Kinetic Basis for the Conjugation of Auxin by a GH3 Family Indole-acetic Acid-Amido Synthetase

The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg²⁺ or Mn²⁺ for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4–9-fold reductions in k_cat/K_m relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action.     

Rapid development of cleaning behaviour by Torresian crows Corvus orru on non-native banteng Bos javanicus in northern Australia

In this paper we report the observation of a rapidly developed vertebrate symbiosis involving ectoparasite cleaning by a native corvid of northern Australia, the Torresian crow Corvus orru , on a recently introduced bovid ungulate, the banteng Bos javanicus . Facultative symbioses benefiting both participants (mutualisms) between birds and mammals appear to be rare, despite the apparent advantages obtained by the participants (ecto-parasite removal from the host and food provision to the cleaner). On three separate dates we observed a total of four C. orru individuals eliciting facilitation behaviours by a total of ten female banteng to assist in the removal of ectoparasites. Our observations document the first-known incidence of facultative cleaning behaviour by a native bird species on a non-native, wild vertebrate that has developed in approximately 150 years since the banteng's introduction to Australia.     

Detection of asymptomatic herpes simplex virus infections after vaccination.

Twenty-two volunteers seronegative for antibodies to herpes simplex virus (HSV) were enrolled in a trial to determine tolerance and immunogenicity of an HSV-2 glycoprotein subunit vaccine. Vaccine was administered at days 0, 28, and 140, and sera were obtained on days 0, 7, 14, 21, 28, 35, 49, 56, 140, 147, and 365 for determination of HSV neutralizing antibody activity and antibody-dependent cell cytotoxicity (ADCC). Sera were also tested by immunoprecipitation of radiolabeled HSV-2-infected cell proteins and polyacrylamide gel electrophoresis to identify the viral proteins which elicited antibody responses in vaccine recipients. After vaccination two male volunteers presented with atypical first-episode genital herpes: patient 1 with a culture-negative genital lesion at day 53 and patient 3 with urethritis at day 68. Seroconversion to wild-type viral proteins not present in the vaccine was detectable by radioimmunoprecipitation-polyacrylamide gel electrophoresis within 10 days in both patients. Two additional volunteers, one a sex contact of patient 1, seroconverted asymptomatically to nonvaccine proteins during the trial. All four vaccine breakthrough patients were indistinguishable from the other volunteers in the time required to develop neutralizing and ADCC antibodies, in the titer of these antibodies, and the time to seroconversion to gB and gD vaccine proteins. However, only one of the four breakthrough patients had antibodies to g80 (a complex of gC-2 and gE) after vaccination as compared with 15 of the other 18 volunteers (P = 0.05). Neither neutralizing antibody nor ADCC titers consistently identified acquisition of wild-type viral infection; therefore, protein-specific serologies were required to detect wild-type antibodies in these four patients. These data underscore the importance of using serologic assays which will distinguish naturally acquired infection from the immune response to vaccination.     

Efficient methods for attachment of thiol specific probes to the 3''-ends of synthetic oligodeoxyribonucleotides.

Methodology is described for the synthesis of DNA oligomers containing a free 3'-thiol group which can be selectively crosslinked with a wide variety of probes. This chemistry is compatible with both phosphotriester and phosphoramidite solid phase chemistry. Moreover, the sulphydryl group is introduced into the 3'-nucleoside solid support linkage prior to oligonucleotide synthesis. Consequently, no additional coupling steps are required after oligonucleotide synthesis, and isolation of the 3'-thiol oligonucleotide requires only one additional deprotection step. Cross-linking of the thiol-containing oligonucleotide to a fluorescent probe was carried out with high selectivity, in high yield, and under mild conditions.     

Ion channel expression by white matter glia: the type-1 astrocyte.

We describe the electrophysiological properties of acutely isolated type-1 astrocytes using a new "tissue print" dissociation procedure. Because the enzymes used did not destroy or modify the ion channels, and the cells retained many processes, the properties may reflect those in vivo. The types of ion channels in type-1 astrocytes changed rapidly during the first 10 postnatal days, when they attained their adult phenotype. This change was dependent on the presence of neurons. In culture, most of these channel types were not expressed, but a phenotype more typical of that in vivo could be induced by co-culture with neurons. The electrophysiological properties of astrocytes make some existing hypotheses of astrocyte function less likely.     

Granulocyte-macrophage colony-stimulating factor primes neutrophils by activating a pertussis toxin-sensitive G protein not associated with phosphatidylinositol turnover

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine which produces diverse biological effects in target cells of myeloid origin. GM-CSF enhances the production of superoxide anion (O2-) by mature neutrophils in response to chemotactic peptides such as formyl-methionyl-leucyl-phenylalanine (fMLP), but alone it has no effect on this system. This process has been termed "priming." fMLP activates neutrophils via a pertussis toxin-sensitive GTP-binding protein, leading to the rapid production of the second messengers diacylglycerol (DAG) and inositol trisphosphate, via phosphatidylinositol turnover, and arachidonic acid (AA) by a presumptive phospholipase A2-mediated mechanism. All three second messengers may lead to the generation of O2-. We investigated the effect of priming of GM-CSF on these systems. GM-CSF had no effect on fMLP-stimulated DAG and inositol trisphosphate levels, nor did it amplify the response to exogenously added phorbol ester (to mimic the action of DAG) or calcium ionophore. Neutrophils primed with the cytokine showed a small, but significant, enhancement of fMLP-stimulated AA release. Compared with unprimed controls, primed neutrophils also showed a significant increase in O2- production when stimulated with either AA or the nonhydrolyzable GTP analogue, GTP-gamma-S. The magnitude of enhanced O2- production was similar to that observed after fMLP treatment of primed cells. All of these effects, including the increased sensitivity to AA treatment, were inhibited by pertussis toxin. These data show that GM-CSF primes neutrophils by modulating the activity of at least one pertussis toxin-sensitive G protein coupled to a metabolic pathway that mobilizes and utilizes arachidonic acid.     

Genetic determination of exocrine pancreatic function in cystic fibrosis.

We showed elsewhere that the pancreatic function status of cystic fibrosis (CF) patients could be correlated to mutations in the CF transmembrane conductance regulator (CFTR) gene. Although the majority of CF mutations--including the most common, delta F508--strongly correlated with pancreatic insufficiency (PI), approximately 10% of the mutant alleles may confer pancreatic sufficiency (PS). To extend this observation, genomic DNA of 538 CF patients with well-documented pancreatic function status were analyzed for a series of known mutations in their CFTR genes. Only 20 of the 25 mutations tested were found in this population. They accounted for 84% of the CF chromosomes, with delta F508 being the most frequent (71%), and the other mutations accounted for less than 5% each. A total of 30 different, complete genotypes could be determined in 394 (73%) of the patients. The data showed that each genotype was associated only with PI or only with PS, but not with both. This result is thus consistent with the hypothesis that PI and PS in CF are predisposed by the genotype at the CFTR locus; the PS phenotype occurs in patients who have one or two mild CFTR mutations, such as R117H, R334W, R347P, A455E, and P574H, whereas the PI phenotype occurs in patients with two severe alleles, such as delta F508, delta I507, Q493X, G542X, R553X, W1282X, 621 + 1G----T, 1717-1G----A, 556delA, 3659delC, I148T, G480C, V520F, G551D, and R560T.