses to the physiological agonist ATP. We report a novel means to determine this by exploiting the differential activation of an autocrine/paracrine signaling pathway. We used Madin-Darby canine kidney epithelial cells (MDCK-D1) and assessed the regulation of cAMP formation by nucleotides. We found that ATP, 2-methylthio-ATP (MT-ATP) and UTP increase cAMP production. The cyclooxygenase inhibitor indomethacin completely inhibited UTP-stimulated, did not inhibit MT-ATP-stimulated, and only partially blocked ATP-stimulated cAMP formation. In parallel studies, ATP and UTP but not MT-ATP stimulated prostaglandin production. By pretreating cells with indomethacin to eliminate the P2Y2/prostaglandin component of cAMP formation, we could assess the indomethacin-insensitive P2 receptor component. Under these conditions, ATP displayed a ten-fold lower potency for stimulation of cAMP formation compared with untreated cells. These data indicate that ATP preferentially activates P2Y2 relative to other P2 receptors in MDCK-D1 cells (P2Y1 and P2Y11, as shown by reverse transcriptase polymerase chain reaction) and that P2Y2 receptor activation is the principal means by which ATP increases cAMP formation in these cells. Blockade of autocrine/paracrine signaling can aid in dissecting the contribution of multiple receptor subtypes activated by an agonist.

Adenosine Triphosphate↗

PubMed1998

Inhibition of a naturally occurring EGFR oncoprotein by the p185neu ectodomain: implications for subdomain contributions to receptor assembly.

Mutant Epidermal Growth Factor Receptor (EGFR) oncoproteins lacking most of subdomains I and II of the extracellular region, a deletion which includes most of the first of two cysteine-rich sequences, have been observed in multiple human epithelial tumors, including malignant gliomas. These EGFR oncoproteins, designated deltaEGFR or EGFRvIII, confer increased tumorigenicity in vivo and are often coexpressed with full-length EGFR in human tumors. We have expressed an ectodomain-derived, carboxyl-terminal deletion mutant of the p185neu oncogene (T691stop) in human glioblastoma cells coexpressing endogenous EGFR and activated deltaEGFR oncoproteins. The p185neu ectodomain-derived mutant forms heterodimers with deltaEGFR proteins and reduces the phosphotyrosine content and kinase activity of deltaEGFR monomers. As a consequence of T691stop neu expression and surface localization, cell proliferation in conditions of full growth and reduced serum and anchorage-independent growth in soft agar was reduced in glioblastoma cells expressing either endogenous EGFR alone or coexpressing EGFR and elevated levels of deltaEGFRs. T691stop neu mutant receptors abrogate the dramatic growth advantage conferred by deltaEGFR in vivo, suggesting that physical associations primarily between subdomains III and IV of the p185neu and EGFR ectodomains are sufficient to modulate signaling from activated EGFR complexes. Receptor-based inhibitory strategies exploit the thermodynamic preference for erbB ectodomains to heterodimerize, thereby creating erbB receptor assemblies which are defective in signaling and do not internalize. Pharmaceuticals which mimic the p185neu ectodomain may therefore have important therapeutic applications in advanced human malignancies expressing erbB receptors.

Animals↗

PubMed1998

Gingival crevicular fluid lactoferrin levels in adult periodontitis patients.

The present study was designed to determine in a cross-sectional study whether there was any relationship between the levels of lactoferrin in gingival crevicular fluid and clinical periodontal parameters. Crevicular fluid was collected from individual sites using standardized filter paper strips (clinically healthy sites, N = 23; periodontitis sites, n = 66) and evaluated for lactoferrin by enzyme-linked immunosorbent assay. The data showed that: (1) the total amounts of lactoferrin were 0.003-0.021 ng (30 second sample) (average 0.009 +/- 0.005 ng) in a clinically healthy periodontium group and 0.016-3.847 ng (30 second sample) (average 0.575 +/- 0.069 ng) in adult periodontitis patients (statistically significantly higher in adult periodontitis patients); and (2) the total amounts of lactoferrin were significantly correlated with clinical parameters, especially a strong positive correlation with gingival crevicular fluid volume (r = 0.85, p < 0.01) and with probing depth (r = 0.71, p < 0.01). These results indicated that quantification of lactoferrin in gingival crevicular fluid may be a more sensitive indicator of periodontal pathology than traditional clinical indices.

Adult↗

PubMed1998

EpsR modulates production of extracellular polysaccharides in the bacterial wilt pathogen Ralstonia (Pseudomonas) solanacearum.

Ralstonia solanacearum is the causal agent of bacterial wilt of many agriculturally important crops. Exopolysaccharide synthesized by products of the epsI operon is the major virulence factor for R. solanacearum. Expression of epsI has been demonstrated to be under the control of several proteins, including several two-component regulators. Overexpression of EpsR was found previously to reduce the amount of synthesis specifically from the epsI promoter. Here we present data that a single chromosomal copy of epsR activates the epsI promoter, suggesting that EpsR is a concentration-dependent effector of epsI gene expression. Furthermore, the ability of EpsR to modulate epsI expression is dependent on the phosphorylation state of EpsR. Gel mobility shift assays suggest that EpsR can specifically bind the epsI promoter and that this binding requires a phosphorylated form of EpsR.

