Functional analysis of the invariant residue G791 of Escherichia coli 16S rRNA

The nucleotide at position 791(G791) of E. coli 16S rRNA was previously identified as an invariant residue for ribosomal function. In order to characterize the functional role of G791, base substitutions were introduced at this position, and mutant ribosomes were analyzed with regard to their protein synthesis ability, via the use of a specialized ribosome system. These ribosomal RNA mutations attenuated the ability of ribosomes to conduct protein synthesis by more than 65%. A transition mutation (G to A) exerted a moderate effect on ribosomal function, whereas a transversion mutation (G to C or U) resulted in a loss of protein synthesis ability of more than 90%. The sucrose gradient profiles of ribosomes and primer extension analysis showed that the loss of protein-synthesis ability of mutant ribosomes harboring a base substitution from G to U at position 791 stems partially from its inability to form 70S ribosomes. These findings show the involvement of the nucleotide at position 791 in the association of ribosomal subunits and protein synthesis steps after 70S formation, as well as the possibility of using 16S rRNA mutated at position 791 for the selection of second-site revertants in order to identify ligands that interact with G791 in protein synthesis.     

Characterization of recombinant CDR1, an Arabidopsis aspartic proteinase involved in disease resistance

The Arabidopsis thaliana constitutive disease resistance 1 (CDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling (Xia, Y., Suzuki, H., Borevitz, J., Blount, J., Guo, Z., Patel, K., Dixon, R. A., and Lamb, C. (2004) EMBO J. 23, 980-988). This apoplastic enzyme is a member of the group of "atypical" plant aspartic proteinases. As for other enzymes of this subtype, CDR1 has remained elusive until recently as a result of its unusual properties and localization. Here we report on the heterologous expression and characterization of recombinant CDR1, which displays unique enzymatic properties among plant aspartic proteinases. The highly restricted specificity requirements, insensitivity toward the typical aspartic proteinase inhibitor pepstatin A, an unusually high optimal pH of 6.0-6.5, proteinase activity without irreversible prosegment removal, and dependence of catalytic activity on formation of a homo-dimer are some of the unusual properties observed for recombinant CDR1. These findings unveil a pattern of unprecedented functional complexity for Arabidopsis CDR1 and are consistent with a highly specific and regulated biological function.     

Within-host competition in genetically diverse malaria infections: parasite virulence and competitive success

Humans and animals often become coinfected with pathogen strains that differ in virulence. The ensuing interaction between these strains can, in theory, be a major determinant of the direction of selection on virulence genes in pathogen populations. Many mathematical analyses of this assume that virulent pathogen lineages have a competitive advantage within coinfected hosts and thus predict that pathogens will evolve to become more virulent where genetically diverse infections are common. Although the implications of these studies are relevant to both fundamental biology and medical science, direct empirical tests for relationships between virulence and competitive ability are lacking. Here we use newly developed strain-specific real-time quantitative polymerase chain reaction protocols to determine the pairwise competitiveness of genetically divergent Plasmodium chabaudi clones that represent a wide range of innate virulences in their rodent host. We found that even against their background of widely varying genotypic and antigenic properties, virulent clones had a competitive advantage in the acute phase of mixed infections. The more virulent a clone was relative to its competitor, the less it suffered from competition. This result confirms our earlier work with parasite lines derived from a single clonal lineage by serial passage and supports the virulence-competitive ability assumption of many theoretical models. To the extent that our rodent model captures the essence of the natural history of malaria parasites, public health interventions which reduce the incidence of mixed malaria infections should have beneficial consequences by reducing the selection for high virulence.     

Inhibition of mycobacterial arylamine N-acetyltransferase contributes to anti-mycobacterial activity of Warburgia salutaris

In this study, we show that extracts and a purified compound of Warburgia salutaris exhibit anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG Pasteur. The extracts did not inhibit growth of Escherichia coli and were not toxic to cultured mammalian macrophage cells at the concentrations at which anti-mycobacterial activity was observed. The extract and pure compound inhibited pure recombinant arylamine N-acetyltransferase (NAT), an enzyme involved in mycobacterial cell wall lipid synthesis. Moreover, neither extract nor pure compound inhibited growth of a strain of M. bovis BCG in which nat has been deleted suggesting that NAT may indeed be a target within the mycobacterial cell. The purified compound is a novel drimane sesquiterpenoid lactone, 11alpha-hydroxycinnamosmolide. These studies show that W. salutaris is a useful source of anti-tubercular compounds for further analysis and supports the hypothesis of a link between NAT inhibition and anti-mycobacterial activity.     

