Alterations in cerebrospinal fluid uridine,hypoxanthine, and xanthine in head-injured patients

1. Examination of the cerebrospinal fluid (CSF) of head-injured patients reveals that the concentration of intraventricular xanthine is elevated and that of uridine is decreased relative to those of adult lumbar CSF. 2. No correlations were observed between CSF lactate and CSF hypoxanthine, xanthine, or uridine, suggesting that changes in purine metabolites and the pyrimidine nucleoside do not index similar cellular events as does lactic acid production. 3. Ventricular CSF from hydrocephalic infants had uridine and hypoxanthine concentrations not significantly different from those of normal adult lumbar CSF, but xanthine was significantly elevated. 4. Since uridine has anticonvulsant properties and is a crucial substrate for cerebral metabolism, it may be useful to evaluate this pyrimidine for use in the management of patients with head injury.     

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.     

Microbially mediated growth suppression and death of salmonella in composted sewage sludge

The role of compost microflora in the suppression of salmonella regrowth in composted sewage sludge was investigated. Microbial inhibition studies of salmonella growth were conducted on nutrient agar, in composts that had been subjected to different temperatures in compost piles, and in radiation sterilized composts inoculated with selected fractions of the compost microflora. Agar assays of inhibition indicated that bacteria and actinomycetes were not suppressive to salmonellae, but a few fungi were. However, compost inoculation assays showed consistently that fungi were not suppressive, but bacteria and actinomycetes were. In compost inoculation assays, microbial antagonists, when present, either killed salmonellae or reduced their growth rate. No suppression of salmonellae occurred in compost taken from 70°C compost-pile zones despite the presence and growth of many types of microbes. With greater numbers and kinds of microbes in 55°C compost, salmonella growth was suppressed 100–10,000-fold. Salmonellae died when inoculated into compost from unheated zones (25–40°C) of piles. Prior colonization of compost with only noncoliform gram-negative bacteria suppressed salmonellae growth 3,000-fold. Coliforms when inoculated prior to salmonellae accounted for 75% of salmonella die-off. Mesophilic curing to allow colonization of curing piles in their entirety by gram-negative bacteria, especially coliforms, should be an effective way to prevent repopulation by salmonellae.     

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.