Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering

Details of the recently elucidated biosynthetic pathways of caffeine and related purine alkaloids are reviewed. The main caffeine biosynthetic pathway is a sequence consisting of xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine. Genes encoding N-methyltransferases involved in three of these four reactions have been isolated and the molecular structure of N-methyltransferases investigated. Pathways for the catabolism of caffeine have also been studied, although there are currently no reports of enzymatic and genetic studies having been successfully carried out. Metabolism of purine alkaloids in species including Camellia, Coffea, Theobroma and Ilex plants is summarised, and evidence for the involvement of caffeine in chemical defense and allelopathy is discussed. Finally, information is presented on metabolic engineering that has produced coffee seedlings with reduced caffeine content, and transgenic caffeine-producing tobacco plants with enhanced disease resistance.     

An evaluation of two biochemical methods of age determination in insects (pteridines and lipofuscins) using the ant Polyrhachis sexpinosa Latrielle (Hymenoptera: Formicidae)

Accurate information on the age of wild-caught animals is valuable for a variety of areas, but can be particularly difficult to obtain for small holometabolous insects, whose body size is fixed at the time of pupal eclosion. A variety of chemical groups, such as lipofuscins and pteridines accumulate in body tissues through time and can be used to predict age in a variety of arthropod taxa. Here we use spectrofluorometry to confirm the presence of extractable levels of lipofuscins and pteridines in individual social insects (using the ant Polyrhachis sexpinosa Latrielle, average body size 25 mg, as an example) and evaluate their ability to predict age. Pteridine levels were independent of age but lipofuscin levels increased with age in a predictable manner ( r ² = 72.8%). Lipofuscin levels therefore represent a new method of age determination for social insects that should be applicable to both individual laboratory and wild-caught animals.     

Reducing the Activity and Secretion of Microbial Antioxidants Enhances the Immunogenicity of BCG

Background

In early clinical studies, the live tuberculosis vaccine Mycobacterium bovis BCG exhibited 80% protective efficacy against pulmonary tuberculosis (TB). Although BCG still exhibits reliable protection against TB meningitis and miliary TB in early childhood it has become less reliable in protecting against pulmonary TB. During decades of in vitro cultivation BCG not only lost some genes due to deletions of regions of the chromosome but also underwent gene duplication and other mutations resulting in increased antioxidant production.

Methodology/Principal Findings

To determine whether microbial antioxidants influence vaccine immunogenicity, we eliminated duplicated alleles encoding the oxidative stress sigma factor SigH in BCG Tice and reduced the activity and secretion of iron co-factored superoxide dismutase. We then used assays of gene expression and flow cytometry with intracellular cytokine staining to compare BCG-specific immune responses in mice after vaccination with BCG Tice or the modified BCG vaccine. Compared to BCG, the modified vaccine induced greater IL-12p40, RANTES, and IL-21 mRNA in the spleens of mice at three days post-immunization, more cytokine-producing CD8+ lymphocytes at the peak of the primary immune response, and more IL-2-producing CD4+ lymphocytes during the memory phase. The modified vaccine also induced stronger secondary CD4+ lymphocyte responses and greater clearance of challenge bacilli.

Conclusions/Significance

We conclude that antioxidants produced by BCG suppress host immune responses. These findings challenge the hypothesis that the failure of extensively cultivated BCG vaccines to prevent pulmonary tuberculosis is due to over-attenuation and suggest instead a new model in which BCG evolved to produce more immunity-suppressing antioxidants. By targeting these antioxidants it may be possible to restore BCG''s ability to protect against pulmonary TB.     

Characterization of expressed sequence tag (EST)-derived microsatellite loci in the fire ant Solenopsis invicta (Hymenoptera: Formicidae)

The fire ant Solenopsis invicta has received considerable attention as a damaging invasive pest. In the present study, 12 polymorphic microsatellite loci were developed from publicly available expressed sequence tags (ESTs) of S. invicta. The number of alleles varied between 3 and 12, with an average of 6.42 per locus. The observed and expected heterozygosities ranged from 0.4167 to 1.0000 and from 0.3446 to 0.8543, respectively. These informative EST-derived microsatellite loci provide a powerful complementary tool for genetic studies of S. invicta.     

Expression of the p12 subunit of human DNA polymerase δ (Pol δ), CDK inhibitor p21WAF1, Cdt1, cyclin A,PCNA and Ki-67 in relation to DNA replication in individual cells

We recently reported that the p12 subunit of human DNA polymerase δ (Pol δ4) is degraded by CRL4^Cdt2 which regulates the licensing factor Cdt1 and p21^WAF1 during the G₁ to S transition. Presently, we performed multiparameter laser scanning cytometric analyses of changes in levels of p12, Cdt1 and p21^WAF1, detected immunocytochemically in individual cells, vis-à-vis the initiation and completion of DNA replication. The latter was assessed by pulse-labeling A549 cells with the DNA precursor ethynyl-2′-deoxyribose (EdU). The loss of p12 preceded the initiation of DNA replication and essentially all cells incorporating EdU were p12 negative. Completion of DNA replication and transition to G₂ phase coincided with the re-appearance and rapid rise of p12 levels. Similar to p12 a decline of p21^WAF1 and Cdt1 was seen at the end of G₁ phase and all DNA replicating cells were p21^WAF1 and Cdt1 negative. The loss of p21^WAF1 preceded that of Cdt1 and p12 and the disappearance of the latter coincided with the onset of DNA replication. Loss of p12 leads to conversion of Pol δ4 to its trimeric form, Pol δ3, so that the results provide strong support to the notion that Pol δ3 is engaged in DNA replication during unperturbed progression through the S phase of cell cycle. Also assessed was a correlation between EdU incorporation, likely reflecting the rate of DNA replication in individual cells, and the level of expression of positive biomarkers of replication cyclin A, PCNA and Ki-67 in these cells. Of interest was the observation of stronger correlation between EdU incorporation and expression of PCNA (r = 0.73) than expression of cyclin A (r = 0.47) or Ki-67 (r = 0.47).     

