Patricia Lanthier

Proteomic Analysis of Campylobacter jejuni 11168 Biofilms Reveals a Role for the Motility Complex in Biofilm Formation

Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in developed countries, and yet little is known concerning the mechanisms by which this fastidious organism survives within its environment. We have demonstrated that C. jejuni 11168 can form biofilms on a variety of surfaces. Proteomic analyses of planktonic and biofilm-grown cells demonstrated differences in protein expression profiles between the two growth modes. Proteins involved in the motility complex, including the flagellins (FlaA, FlaB), the filament cap (FliD), the basal body (FlgG, FlgG2), and the chemotactic protein (CheA), all exhibited higher levels of expression in biofilms than found in stationary-phase planktonic cells. Additional proteins with enhanced expression included those involved in the general (GroEL, GroES) and oxidative (Tpx, Ahp) stress responses, two known adhesins (Peb1, FlaC), and proteins involved in biosynthesis, energy generation, and catabolic functions. An aflagellate flhA mutant not only lost the ability to attach to a solid matrix and form a biofilm but could no longer form a pellicle at the air-liquid interface of a liquid culture. Insertional inactivation of genes that affect the flagellar filament (fliA, flaA, flaB, flaG) or the expression of the cell adhesin (flaC) also resulted in a delay in pellicle formation. These findings demonstrate that the flagellar motility complex plays a crucial role in the initial attachment of C. jejuni 11168 to solid surfaces during biofilm formation as well as in the cell-to-cell interactions required for pellicle formation. Continued expression of the motility complex in mature biofilms is unusual and suggests a role for the flagellar apparatus in the biofilm phenotype.

Differentiation of mouse Neuro 2A cells into dopamine neurons.

Neuro 2A (N2a) is a mouse neural crest-derived cell line that has been extensively used to study neuronal differentiation, axonal growth and signaling pathways. A convenient characteristic of these cells is their ability to differentiate into neurons within a few days. However, most differentiation methods reported for N2a cells do not provide information about the neuronal types obtained after each treatment. In this study, we evaluated the generation of N2a dopamine neurons following treatment with a number of factors known to induce neuronal differentiation. Our results showed that N2a cells express Nurr-related factor 1 (Nurr1) and produce low levels of tyrosine hydroxylase (TH) and dopamine. Both TH and dopamine levels were significantly enhanced in the presence of dibutyryl cyclic adenosine monophosphate (dbcAMP), as evidenced by Western blot, immunocytochemistry and high performance liquid chromatography (HPLC). In contrast to dbcAMP, other factors such as transforming growth factor beta1 (TGF beta 1), bone morphogenetic protein 4 (BMP4), glial cell-derived neurotrophic factor (GDNF) and retinoic acid (RA) did not increase TH expression. Further investigation confirmed that the effect of dbcAMP on production of TH-positive neurons was mediated through cyclic AMP (cAMP) responsive element binding protein (CREB) and it was antagonized by RA. Thus, although various treatments can be used to generate N2a neurons, only dbcAMP significantly enhanced the formation of dopamine neurons. Taken together, this study provided a simple and reliable method to generate dopamine neurons for rapid and efficient physiological and pharmacological assays.

MALDI mass spectrometry imaging of gangliosides in mouse brain using ionic liquid matrix.

Mass spectrometry imaging has emerged as a powerful tool for the direct detection of biomolecules, mainly phospholipids, proteins and peptides, in tissue samples. To date, there is very little information available on the direct analysis of gangliosides in brain tissue. One major hurdle for imaging gangliosides in tissue using mass spectrometry is that sialic acid residues can be dissociated in ionization process. In this report, we investigated an ionic liquid matrix for mass spectrometry imaging of gangliosides. This ionic liquid matrix offered excellent sensitivity for detection gangliosides without significant loss of sialic acid residues. Thus, it can be used to study the abundance and anatomical localization of gangliosides in mouse brain using mass spectrometry imaging technique. Mass spectrometry image analyses of the mouse brain tissue sections demonstrated that the N-fatty acyl chains of gangliosides were differentially distributed in mouse hippocampal regions, whereby the gangliosides with N-C(18) acyl chain were enriched in CA1 region, while gangliosides with N-C(20) acyl chain were enriched in dentate gyrus. In addition, this observation is true for mono-, di- and tri-sialylated gangliosides. Although the linkage information was not determined, the mass spectrometry imaging technique was capable of spatial tissue mapping of ceramide structures in gangliosides.

