A 5-formyltetrahydrofolate cycloligase paralog from all domains of life: comparative genomic and experimental evidence for a cryptic role in thiamin metabolism

A paralog (here termed COG0212) of the ATP-dependent folate salvage enzyme 5-formyltetrahydrofolate cycloligase (5-FCL) occurs in all domains of life and, although typically annotated as 5-FCL in pro- and eukaryotic genomes, is of unknown function. COG0212 is similar in overall structure to 5-FCL, particularly in the substrate binding region, and has distant similarity to other kinases. The Arabidopsis thaliana COG0212 protein was shown to be targeted to chloroplasts and to be required for embryo viability. Comparative genomic analysis revealed that a high proportion (19%) of archaeal and bacterial COG0212 genes are clustered on the chromosome with various genes implicated in thiamin metabolism or transport but showed no such association between COG0212 and folate metabolism. Consistent with the bioinformatic evidence for a role in thiamin metabolism, ablating COG0212 in the archaeon Haloferax volcanii caused accumulation of thiamin monophosphate. Biochemical and functional complementation tests of several known and hypothetical thiamin-related activities (involving thiamin, its breakdown products, and their phosphates) were, however, negative. Also consistent with the bioinformatic evidence, the COG0212 proteins from A. thaliana and prokaryote sources lacked 5-FCL activity in vitro and did not complement the growth defect or the characteristic 5-formyltetrahydrofolate accumulation of a 5-FCL-deficient (ΔygfA) Escherichia coli strain. We therefore propose (a) that COG0212 has an unrecognized yet sometimes crucial role in thiamin metabolism, most probably in salvage or detoxification, and (b) that is not a 5-FCL and should no longer be so annotated.     

Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration

Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. Here we show that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, we propose that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubule behaviours involved in cell migration.     

Engaging neuroscience to advance translational research in brain barrier biology

The delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by brain barriers, of which the most well known are the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood flow, and--when barrier integrity is impaired--in the pathology of many common CNS disorders such as Alzheimer's disease, Parkinson's disease and stroke.     

Rapid immunomagnetic negative enrichment of neutrophil granulocytes from murine bone marrow for functional studies in vitro and in vivo

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*10⁶ PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.     

Studies on the antibacterial effects of statins--in vitro and in vivo

Background

Statin treatment has been associated with a beneficial outcome on respiratory tract infections. In addition, previous in vitro and in vivo experiments have indicated favorable effects of statins in bacterial infections.

Aim

The aim of the present study was to elucidate possible antibacterial effects of statins against primary pathogens of the respiratory tract.

Methods

MIC-values for simvastatin, fluvastatin and pravastatin against S. pneumoniae, M. catarrhalis and H. influenzae were determined by traditional antibacterial assays. A BioScreen instrument was used to monitor effects of statins on bacterial growth and to assess possible synergistic effects with penicillin. Bacterial growth in whole blood and serum from healthy volunteers before and after a single dose of simvastatin, fluvastatin and penicillin (positive control) was determined using a blood culture system (BactAlert).

Findings

The MIC-value for simvastatin against S pneumoniae and M catarrhalis was 15 µg/mL (36 mmol/L). Fluvastatin and Pravastatin showed no antibacterial effect in concentrations up to 100 µg/mL (230 µmol/L). Statins did not affect growth or viability of H influenzae. Single doses of statins given to healthy volunteers did not affect growth of pneumococci, whereas penicillin efficiently killed all bacteria.

Conclusions

Simvastatin at high concentrations 15 µg/mL (36 µmol/L) rapidly kills S pneumoniae and M catarrhalis. However, these concentrations by far exceed the concentrations detected in human blood during simvastatin therapy (1–15 nmol/L) and single doses of statins given to healthy volunteers did not improve antibacterial effects of whole blood. Thus, a direct bactericidal effect of statins in vivo is probably not the mechanism behind the observed beneficial effect of statins against various infections.     

Replication of extended lifespan phenotype in mice with deletion of insulin receptor substrate 1

We previously reported that global deletion of insulin receptor substrate protein 1 (Irs1) extends lifespan and increases resistance to several age-related pathologies in female mice. However, no effect on lifespan was observed in male Irs1 null mice. We suggested at the time that the lack of any effect in males might have been due to a sample size issue. While such lifespan studies are essential to our understanding of the aging process, they are generally based on survival curves derived from single experiments, primarily due to time and economic constraints. Consequently, the robustness of such findings as a basis for further investigation has been questioned. We have therefore measured lifespan in a second, separate cohort of Irs1 null female mice, and show that, consistent with our previous finding, global deletion of Irs1 significantly extends lifespan in female mice. In addition, an augmented and completed study demonstrates lifespan extension in male Irs1 null mice. Therefore, we show that reduced IRS1-dependent signalling is a robust mechanism through which mammalian lifespan can be modulated.     

