We used ultra-high field MRI to visualize cortical lesion types described by neuropathology in 16 patients with multiple sclerosis (MS) compared with 8 age-matched controls; to characterize the ...contrast properties of cortical lesions including T2*, T2, T1, and phase images; and to investigate the relationship between cortical lesion types and clinical data.
We collected, on a 7-T scanner, 2-dimensional fast low-angle shot (FLASH)-T2*-weighted spoiled gradient-echo, T2-weighted turbo spin-echo (TSE) images (0.33 x 033 x 1 mm(3)), and a 3-dimensional magnetization-prepared rapid gradient echo.
Overall, 199 cortical lesions were detected in patients on both FLASH-T2* and T2-TSE scans. Seven-tesla MRI allowed for characterization of cortical plaques into type I (leukocortical), type II (intracortical), and type III/IV (subpial extending partly or completely through the cortical width) lesions as described histopathologically. Types III and IV were the most frequent type of cortical plaques (50.2%), followed by type I (36.2%) and type II (13.6%) lesions. Each lesion type was more frequent in secondary progressive than in relapsing-remitting MS. This difference, however, was significant only for type III/IV lesions. T2*-weighted images showed the highest, while phase images showed the lowest, contrast-to-noise ratio for all cortical lesion types. In patients, the number of type III/IV lesions was associated with greater disability (p < 0.02 by Spearman test) and older age (p < 0.04 by Spearman test).
Seven-tesla MRI detected different histologic cortical lesion types in our small multiple sclerosis (MS) sample, suggesting, if validated in a larger population, that it may prove a valuable tool to assess the contribution of cortical MS pathology to clinical disability.
Cortical subpial demyelination is frequent in multiple sclerosis (MS) and is closely associated with disease progression and poor neurological outcome. Although cortical lesions have been difficult ...to detect using conventional MRI, preliminary data using T2*-weighted imaging at ultra-high field 7T MRI showed improved sensitivity for detecting and categorizing different histological types of cortical MS lesions. In this study we combined high-resolution 7T MRI with a surface-based analysis technique to quantify and map subpial T2*-weighted signal changes in seventeen patients with MS. We applied a robust method to register 7T data with the reconstructed cortical surface of each individual and used a general linear model to assess in vivo an increase in subpial T2*-weighted signal in patients versus age-matched controls, and to investigate the spatial distribution of cortical subpial changes across the cortical ribbon. We also assessed the relationship between subpial T2* signal changes at 7T, Expanded Disability Status Scale (EDSS) score and white matter lesion load (WMLL). Patients with MS showed significant T2*-weighted signal increase in the frontal lobes (parsopercularis, precentral gyrus, middle and superior frontal gyrus, orbitofrontal cortex), anterior cingulate, temporal (superior, middle and inferior temporal gyri), and parietal cortices (superior and inferior parietal cortex, precuneus), but also in occipital regions of the left hemisphere. We found significant correlations between subpial T2*-weighted signal and EDSS score in the precentral gyrus (ρ=0.56, P=0.02) and between T2*-weighted signal and WMLL in the lateral orbitofrontal, superior parietal, cuneus, precentral and superior frontal regions. Our data support the presence of disseminated subpial increases in T2* signal in subjects with MS, which may reflect the diffuse subpial pathology described in neuropathology.
The
IPC1 gene from
Saccharomyces cerevisiae, which encodes inositolphosphorylceramide (IPC) synthase, was first identified as a novel and essential gene encoding resistance to the natural product ...antifungal aureobasidin A (
AUR1). The formation of IPC in fungi is essential for viability, suggesting inhibitors of IPC1p function would make ideal antifungal drug candidates. Homologs of the
AUR1/IPC1 gene were identified from a number of human pathogenic fungi,
Candida glabrata,
Candida krusei,
Candida parapsilosis,
Candida tropicalis and
Cryptococcus neoformans. Comparison of these genes with other homologous genes from
Candida albicans,
Aspergillus fumigatus,
Aspergillus nidulans,
Saccharomyces cerevisiae and
Schizosaccharomyces pombe reveals a conserved structural motif for inositolphosphoryl transferases which is similar to a motif recently described for lipid phosphatases, but with unique characteristics.
Protein phosphorylation is a primary form of information transfer in cell signaling pathways and plays a crucial role in regulating biological responses. Aberrant phosphorylation has been implicated ...in a number of diseases, and kinases and phosphatases, the cellular enzymes that control dynamic phosphorylation events, present attractive therapeutic targets. However, the innate complexity of signaling networks has presented many challenges to therapeutic target selection and successful drug development. Approaches in phosphoproteomics can contribute functional, systems-level datasets across signaling networks that can provide insight into suitable drug targets, more broadly profile compound activities, and identify key biomarkers to assess clinical outcomes. Advances in MS-based phosphoproteomics efforts now provide the ability to quantitate phosphorylation with throughput and sensitivity to sample a significant portion of the phosphoproteome in clinically relevant systems. This review will discuss recent work and examples of application data that demonstrate the utility of MS, with a particular focus on the use of quantitative phosphoproteomics and phosphotyrosine-directed signaling analyses to provide robust measurement for functional biological interpretation of drug action on signaling and phenotypic outcomes.