Astrocyte-secreted GDNF and glutathione antioxidant system protect neurons against 6OHDA cytotoxicity

In recent years, GDNF has emerged as a protective and restorative agent in several models of neurodegeneration; however, the exact molecular mechanisms responsible for these effects are not yet fully understood. Here we examined the effects of astrocytes secreting GDNF on neurons subjected to 6OHDA toxicity using in vitro neuron-astroglia co-cultures. Astrocytes were transduced with lentiviral vectors carrying the GDNF gene under the control of either human glial fibrillary acidic protein or cytomegalovirus promoters. The overexpression of GDNF, regardless of the promoter employed, had no obvious adverse effects on astroglia and the engineered cells stably produced and secreted GDNF for extended periods of time (> or =3 weeks). These astrocytes very effectively protected neurons against 6OHDA, in both mouse and human co-culture systems. The neuroprotective effects were mediated not only by GDNF, but also by the antioxidant GSH since its depletion reduced the level of GDNF protection. Furthermore, neurons and astrocytes expressed different components of GDNF signaling complex, suggesting that they might utilize separate pathways to mediate autocrine and paracrine effects of GDNF.

Induction and stability of alcohol oxidase in the methylotrophic yeast Pichia pastoris

SummaryAlcohol oxidase biosynthesis was induced when Pichia pastoris was grown in a medium containing methanol as the sole carbon and energy source. Specific activity was highest during the logarithmic phase of growth (1.22 g acetaldehyde produced/g cell dry wt. per hour), and declined steadily thereafter. The addition of 0.1% (w/v) yeast extract to the methanol growth medium promoted higher biomass production, increased alcohol oxidase specific activity, and contributed to increased enzyme stability under use conditions. When P. pastoris was used for wholecell bioconversions, 30.2 g of ethanol were oxidized to 28 g acetaldehyde in 12 h, at a carbon recovery of 97%. Acetaldehyde concentrations in excess of 1 M were achieved when the concentration of the TRIS buffer, used to chemically trap the acetaldehyde, was increased to 1 M.

IDENTIFICATION OF ATAXIA-ASSOCIATED mtDNA MUTATIONS (m.4052T>C and m.9035T>C) AND EVALUATION OF THEIR PATHOGENICITY IN TRANSMITOCHONDRIAL CYBRIDS

The potential pathogenicity of two homoplasmic mtDNA point mutations, 9035T>C and 4452T>C, found in a family afflicted with maternally transmitted cognitive developmental delay, learning disability, and progressive ataxia was evaluated using transmitochondrial cybrids. We confirmed that the 4452T>C transition in tRNAMet represented a polymorphism; however, 9035T>C conversion in the ATP6 gene was responsible for a defective F0‐ATPase. Accordingly, mutant cybrids had a reduced oligomycin‐sensitive ATP hydrolyzing activity. They had less than half of the steady‐state content of ATP and nearly an 8‐fold higher basal level of reactive oxygen species (ROS). Mutant cybrids were unable to cope with additional insults, i.e., glucose deprivation or tertiary‐butyl hydroperoxide, and they succumbed to either apoptotic or necrotic cell death. Both of these outcomes were prevented by the antioxidants CoQ10 and vitamin E, suggesting that the abnormally high levels of ROS were the triggers of cell death. In conclusion, the principal metabolic defects, i.e., energy deficiency and ROS burden, resulted from the 9035T>C mutation and could be responsible for the development of clinical symptoms in this family. Furthermore, antioxidant therapy might prove helpful in the management of this disease. Muscle Nerve, 2009

Biological upgrading of dilute ethanol streams to acetaldehyde for low-temperature recovery☆

Abstract A whole-cell process, using the highly active alcohol oxidase enzyme system of methylotrophic yeasts, was developed for the biological upgrading of dilute ethanol streams to the more valuable, more easily recoverable product, acetaldehyde. Of five methylotrophic yeasts, Pichia pastoris exhibited the highest level of oxidative activity (1·22 g acetaldehyde g cells dry wt −1 h −1 ). Under optimized conditions, 95% of a dilute 18 g l −1 ethanol solution was oxidized to acetaldehyde at 100% of theoretical yield. The alcohol oxidase enzyme system of P. pastoris was psychrotolerant, and showed only a 32·5% decrease in activity when the bioconversion temperature was lowered from 30 to 3°C. Under practical applied conditions, dilute ethanol solutions ranging from 0·5 to 3·0% (w/v) were converted to acetaldehyde at process efficiencies of 73 to 61%, respectively. Acetaldehyde readily evaporated from reaction media at 22°C, offering an attractive alternative to the expense of ethanol distillation.