Aeromonas caviae exhibits aggregative adherence to HEp-2 cells.
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Aeromonas↗
Biomedical subjects
J G Shaw
Publications and source records attributed to J G Shaw.
The M gamma chain of human fetal hemoglobin is an A gamma chain with an in vitro modification of gamma 141 leucine to hydroxyleucine.
We have reanalyzed the structure of the gamma T-15 peptide from the minor M gamma chain of human hemoglobin (Hb) F. Amino acid analysis confirmed that the Leu 141 residue was missing from position 9 of this peptide, and liquid secondary ion mass spectrometry indicated that it was replaced, not by methionine (residue mass 131) as previously believed, but by an amino acid of mass 129. By analogy with the recently reported oxidation of the corresponding leucine at position gamma 141 of the unstable Hb Atlanta, it appears that the M gamma chain also results from the oxidation of gamma 141 to hydroxyleucine (residue mass 129). The finding that the proportion of the M gamma chain increased when red cell lysates were prepared with carbon tetrachloride prompted us to reinvestigate the oxidation mechanism involved in the formation of beta 141 hydroxyleucine in Hb Atlanta. Oxidation of the beta 141 residue could be detected when carbon tetrachloride was used in the lysis protocol, while conversion of oxyhemoglobin to carbon monoxyhemoglobin prior to carbon tetrachloride treatment prevented oxidation. It therefore appears that the hydroxylation of Leu 141 is not an in vivo process in the circulating red cell. Perhaps leucine at position 141 of the beta, gamma, and delta chains (and at position 136 of the alpha chain), which forms a contact with heme and is located directly across the heme plate from the E helix, is oxidized to hydroxyleucine at a very low rate forming minute amounts of modified chains; this process is accelerated by treatment with agents such as carbon tetrachloride and prolonged exposure to air.
Adult↗
Plants that express a potyvirus proteinase gene are resistant to virus infection.
Transgenic tobacco plants that express the genome-linked protein/proteinase-coding region of the potyvirus tobacco vein mottling virus (TVMV) were produced and tested for their reaction to inoculation with TVMV and two other potyviruses. These plants did not develop disease symptoms after being inoculated with large doses of TVMV but were as susceptible to infection by the other potyviruses as were control plants. Lines of tobacco that express the coat protein- or the nonstructural cylindrical inclusion protein-coding regions were also produced. The coat protein transgenic plants were protected against all three potyviruses, and the cylindrical inclusion transgenic plants were susceptible to all three potyviruses. These results indicate that some, but not all, TVMV genes can be used to confer protection against potyviruses in plants. The results also suggest that combinations of viral genes in transgenic plants might improve protection against potyviruses.
Base Sequence↗
Sequence analysis and interposon mutagenesis of a sensor-kinase (DctS) and response-regulator (DctR) controlling synthesis of the high-affinity C4-dicarboxylate transport system in Rhodobacter capsulatus.
A two-component sensor-regulator system has been identified in the purple photosynthetic bacterium Rhodobacter capsulatus, which controls the expression of high-affinity C4-dicarboxylate transport activity in these cells. Nucleotide sequencing has revealed the existence of two genes, dctS and dctR, which together form an operon linked to, but divergently transcribed from, the previously identified dctP gene, which encodes the periplasmic binding protein of the transport system. The DctS protein is predicted to be a membrane-bound sensor-kinase with two potential membrane-spanning sequences in the N-terminal region. DctR was found to have sequence similarity throughout its entire length with proteins in the FixJ subfamily of response-regulators, especially to FixJ itself (42% identical residues). Insertional inactivation of the dctS and dctR genes resulted in the inability of the resulting mutants to grow on or transport malate, succinate or fumarate under aerobic conditions in the dark, and such mutants did not express the DctP protein. The mutants were complemented in trans by plasmids containing intact copies of the dctS and dctR genes.
Amino Acid Sequence↗
Association of the non-structural P3 viral protein with cylindrical inclusions in potyvirus-infected cells.
Antibodies raised against the 42K non-structural P3 protein encoded by the RNA genome of a potyvirus (tobacco vein mottling virus, TVMV) have been used with immunogold labelling procedures to determine the subcellular location of P3 in infected Nicotiana tabacum cells. P3-specific gold label was found almost exclusively associated with the cylindrical inclusions typically formed in the cytoplasm of potyvirus-infected cells by another non-structural viral protein, the cylindrical inclusion protein. The P3 antibodies reacted with inclusions in both the transverse (pinwheel-like) and longitudinal (bundle-like) orientations of these structures. Immunocytological examination of TVMV-infected protoplasts showed the association of P3 with cylindrical inclusions during the early stages of formation of these structures and suggests that the P3 may be involved in the replication of potyviral RNA.