Bacterial Proteins↗

PubMed1998

Mutations in the N terminus of the brome mosaic virus polymerase affect genetic RNA-RNA recombination.

Previously, we have observed that mutations in proteins 1a and 2a, the two virally encoded components of the brome mosaic virus (BMV) replicase, can affect the frequency of recombination and the locations of RNA recombination sites (P. D. Nagy, A. Dzianott, P. Ahlquist, and J. J. Bujarski, J. Virol. 69:2547-2556, 1995; M. Figlerowicz, P. D. Nagy, and J. J. Bujarski, Proc. Natl. Acad. Sci. USA 94:2073-2078, 1997). Also, it was found before that the N-terminal domain of 2a, the putative RNA polymerase protein, participates in the interactions between 1a and 2a (C. C. Kao, R. Quadt, R. P. Hershberger, and P. Ahlquist, J. Virol. 66:6322-6329, 1992; E. O'Reilly, J. Paul, and C. C. Kao, J. Virol. 71:7526-7532, 1997). In this work, we examine how mutations within the N terminus of 2a influence RNA recombination in BMV. Because of the likely electrostatic character of 1a-2a interactions, five 2a mutants, MF1 to MF5, were generated by replacing clusters of acidic amino acids with their neutral counterparts. MF2 and MF5 retained nearly wild-type levels of 1a-2a interaction and were infectious in Chenopodium quinoa. However, compared to that in wild-type virus, the frequency of nonhomologous recombination in both MF2 and MF5 was markedly decreased. Only in MF2 was the frequency of homologous recombination reduced and the occurrence of imprecise homologous recombination increased. In MF5 there was also a 3' shift in the positions of homologous crossovers. The observed effects of MF2 and MF5 reveal that the 2a N-terminal domain participates in different ways in homologous and in nonhomologous BMV RNA recombination. This work maps specific locations within the N terminus involved in 1a-2a interaction and in recombination and further suggests that the mechanisms of the two types of crossovers in BMV are different.

Amino Acid Sequence↗

PubMed1998

Interactions between the structural domains of the RNA replication proteins of plant-infecting RNA viruses.

Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.

Animals↗

PubMed1998

Mechanistic analysis of RNA synthesis by RNA-dependent RNA polymerase from two promoters reveals similarities to DNA-dependent RNA polymerase.

The brome mosaic virus (BMV) RNA-dependent RNA polymerase (RdRp) directs template-specific synthesis of (-)-strand genomic and (+)-strand subgenomic RNAs in vitro. Although the requirements for (-)-strand RNA synthesis have been characterized previously, the mechanism of subgenomic RNA synthesis has not. Mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for RNA synthesis. Unlike (-)-strand RNA synthesis, which required only a high GTP concentration, subgenomic RNA synthesis required high concentrations of both GTP and UTP. Phylogenetic analysis of the sequences surrounding the initiation sites for subgenomic and genomic (+)-strand RNA synthesis in representative members of the alphavirus-like superfamily revealed that the +1 and +2 positions are highly conserved as a pyrimidine-adenylate. GDP and dinucleotide primers were able to more efficiently stimulate (-)-strand synthesis than subgenomic synthesis under conditions of limiting GTP. Oligonucleotide products of 6-, 7-, and 9-nt were synthesized and released by RdRp in 3-20-fold molar excess to full-length subgenomic RNA. Termination of RNA synthesis by RdRp was not induced by template sequence alone. Our characterization of the stepwise mechanism of subgenomic and (-)-strand RNA synthesis by RdRp permits comparisons to the mechanism of DNA-dependent RNA synthesis.

Alphavirus↗

PubMed1997

Sequence-specific recognition of a subgenomic RNA promoter by a viral RNA polymerase.

RNA templates of 33 nucleotides containing the brome mosaic virus (BMV) core subgenomic promoter were used to determine the promoter elements recognized by the BMV RNA-dependent RNA polymerase (RdRp) to initiate RNA synthesis. Nucleotides at positions -17, -14, -13, and -11 relative to the subgenomic initiation site must be maintained for interaction with the RdRp. Changes to every other nucleotide at these four positions allow predictions for the base-specific functional groups required for RdRp recognition. RdRp contact of the nucleotide at position -17 was suggested with a template competition assay. Comparison of the BMV subgenomic promoter to those from other plant and animal alphaviruses shows a remarkable degree of conservation of the nucleotides required for BMV subgenomic RNA synthesis. We show that the RdRp of the plant-infecting BMV is capable of accurately, albeit inefficiently, initiating RNA synthesis from the subgenomic promoter of the animal-infecting Semliki Forest virus. The sequence-specific recognition of RNA by the BMV RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.

Animals↗

PubMed1997

Characterization of RNA products associated with or aborted by a viral RNA-dependent RNA polymerase.

