Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei

Background

Penicillium marneffei is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37°C), a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in P. marneffei that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of P. marneffei.

Results

Whole cell proteins from the early stages of mould and yeast development in P. marneffei were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated RanA, was subsequently cloned and characterized. The P. marneffei RanA protein sequence, which contained the signature motif of Ran-GTPases, exhibited 90% homology to homologous Aspergillus proteins.

Conclusion

This study clearly demonstrates the utility of proteomic approaches to studying dimorphism in P. marneffei. Moreover, this strategy complements and extends current genetic methodologies directed towards understanding the molecular mechanisms of phase transition. Finally, the documented increased levels of RanA expression suggest that cellular development in this fungus involves additional signaling mechanisms than have been previously described in P. marneffei.     

Embryonic rotational behaviour in the pond snail Lymnaea stagnalis: influences of environmental oxygen and development stage

Responses of freshwater organisms to environmental oxygen tensions (PO₂) have focused on adult (i.e. late developmental) stages, yet responses of embryonic stages to changes in environmental PO₂ must also have implications for organismal biology. Here we assess how the rotational behaviour of the freshwater snail Lymnaea stagnalis changes during development in response to conditions of hypoxia and hyperoxia. As rotation rate is linked to gas mixing in the fluid surrounding the embryo, we predicted that it would increase under hypoxic conditions but decrease under hyperoxia. Contrary to predictions, early, veliger stage embryos showed no change in their rotation rate under hyperoxia, and later, hippo stage embryos showed only a marginally significant increase in rotation under these conditions. Predictions for hypoxia were broadly supported, however, with both veliger and hippo stages showing a marked hypoxia-related increase in their rotation rates. There were also subtle differences between developmental stages, with hippos responding at PO₂s (50% air saturation) greater than those required to elicit a similar response in veligers (20% air saturation). Differences between developmental stages also occurred on return to normoxic conditions following hypoxia: rotation in veligers returned to pre-exposure levels, whereas there was a virtual cessation in embryos at the hippo stage, likely the result of overstimulation of oxygen sensors driving ciliary movement in later, more developed embryos. Together, these findings suggest that the spinning activity of L. stagnalis embryos varies depending on environmental PO₂s and developmental stage, increasing during hypoxia to mix capsular contents and maintain a diffusive gradient for oxygen entry into the capsule from the external environment (“stir-bar” theory of embryonic rotational behaviour).     

Proof-of-concept human laboratory study for protracted abstinence in alcohol dependence: effects of gabapentin

There is a need for safe medications that can effectively support recovery by treating symptoms of protracted abstinence that may precipitate relapse in alcoholics, e.g. craving and disturbances in sleep and mood. This proof-of-concept study reports on the effectiveness of gabapentin 1200 mg for attenuating these symptoms in a non-treatment-seeking sample of cue-reactive, alcohol-dependent individuals. Subjects were 33 paid volunteers with current Diagnostic and Statistical Manual of Mental Disorders-IV alcohol dependence and a strength of craving rating 1 SD or greater for alcohol than water cues. Subjects were randomly assigned to gabapentin or placebo for 1 week and then participated in a within-subjects trial where each was exposed to standardized sets of pleasant, neutral and unpleasant visual stimuli followed by alcohol or water cues. Gabapentin was associated with significantly greater reductions than placebo on several measures of subjective craving for alcohol as well as for affectively evoked craving. Gabapentin was also associated with significant improvement on several measures of sleep quality. Side effects were minimal, and gabapentin effects were not found to resemble any major classes of abused drugs. Results suggest that gabapentin may be effective for treating the protracted abstinence phase in alcohol dependence and that a randomized clinical trial would be an appropriate next step. The study also suggests the value of cue-reactivity studies as proof-of-concept screens for potential antirelapse drugs.     

Extremely short duration high intensity interval training substantially improves insulin action in young healthy males

Background

Impaired glucose tolerance (IGT) is a prediabetic state. If IGT can be prevented from progressing to overt diabetes, hyperglycemia-related complications can be avoided. The purpose of the present study was to examine whether pioglitazone (ACTOS^®) can prevent progression of IGT to type 2 diabetes mellitus (T2DM) in a prospective randomized, double blind, placebo controlled trial.