Genome, Viral↗
Posttranslational modification of beta 141 Leu associated with the beta 75(E19)Leu-->Pro mutation in Hb Atlanta.
The recent report of the oxidation of beta 141 Leu in a New Zealand family with Hb Atlanta [ beta 75(E19)Leu-->Pro] prompted us to reinvestigate the original Hb Atlanta case from Georgia. Tryptic peptide maps showed that the modified beta CoT-14 peptide was present together with beta AT-14 in isopropanol precipitates of Hb Atlanta. Amino acid analysis confirmed that beta CoT-14 lacked leucine and mass spectrometry indicated that it had an increased mass of 16 Daltons. These findings support the proposition that the beta 75 Leu-->Pro substitution in the E helix is the direct cause of the beta 141 Leu oxidation.
Amino Acid Sequence↗
Perturbation of the equilibrium between open and closed conformations of the periplasmic C4-dicarboxylate binding protein from Rhodobacter capsulatus.
A kinetic and thermodynamic analysis has been carried out on the conformational transitions of the periplasmic C4-dicarboxylate binding protein (DctP) from the photosynthetic bacterium Rhodobacter capsulatus. This protein is distinct from other periplasmic binding proteins characterized to date in that the transition between the putative closed-unliganded (BP1) and open-unliganded (BP2) conformations is slow compared to the rate of ligand binding [Walmsley, A. R., Shaw, J. G., & Kelly, D. J. (1992) J. Biol. Chem. 276, 8064-8072]. Using stopped-flow fluorescence techniques, we have probed the conformational dynamics of the closed to open transition of DctP in the absence and presence of ligand. Both the forward rate constant for the BP1 to BP2 interconversion (k1) and the fumarate dissociation rate constant (k-3) were found to increase in a biphasic manner between pH 5 and pH 11. The data were fitted to a two-pKa function which gave pKa values of 10.3 and 5.4 for the BP1 to BP2 interconversion and 8.9 and 4.5 for the closed-liganded (BP3L) to open-liganded (BP2L) transition. An increase in ionic strength at constant pH resulted in a hyperbolic increase in both k1 and k-3 to maximal rates that were similar in each case to the values obtained in pH variation experiments. Measurement of the temperature dependencies of k1 and k-3 also gave similar activation energies. Gibbs free energy, enthalpy, and entropy changes were determined for the open to closed transitions of DctP in both the presence and absence of ligand.(ABSTRACT TRUNCATED AT 250 WORDS)
Carrier Proteins↗
The mechanism of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus.
The kinetics of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus were investigated by exploiting the changes in the intrinsic fluorescence of the protein upon binding ligands. Steady state measurements have shown that L-malate, succinate, and fumarate are all bound with sub-micromolar Kd values, whereas D-malate is bound 2 orders of magnitude more weakly. Stopped-flow studies have revealed that the binding process involves at least three steps. In the absence of ligand, the protein is in equilibrium between an essentially nonbinding form, BP1, and the binding form, BP2. Ligands bind to the BP2 form, shifting the equilibrium toward the BP2-L conformation, and also inducing a further isomerization of the protein, to the BP3-L form. The kinetic properties of the four different conformational states of the DctP protein identified in this study would be consistent with their identification as the closed-conformation, the open-conformation, an open-liganded conformation, and a closed-liganded conformation. The latter three states have been identified by x-ray crystallographic studies of binding proteins, but no kinetic or structural data have been presented previously to support the possibility of a closed but unliganded conformation.
Bacterial Proteins↗
A determinant of disease symptom severity is located in the 3'-terminal noncoding region of the RNA of a plant virus.