Differential crop damage by healthy and nucleopolyhedrovirus-infected Mamestra brassicae L. (Lepidoptera: Noctuidae) larvae: a field examination

Baculovirus infection in Lepidoptera can alter both larval mobility and feeding rates, which can in turn affect pathogen transmission and dispersal in the field. We compared the damage to cabbage plants in the field caused by healthy and nucleopolyhedrovirus-infected Mamestra brassicae L. (Lepidoptera: Noctuidae) larvae released as second and fourth instars. There was no significant difference in plant consumption by healthy and infected larvae for the first 4 days after release. From day 5 onwards, infected larvae caused significantly less defoliation. This pattern was similar for larvae at both larval instars. Defoliation was greater for fourth instars throughout the experiment.     

Use of the paired samples (cerebrospinal fluid and serum) in immunodiagnostic of active and inactive human neurocysticercosis

Paired samples of cerebrospinal fluid (CSF) and serum of 30 patients--10 with active, 10 with inactive neurocysticercosis (NCC), and 10 control subjects--were evaluated by enzyme-linked immunosorbent assay (ELISA) using two Taenia crassiceps metacestode extracts as antigen in order to detect IgG antibodies. In active NCC, high levels of IgG were detected (p < 0.05). The CSF samples showed 80% (CI 72-88) of reactivity in the saline extract (S) and 90% (CI 84-95) in sodium dodecyl sulphate (SDS) and the serum samples were reactive in 90% (CI 84-95) and 100% (CI 98-100) in the S and SDS antigenic extracts, respectively. The use of the paired samples of CSF and serum in active NCC showed equivalent results suggesting that the serum samples could be used as a screening in those patients whose CSF puncture is counter-indicated.     

Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Isolation and characterization of microsatellite loci in Pimelodus microstoma (Siluriformes: Pimelodidae)

Ten microsatellite loci were developed and described for Pimelodus microstoma, identified in eggs collected from the lower Das Cinzas river. Nine loci were polymorphic, with 02–19 alleles per locus. The observed heterozygosity values were up to 0.898. Five species of the Pimelodidae family were also tested for amplification of loci, among which Pimelodus maculatus was the most successful in the transferability of primers. These loci have proven to be useful for studying the variability and structuring of populations of Pimelodus microstoma.     

Monthly Occurrence of Vectors and Reservoir Rodents of Scrub Typhus in an Endemic Area of Jeollanam-do, Korea

Monthly surveys were conducted to investigate the occurrence of chigger mites and seroprevalence of scrub typhus among small mammals in Jeollanam-do, the southwestern part of Korea, from November 2006 through October 2007. Fifty-eight small mammals, including 57 Apodemus agrarius (98.3%) and 1 Crocidura lasiura (1.7%), were captured, and a total of 4,675 chigger mites representing 4 genera and 8 species were collected from them. The chigger infestation rate among small mammals was 69.0%. The most predominant species in A. agrarius was Leptotrombidium scutellare (54.0%), followed by Leptotrombidium pallidum (39.4%), Leptotrombidium orientale (4.4%), Leptotrombidium palpale (1.1%), Neotrombicula tamiyai (0.6%), Eushoengastia koreaensis (0.3%), Neotrombicula gardellai (0.3%), and Cheladonta ikaoensis (<0.1%). The chigger index of A. agrarius was the highest in October (740.0), followed by November (242.0), September (134.6), March (98.3), February (38.2), January (35.3), December (34.5), April (30.8), and May (1.7). The average antibody positive rate of scrub typhus in wild rodents was 50.0%. The seropositive rates were high in October (100.0%) and November (83.3%), whereas those in other months were relatively low (28.6-57.1%). The chigger index of L. scutellare rapidly increased in September to form an acuminate peak in October, followed by a gradual decline. These results suggest that the outbreak of scrub typhus in the southwestern part of Korean peninsula is mostly due to L. scutellare.