Characterisation of the Candida albicans Phosphopantetheinyl Transferase Ppt2 as a Potential Antifungal Drug Target

Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds.     

Glucose and pyruvate metabolism in severe chronic obstructive pulmonary disease

The mechanisms leading to weight loss in patients with chronic obstructive pulmonary disease (COPD) are poorly understood but may involve alterations in macronutrient metabolism. Changes in muscle oxidative capacity and lactate production during exercise suggest glucose metabolism may be altered in COPD subjects. The objective of this study was to determine differences in the rates of glucose production and clearance, the rate of glycolysis (pyruvate production), and oxidative and nonoxidative pyruvate disposal in subjects with severe COPD compared with healthy controls. The in vivo rates of glucose production and clearance were measured in 14 stable outpatients with severe COPD (seven with low and seven with preserved body mass indexes) and 7 healthy controls using an intravenous infusion of [(2)H(2)]glucose. Additionally, pyruvate production and oxidative and non-oxidative pyruvate disposal were measured using intravenous infusions of [(13)C]bicarbonate and [(13)C]pyruvate. Endogenous glucose flux and glucose clearance were significantly faster in the combined COPD subjects (P = 0.002 and P < 0.001, respectively). This difference remained significant when COPD subjects were separated by body mass index. Pyruvate flux and oxidation were significantly higher in the combined COPD subjects than controls (P = 0.02 for both), but there was no difference in nonoxidative pyruvate disposal or plasma lactate concentrations between the two groups. In subjects with severe COPD, there are alterations in glucose metabolism leading to increased glucose production and faster glucose metabolism by glycolysis and oxidation compared with controls. However, no difference in glucose conversion to lactate via pyruvate reduction is observed.     

An NCAM Mimetic, FGL, Alters Hippocampal Cellular Morphometry in Young Adult (4 Month-Old) Rats

The neural cell adhesion molecule, NCAM, is ubiquitously expressed within the CNS and has roles in development, cognition, neural plasticity and regulation of the immune system. NCAM is thus potentially an important pharmacological target for treatment of brain diseases. A cell adhesion mimetic FGL, a 15 amino-acid peptide derived from the second fibronectin type-III module of NCAM, has been shown to act as a neuroprotective agent in experimental disease and ageing models, restoring hippocampal/cognitive function and markedly alleviating deleterious changes in the CNS. However, the effects of FGL on the hippocampus of young healthy rats are unknown. The present study has examined the cellular neurobiological consequences of subcutaneous injections of FGL, on hippocampal cell morphometry in young (4 month-old) rats. We determined the effects of FGL on hippocampal volume, pyramidal neuron number/density (using unbiased quantitative stereology), and examined aspects of neurogenesis (using 2D morphometric analyses). FGL treatment reduced total volume of the dorsal hippocampus (associated with a decrease in total pyramidal neuron numbers in CA1 and CA3), and elevated the number of doublecortin immunolabeled neurons in the dentate gyrus, indicating a likely influence on neurogenesis in young healthy rats. These data indicate that FGL has a specific age dependent effect on the hippocampus, differing according to the development and maturity of the CNS.     

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.     

Analysis of fimbrial gene clusters and their expression in enterohaemorrhagic Escherichia coli O157:H7

The sequence of two enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains reveals the possession of at least 16 fimbrial gene clusters, many of the chaperone/usher class. The first part of this study examined the distribution of these clusters in a selection of EHEC/EPEC (enteropathogenic E. coli) serotypes to determine if any were likely to be unique to E. coli O157:H7. Six of the clusters, as determined by the presence of amplified main subunit or usher gene sequences, were detected only in the E. coli O157 and O145 serotypes tested. With the exception of one serotype O103 strain that contained an lpf2 cluster, lpf sequences were only detected in E. coli O157 of the serotypes tested. Expression from each cluster was measured by the construction of chromosomally integrated lacZ promoter fusions and plasmid-based eGFP fusions in E. coli O157:H7. This analysis demonstrated that the majority (11/15) of main fimbrial subunit genes were not expressed under the majority of conditions tested in vitro. One of the clusters showing promoter activity, loc8, has a temperature expression optimum indicating a possible role outside the host. From the presence of pseudogenes in three of the clusters, the lack of FimH-like minor adhesins in the clusters and their limited expression in vitro, it would appear that E. coli O157:H7 has a limited repertoire of expressed functional fimbriae. This restricted selection of fimbriae may be important in bringing about the tropism E. coli O157:H7 demonstrates for the terminal rectum of cattle.