Inoculation of Nicotiana tabacum plants with RNA transcribed in vitro from a variant (pXBS8) of a cloned full-length DNA copy of tobacco vein mottling virus (TVMV) RNA resulted in attenuation of the vein mottling and blotching symptoms typically produced by transcripts of cloned wild-type cDNA (pXBS7). Similar amounts of virus were detected by ELISA (using anti-TVMV coat protein serum) in systemically infected leaves of plants inoculated with pXBS7 or pXBS8 transcripts. pXBS8 was shown to contain a 58-nucleotide segment in the sequence corresponding to the 3'-terminal untranslated region of TVMV RNA that was not present in pXBS7. This segment resulted in the appearance in pXBS8 transcripts of four adjacent direct repeats of a 14-nucleotide sequence, AUAAUUAUAUAUAU, that is present in the 3'-untranslated region of TVMV RNA, with two additional nucleotides (AU) between the first and second repeats. Insertion of restriction fragments containing the segment into pXBS7 and inoculation of plants with transcripts of the chimeric construct (pXBS78) resulted in the attenuated-symptom phenotype and was not accompanied by a reduced accumulation of virus in the plant as determined by ELISA and Northern blot analysis. When the extra nucleotides were removed from the variant clone, symptoms induced by transcripts of the cDNA (pXBS87) resembled those induced by wild-type transcripts. The results indicate that a noncoding region of the genome can have a direct effect on the induction of disease symptoms by an RNA virus.
Base Sequence↗
Two newly detected nonstructural viral proteins in potyvirus-infected cells.
The existence of two viral RNA-encoded proteins in cells infected with tobacco vein mottling potyvirus (TVMV) has been demonstrated. One of the proteins (named 34K) maps at the N-terminus of the TVMV polyprotein and the other (42K) between the helper component and cylindrical inclusion proteins; both had previously been predicted in the consensus potyviral genetic map. The 34K and 42K coding regions of TVMV were cloned separately in a bacterial expression vector and the proteins were isolated from transformed Escherichia coli. These were used to raise polyclonal antibodies which reacted specifically with proteins of the expected size in immunoblots of extracts of TVMV-infected tobacco leaves and protoplasts. In addition to 42K, the anti-42K serum detected similar amounts of a second protein of apparent size 37 kDa that was absent in 42K-expressing bacteria. Both 34K and 42K were present predominantly in membrane-enriched fractions of extracts of TVMV-infected tobacco leaves. Computer analysis of the deduced amino acid sequence of 42K suggests that this viral protein may be an integral transmembrane protein.
Base Sequence↗
VPg-mediated aggregation of potyviral RNA.
RNA prepared from the potyvirus tobacco vein mottling virus contained aggregates of the 9.5 kb genomic RNA with electrophoretic mobilities corresponding to 20 and 41 kb species. Similar aggregates were present in preparations of the RNAs of two other potyviruses. Aggregation occurred during or after purification of the RNA by sucrose gradient centrifugation and alcohol precipitation and was dependent upon the presence of a protein apparently bound covalently to a region at or near the 5' terminus of the viral RNA. This protein is probably the VPg. The RNAs of tobacco mosaic virus and cowpea mosaic virus did not form aggregates when isolated from purified virus by similar procedures.
Electrophoresis, Agar Gel↗
Purification, characterization and nucleotide sequence of the periplasmic C4-dicarboxylate-binding protein (DctP) from Rhodobacter capsulatus.
A periplasmic binding protein essential for high-affinity transport of the C4-dicarboxylates malate, succinate and fumarate across the cytoplasmic membrane of the purple photosynthetic bacterium Rhodobacter capsulatus has been purified to homogeneity and some of its ligand-binding properties characterized. The protein was not produced in a Tn5 insertion mutant unable to transport C4-dicarboxylates under aerobic conditions in the dark. Wild-type DNA corresponding to the location of the transposon insertion site was subcloned and a 1.5 kb section sequenced. A complete open reading frame of 999 bp was identified that encoded a 333-residue protein (DctP) with a molecular weight of 36,128 with a 26-residue amino-terminal signal peptide. The identify of this protein with the purified dicarboxylate-binding protein and the position of the predicted signal peptide cleavage site was confirmed by N-terminal sequencing. No significant homology with other proteins was detected in database searches. A GC-rich region of dyad symmetry was located 7 bp downstream of the dctP translational stop codon. This structure may be of significance in regulating the relative abundance of DctP and other dct gene products which comprise the high-affinity dicarboxylate transport system in this bacterium.
Amino Acid Sequence↗
A tyrosine residue in the small nuclear inclusion protein of tobacco vein mottling virus links the VPg to the viral RNA.