Symbiotic effectiveness of rhizobial mutualists varies in interactions with native Australian legume genera

Background and Objectives

Interactions between plants and beneficial soil organisms (e.g. rhizobial bacteria, mycorrhizal fungi) are models for investigating the ecological impacts of such associations in plant communities, and the evolution and maintenance of variation in mutualisms (e.g. host specificity and the level of benefits provided). With relatively few exceptions, variation in symbiotic effectiveness across wild host species is largely unexplored.

Methods

We evaluated these associations using representatives of several legume genera which commonly co-occur in natural ecosystems in south-eastern Australia and an extensive set of rhizobial strains isolated from these hosts. These strains had been previously assigned to specific phylotypes on the basis of molecular analyses. In the first of two inoculation experiments, the growth responses of each host species was evaluated with rhizobial strains isolated from that species. The second experiment assessed performance across genera and the extent of host specificity using a subset of these strains.

Results

While host growth responses to their own (sympatric) isolates varied considerably, rhizobial phylotype was a significant predictor of symbiotic performance, indicating that bacterial species designations on the basis of molecular markers have ecological importance. Hosts responded in qualitatively different ways to sympatric and allopatric strains of rhizobia, ranging from species with a clear preference for their own strains, to those that were broad generalists, through to species that grew significantly better with allopatric strains.

Conclusion

Theory has focused on trade-offs between the provision of benefits and symbiont competitive ability that might explain the persistence of less beneficial strains. However, differences in performance among co-occurring host species could also drive such patterns. Our results thus highlight the likely importance of plant community structure in maintaining variation in symbiotic effectiveness.     

Human papillomavirus infection and anxiety: analyses in women with low-grade cervical cytological abnormalities unaware of their infection status

Background

Women testing positive for human papillomavirus (HPV) infection experience increased levels of anxiety that have been attributed to fears of stigmatization and developing cervical cancer. The objective of this study was to investigate the association between HPV infection and anxiety in women who were unaware they had been tested specifically for HPV, to determine if any anxiety experienced by HPV-positive women could be due to causes other than learning of test results.

Methods

This study was nested within a randomised controlled trial of management of women with abnormal cervical cytology conducted in the United Kingdom with recruitment between 1999 and 2002. At baseline, prior to having a sample taken for HPV testing, the results of which were not disclosed, women were assessed for anxiety using the Hospital Anxiety and Depression Scale and asked about fears of developing cervical cancer (“cancer worries”); this assessment was repeated at 12, 18, 24, and 30 months of follow-up. Logistic regression and generalized estimating equations were used for the cross-sectional (baseline) and longitudinal analyses, respectively.

Results

Among the 2842 participants, there was no association between HPV status and anxiety among white women. Among non-white women, however, anxiety was less common among HPV-positive than HPV-negative women (adjusted odds ratio 0.41, 95% confidence interval 0.22 to 0.77). Among non-smokers, cancer worry was more common in HPV-positive than HPV-negative women; the opposite association was observed among ex-smokers.

Conclusions

Associations between HPV status and anxiety may be explained by factors other than learning of test results and may vary by ethnicity and lifestyle factors.     

Comprehensive analysis of 5-aminolevulinic acid dehydrogenase (ALAD) variants and renal cell carcinoma risk among individuals exposed to lead

Background

Epidemiologic studies are reporting associations between lead exposure and human cancers. A polymorphism in the 5-aminolevulinic acid dehydratase (ALAD) gene affects lead toxicokinetics and may modify the adverse effects of lead.

Methods

The objective of this study was to evaluate single-nucleotide polymorphisms (SNPs) tagging the ALAD region among renal cancer cases and controls to determine whether genetic variation alters the relationship between lead and renal cancer. Occupational exposure to lead and risk of cancer was examined in a case-control study of renal cell carcinoma (RCC). Comprehensive analysis of variation across the ALAD gene was assessed using a tagging SNP approach among 987 cases and 1298 controls. Occupational lead exposure was estimated using questionnaire-based exposure assessment and expert review. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression.

Results

The adjusted risk associated with the ALAD variant rs8177796^CT/TT was increased (OR = 1.35, 95%CI = 1.05–1.73, p-value = 0.02) when compared to the major allele, regardless of lead exposure. Joint effects of lead and ALAD rs2761016 suggest an increased RCC risk for the homozygous wild-type and heterozygous alleles (^GGOR = 2.68, 95%CI = 1.17–6.12, p = 0.01; ^GAOR = 1.79, 95%CI = 1.06–3.04 with an interaction approaching significance (p_int = 0.06).. No significant modification in RCC risk was observed for the functional variant rs1800435^(K68N). Haplotype analysis identified a region associated with risk supporting tagging SNP results.

Conclusion

A common genetic variation in ALAD may alter the risk of RCC overall, and among individuals occupationally exposed to lead. Further work in larger exposed populations is warranted to determine if ALAD modifies RCC risk associated with lead exposure.