Methods/Design

602 IGT subjects were identified with OGTT (2-hour plasma glucose = 140–199 mg/dl). In addition, IGT subjects were required to have FPG = 95–125 mg/dl and at least one other high risk characteristic. Prior to randomization all subjects had measurement of ankle-arm blood pressure, systolic/diastolic blood pressure, HbA_1C, lipid profile and a subset had frequently sampled intravenous glucose tolerance test (FSIVGTT), DEXA, and ultrasound determination of carotid intima-media thickness (IMT). Following this, subjects were randomized to receive pioglitazone (45 mg/day) or placebo, and returned every 2–3 months for FPG determination and annually for OGTT. Repeat carotid IMT measurement was performed at 18 months and study end. Recruitment took place over 24 months, and subjects were followed for an additional 24 months. At study end (48 months) or at time of diagnosis of diabetes the OGTT, FSIVGTT, DEXA, carotid IMT, and all other measurements were repeated. Primary endpoint is conversion of IGT to T2DM based upon FPG ≥ 126 or 2-hour PG ≥ 200 mg/dl. Secondary endpoints include whether pioglitazone can: (i) improve glycemic control (ii) enhance insulin sensitivity, (iii) augment beta cell function, (iv) improve risk factors for cardiovascular disease, (v) cause regression/slow progression of carotid IMT, (vi) revert newly diagnosed diabetes to normal glucose tolerance.

Conclusion

ACT NOW is designed to determine if pioglitazone can prevent/delay progression to diabetes in high risk IGT subjects, and to define the mechanisms (improved insulin sensitivity and/or enhanced beta cell function) via which pioglitazone exerts its beneficial effect on glucose metabolism to prevent/delay onset of T2DM.

Trial Registration

clinical trials.gov identifier: NCT00220961     

The products of the herpes simplex virus type 1 immediate-early US1/US1.5 genes downregulate levels of S-phase-specific cyclins and facilitate virus replication in S-phase Vero cells

Herpes simplex virus type 1 ICP22-/U(S)1.5- mutants initiate viral gene expression in all cells; however, in most cell types, the replication process stalls due to an inability to express gamma2 late proteins. Although the function of ICP22/U(S)1.5 has not been established, it has been suggested that these proteins activate, induce, or repress the activity of cellular proteins during infection. In this study, we hypothesized that cell cycle-associated proteins are targets of ICP22/U(S)1.5. For this purpose, we first isolated and characterized an ICP22-/U(S)1.5- mutant virus, 22/n199. Like other ICP22-/U(S)1.5- mutants, 22/n199 replicates in a cell-type-specific manner and fails to induce efficient gamma2 late gene expression in restrictive cells. Although synchronization of restrictive human embryonic lung cells in each phase of the cell cycle did not overcome the growth restrictions of 22/n199, synchronization of permissive Vero cells in S phase rendered them less able to support 22/n199 plaque formation and replication. Consistent with this finding, expression of cellular S-phase cyclins was altered in an ICP22/U(S)1.5-dependent manner specifically when S-phase Vero cells were infected. Collectively, these observations support the notion that ICP22/U(S)1.5 deregulates the cell cycle upon infection of S-phase permissive cells by altering expression of key cell cycle regulatory proteins either directly or indirectly.     

Gene flow and range expansion in a mountain‐dwelling passerine with a fragmented distribution

We studied gene flow and bottleneck events in the population history of locally isolated citril finches endemic to European mountains. For the present study, we used two genetic markers with different rates of evolution: a fast evolving mitochondrial marker (ATPase6/8) and a more slowly evolving nuclear marker (02401). Populations north of the Pyrenees showed in general fewer haplotypes and a considerable lower nucleotide and gene diversity than the Iberian populations. Unexpectedly, we found very little genetic variability in the fast evolving mitochondrial marker, arguing for a strong and relatively recent bottleneck event in the species population history. This pattern potentially reflects a sudden decrease of crucial resources during Mid‐Holocene (mountain pine, Scots pine, and black pine) and a subsequent breakdown of the population. The bottleneck could also have been caused or coincide with a selective sweep in the mitochondrion. By contrast, the slowly evolving nuclear marker showed a much higher variability. This marker probably reflects major gene flow along a potential expansion pathway from the Eastern Pyrenees, northwards to the populations of Central Europe, and southwards to the more fragmented populations of central and southern Spain. The population of the Western Pyrenees (Navarra) appears to be cut‐off from this major gene flow and our data indicate a certain degree of partial isolation, probably reflecting more ancient events (e.g. the separation in distinct refuge sites during the last glacial maximum). © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 707–721.