The identity of the amino acid residue that links the VPg of the potyvirus tobacco vein mottling virus (TVMV) to the viral RNA was determined. 32P-labeled TVMV RNA was digested with RNase A and micrococcal nuclease. The resulting 32P-labeled VPg was isolated and partially hydrolyzed with 6 N HCl at 110 degrees C for 2 h. Analysis by thin-layer electrophoresis revealed the presence of [32P]phosphotyrosine but not [32P]phosphoserine or [32P]phosphothreonine. Another preparation of TVMV RNA was treated with endoproteinase Lys-C, and the resulting peptide-RNA was purified by sodium dodecyl sulfate-sucrose gradient centrifugation. The sequence of the N-terminal 15 amino acid residues of the peptide, when compared with the RNA-derived amino acid sequence of the TVMV polyprotein, demonstrated that the peptide occurs in the small nuclear inclusion protein. These data suggest that Tyr-1860 of the polyprotein is the amino acid residue that links the TVMV VPg to the viral RNA.
Amino Acid Sequence↗
Adaptive behavior of preschool children with developmental delays: parent versus teacher ratings.
Parents and teachers of 20 children with developmental delays, 24 through 38 months of age, from an early education program in Columbus, Ohio, were interviewed using the Adaptive Behavior Scale for Infants and Early Childhood. Significant differences were found between some of the ratings over the 23 domain and 11 subdomain scores, specifically in regard to sex of the child and length of time of the child's participation in the program. Parents provided higher ratings than did teachers on some domains. In general, parents and teachers tended to agree on the overall adaptive functional levels.
Activities of Daily Living↗
Inhibition of uncoating of tobacco mosaic virus particles in protoplasts from transgenic tobacco plants that express the viral coat protein gene.
The uncoating of tobacco mosaic virus (TMV) particles in protoplasts isolated from leaves of transgenic tobacco plants that express the TMV coat protein gene was investigated. Extracts of these protoplasts collected up to 1 hr after inoculation with TMV contained fewer of the complexes ("striposomes") thought to be involved in cotranslational disassembly of virus particles than did extracts of protoplasts that do not express the viral coat protein gene. These results are consistent with the hypothesis that TMV coat protein-mediated resistance is at least in part the result of inhibition of the uncoating of the virus particles in the inoculum.
Capsid↗
The VPg of tobacco etch virus RNA is the 49-kDa proteinase or the N-terminal 24-kDa part of the proteinase.
Preparations of tobacco etch virus (TEV) RNA which were purified by sucrose gradient centrifugation, digested with RNase, and analyzed by SDS-polyacrylamide gel electrophoresis contained proteins of 49, 32, and 24 kDa. The 49- and 24-kDa proteins reacted with polyclonal antiserum to the TEV 49-kDa proteinase while the 32-kDa protein reacted with anti-TEV serum. Further purification of the RNA by centrifugation through CsCl removed the coat protein (32 kDa), but not the 49- and 24-kDa proteins. The 49- and 24-kDa proteins did not migrate into a polyacrylamdie gel when the RNA was not digested with RNase. These results indicate that the VPg of TEV is either the 49-kDa proteinase or the 24 kDa that represents the amino-terminal half thereof.
Centrifugation, Density Gradient↗
Mutagenesis, cloning and complementation analysis of C4-dicarboxylate transport genes from Rhodobacter capsulatus.
Transposon mutagenesis was used to isolate insertion mutants of the photosynthetic bacterium Rhodobacter capsulatus which were unable to grow under aerobic conditions in the dark on malate, succinate or fumarate as sole carbon sources. Of five mutants isolated, all were deficient in C4-dicarboxylate transport. However, these mutants were still capable of photoheterotrophic growth, although at a slower rate than the wild type, on malate and succinate (but not fumarate). The mutated locus (designated dct) was complemented in trans using a cosmid gene bank. Subcloning and complementation analysis indicated that at least three closely linked genes essential for aerobic dicarboxylate transport were contained within an 8.3 kb region of the Rhodobacter capsulatus chromosome.
Biological Transport↗
Expression of a potyvirus non-structural protein in transgenic tobacco.
A cDNA fragment encoding the cytoplasmic inclusion protein of tobacco vein mottling virus was inserted into the plant expression cassette of a Ti plasmid-based binary vector. The vector was transferred to Agrobacterium tumifaciens, and following a modified leaf disc procedure, transformed tobacco plants were obtained. Analysis of poly(A)+ RNA from transgenic plants revealed a novel RNA of approximately 2100 nucleotides possessing tobacco vein mottling virus sequences. Also, immunoprecipitation of protein extracts of [35S]methionine-labeled transformed callus using anti-cytoplasmic inclusion protein antiserum revealed a polypeptide of approximately 70 kDa. This size is consistent with that predicted from the inserted tobacco vein mottling virus coding sequences. Together these data demonstrate the expression of the cytoplasmic inclusion protein in the absence of viral infections.
Blotting, Northern↗