Restoring Savanna Using Fire: Impact on the Breeding Bird Community

Restoration of many terrestrial plant communities involves the reintroduction of fire. However, there have been few studies of the effects of fire on the avifauna during the restoration process. To study the effects of oak savanna restoration on avian communities, breeding birds were censused and the vegetation structure documented in seven experimental burn units (8–18 ha) that had experienced different frequencies of controlled burns during the past 31 years (0–26 burns). Data were analyzed with both direct and indirect gradient analyses using multivariate techniques. The results showed that, as savanna restoration proceeded, there was a general decline in predominantly insectivorous species, particularly those that feed in the upper canopy region (leaves and air space), and a general increase in omnivorous species, particularly those that feed on the ground and in the lower canopy. Insectivorous bark gleaners (woodpeckers) also increased during restoration and were correlated with the increase in standing dead trees resulting from the fires. Overall, savanna restoration resulted in increases in the abundance of many open country bird species, including many species that have been declining in central and eastern North America, including red‐headed woodpecker, Baltimore oriole, eastern kingbird, vesper sparrow, field sparrow, lark sparrow, brown thrasher, American goldfinch, and brown‐headed cowbird. The shifts in species and guilds were correlated with changes in burn frequency and the macro vegetation structure—tree and shrub density, leaf area index, and relative proportion of standing dead trees. The findings show that savanna restoration can increase bird diversity and provide important habitat for uncommon or declining bird species. These birds are most likely attracted to one or more of the distinctive habitat features of the restored savanna environments, including scattered mature trees, standing dead trees and snags, and presence of both shrubby and grassland vegetation. The findings also suggest that restoration ecologists and wildlife biologists will need to work together to achieve desired goals, since different types of savanna restoration efforts may produce different effects on the breeding bird community.     

Application of semi-quantitative electrofishing to juvenile salmonid stock surveys

A semi-quantitative electrofishing technique is described, which has applications for assessing distribution and densities of 0+ salmonids in streams. Linear regressions of logarithmically-transformed data satisfactorily described the relationship between numbers of 0+ Atlantic salmon ( Salmo salar L.) and 0+ brown trout ( Salmo trutta L.) captured in a standard 5 min fishing and quantitative population estimates obtained using a commonly used method. Applications of the semi-quantitative technique to management are discussed and a categorization system proposed.     

Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea (Camellia sinensis L.)

Purine alkaloid catabolism pathways in young, mature and agedleaves of tea (Camellia sinensis L.) were investigated by incubatingleaf sections with ¹⁴C-labelled theobromine, caffeine, theophyllineand xanthine. Incorporation of label into CO₂ was determinedand methanol-soluble metabolites were analysed by high-performanceliquid chromatography-radiocounting and thin layer chro-matography.The data obtained demonstrate that theobromine is the immediateprecursor of caffeine, which accumulates in tea leaves becauseits conversion to theophylline is the rate limiting step inthe purine alkaloid catabolism pathway. The main fate of [8-¹⁴C]theophyllineincubated with mature and aged leaves, and to a lesser extentyoung leaves, is conversion to 3-methylxanthine and onto xanthinewhich is degraded to ¹⁴CO₂ via the purine catabolism pathway.However, with young leaves, sizable amounts of [8-¹⁴C]-theophyllinewere salvaged for the synthesis of caffeine via a 3-methylxanthine     

Endogenous indoles and the biosynthesis and metabolism of indole-3-acetic acid in cultures of Rhizobium phaseoli

Gas chromatography-mass spectrometric analyses of purified extracts from cultures of Rhizobium phaseoli wild-type strain 8002, grown in a non-tryptophan-supplemented liquid medium, demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol (IEt), indole-3-aldehyde and indole-3-methanol (IM). In metabolism studies with ³H-, ¹⁴C- and ²H-labelled substrates the bacterium was shown to convert tryptophan to IEt, IAA and IM; IEt to IAA and IM; and IAA to IM. Indole-3-acetamide (IAAm) could not be detected as either an endogenous constituent or a metabolite of [³H]tryptophan nor did cultures convert [¹⁴C]IAAm to IAA. Biosynthesis of IAA in R. phaseoli, thus, involves a different pathway from that operating in Pseudomonas savastanio and Agrobacterium tumefaciens-induced crown-gall tumours.Abbreviations IAA indole-3-acetic acid - IAld indole-3-aldehyde - IAAm indole-3-acetamide - IEt indole-3-ethanol - IM indole-3-methanol - HPLC-RC high-performance liquid chromatography-radio counting - GC-MS gas chromatography-mass spectrometry