Ternary RNA-dependent RNA polymerase complexes were arrested at various stages of synthesis in vitro by limiting one or more NTPs. The RNAs synthesized prior to/during the arrest of the complex were characterized and the ability of the arrested complexes to continue elongation of these nascent RNAs in the presence of all four NTPs was analyzed. Nascent RNAs of 10 nt and longer remained associated with the RdRp complex and could be extended into full-length RNAs while shorter nascent RNAs were released by the complex. These results establish that previously observed RdRp-synthesized oligoribonucleotides of 8 nt or less are abortive initiation products and confirms that RdRp undergoes a transition from initiation to elongation after the synthesis of 8 nt.

Base Sequence↗

PubMed1997

RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase: transition from initiation to elongation.

The initiation and elongation phases of (-)-strand RNA synthesis in vitro by the brome mosaic virus RNA-dependent RNA polymerase (RdRp) are differentially sensitive to inhibitors. In an attempt to characterize further the transition RdRp makes from initiation to elongation, we determined the conditions needed to pause the ternary complex and complete only one round of RNA synthesis. During the transition we were able to discern step-wise increases in the affinity of RdRp for RNA by measuring sensitivity to heparin and competition for RdRp by an alternative template. Three distinct stability levels of RdRp-template interactions were found. The first stable RdRp-RNA complex was observed when RdRp bound to the RNA template. A further increase occurred when RdRp synthesized the first phosphodiester bond. A final increase occurred upon formation of between 3 and 13 phosphodiester bonds. After this last transition, RdRp appeared to be tightly committed to the template RNA. These results are analogous to the mechanism of action of DNA-dependent RNA polymerases and are relevant to protein-RNA interaction and template switching by an RdRp.

Air Pollutants↗

PubMed1997

Internal mammary silicone lymphadenopathy mimicking recurrent breast cancer.

This case describes a unique complication of silicone breast implantation that is previously undocumented. Internal mammary silicone lymphadenopathy mimicking breast cancer recurrence represents an important new clinical entity. The diagnosis and management of this clinical enigma are challenging. In our patient, videothoracoscopy proved to be minimally invasive and allowed complete resection of the entire chain of pathologic nodes. Explantation of the silicone prostheses and complete capsulectomy are indicated. Light microscopy alone can suggest the diagnosis of silicone lymphadenopathy. Infrared spectral analysis can be a helpful adjunct to allow an unequivocal diagnosis.

Biopsy↗

PubMed1997

Analysis of the interaction of viral RNA replication proteins by using the yeast two-hybrid assay.

The yeast two-hybrid system has been a useful tool in the genetic evaluation of protein-protein interactions. However, the biological relevance of these two-hybrid interactions to viral positive-strand RNA replication has not been demonstrated. The brome mosaic virus (BMV) system has been characterized extensively both genetically and biochemically, providing numerous mutations in the BMV 1a helicase-like and 2a polymerase-like proteins. We have tested wild-type 1a and 18 insertion mutations of 1a and found a perfect correlation between the in planta phenotypes and their ability to interact with 2a in the two-hybrid system. This finding allowed further characterization of the interaction between and among the BMV viral proteins. Using the two-hybrid assay, we have found that the interaction between the helicase-like region of 1a and the N terminus of 2a is stabilized by the presence of the centrally conserved polymerase-like domain of 2a. We have also identified a novel interaction between the 1a helicase-like protein and itself. Additionally, we have found this interaction in two related tripartite RNA viruses, cowpea chlorotic mottle virus and cucumber mosaic virus. We have demonstrated that this protein-protein interaction is specific to homologous pairings of the protein.

Bromovirus↗

PubMed1997

Minimal templates directing accurate initiation of subgenomic RNA synthesis in vitro by the brome mosaic virus RNA-dependent RNA polymerase.

Short regions of (-)-strand brome mosaic virus (BMV) RNA3, proscripts, were shown to direct accurate in vitro synthesis of (+)-strand subgenomic RNA by the BMV RNA-dependent RNA polymerase (RdRp), facilitating characterization of the sequences and/or structures directing subgenomic RNA synthesis. Proscripts retaining fewer than 8-nt of an 18-nt polyuridylate tract located just upstream of the core promoter sequence (French R, Ahlquist P, 1988, J Virol 62:2411-2420; Marsh LE, Dreher TW, Hall TC, 1988, Nucleic Acids Res 16:981-995) directed dramatically less synthesis of 26-nt or longer products. Original levels of RNA synthesis were not restored by replacement of the 3' polyuridylate tract with polyadenylate, polycytidylate, or polyguanylate tracts, or by movement of the polyuridylate tract to the 5' end of the proscript. The polyuridylate tract presumably binds some component(s) required for RdRp activity because the addition of poly(U) [but not poly(C)] RNA to RdRp reactions decreased RNA synthesis significantly. Quite surprisingly, deletions of the polyuridylate tract in proscripts directing synthesis of 24-nt or shorter products had little or no detrimental effect on subgenomic RNA synthesis, correlated with their inability to form a computer-predicted stem-loop present in longer proscripts requiring the polyuridylate tract. Successive 3' and 5' deletions demonstrated that the minimal elements required for accurate initiation of subgenomic RNA synthesis are within a proscript of 22-nt.

Bromovirus↗