The echinocandins are a family of cyclic lipopeptides with potent antifungal activity. These compounds inhibit the synthesis of BETA-1,3-glucan in fungi. The new semisynthetic echinocandin LY303366 ...was derivatized to produce a photoactivatable cross-linking echinocandin analog with antifungal activity. This analog was radioiodinated and used as a probe in microsomal membrane preparations of Candida albicans which contain glucan synthase activity. The photoaffinity probe identified two major proteins of 40 and 18 kDa in both membrane preparations. Labeling of these proteins was specific in that it required irradiation with UV light and was effectively competed against with unlabeled echinocandin analogs. In addition, the abilities of echinocandin analogs to compete with the photoaffinity probe correlated to their relative antifungal potencies and glucan synthase inhibition. The 40-kDa protein was isolated, and partial sequences were obtained from internal peptide fragments of the protein. Analysis of the sequences of these internal peptides of the 40-kDa protein revealed that it was a new protein not previously described as being involved in glucan synthesis or the mode of action of echinocandins.
The Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides. Although closely related, experimental ...evidence suggests that the four Class I PI3Ks may be functionally distinct. To further study their unique biochemical properties, the three human Class Ia PI3K (α, β, and δ) p110 catalytic domains were cloned and co-expressed with the p85α regulatory domain in Sf9 cells. None of the p110 subunits were successfully expressed in the absence of p85α. Successful expression and purification of each p85α/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells. Proteins were purified as the p85α/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient. The purification yields were high using the optimized ratio of p85/p110 vector and small culture volumes, with 24
mg/L cell culture media for p85α/p110α, 17.5
mg/L for p85α/p110δ, and 3.5
mg/L for p85α/p110β. The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting. The activities of the three p85α/p110 proteins and the Class Ib p110γ catalytic domain were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/phosphatidylserine (PS) liposome. All four enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2. The surface concentrations that gave maximal activity for each human isoform with 0.5
mM PIP2 were 2.5
mol% PIP2 for p110γ, 7.5
mol% for p85α/p110β, and 10
mol% PIP2 for p85α/p110α and p85α/p110δ. The specific activity of p85α/p110α was three to five times higher than that of the other human isoforms. These kinetic differences may contribute to the unique roles of these isoforms in cells.
Cortical subpial demyelination is frequent in multiple sclerosis (MS) and is closely associated with disease progression and poor neurological outcome. Although cortical lesions have been difficult ...to detect using conventional MRI, preliminary data using T2*-weighted imaging at ultra-high field 7T MRI showed improved sensitivity for detecting and categorizing different histological types of cortical MS lesions. In this study we combined high-resolution 7T MRI with a surface-based analysis technique to quantify and map subpial T2*-weighted signal changes in seventeen patients with MS. We applied a robust method to register 7T data with the reconstructed cortical surface of each individual and used a general linear model to assess in vivo an increase in subpial T2*-weighted signal in patients versus age-matched controls, and to investigate the spatial distribution of cortical subpial changes across the cortical ribbon. We also assessed the relationship between subpial T2* signal changes at 7T, Expanded Disability Status Scale (EDSS) score and white matter lesion load (WMLL). Patients with MS showed significant T2*-weighted signal increase in the frontal lobes (parsopercularis, precentral gyrus, middle and superior frontal gyrus, orbitofrontal cortex), anterior cingulate, temporal (superior, middle and inferior temporal gyri), and parietal cortices (superior and inferior parietal cortex, precuneus), but also in occipital regions of the left hemisphere. We found significant correlations between subpial T2*-weighted signal and EDSS score in the precentral gyrus (ρ=0.56, P=0.02) and between T2*-weighted signal and WMLL in the lateral orbitofrontal, superior parietal, cuneus, precentral and superior frontal regions. Our data support the presence of disseminated subpial increases in T2* signal in subjects with MS, which may reflect the diffuse subpial pathology described in neuropathology.
► In vivo study of disseminated subpial demyelination in MS using 7T MRI and surface-based analysis. ► T2*-weighted signal increase in the cortex of MS patients versus controls. ► Preferential distribution of demyelinating lesions in specific areas of the brain. ► T2*-weighted signal correlates with EDSS in precentral gyrus.
Syringomycin E is an antifungal cyclic lipodepsinonapeptide produced by
Pseudomonas syringae pv.
syringae. To understand the mechanism of action of syringomycin E, a novel resistant
Saccharomyces ...cerevisiae strain, BW7, was isolated and characterized. Lipid analyses revealed that BW7 contained only the hydrophobic subspecies of sphingolipids that are normally minor components in wild type strains. This aberrant sphingolipid composition was the result of lack of α-hydroxylation of the amide-linked very long chain fatty acids, suggesting a defective sphingolipid α-hydroxylase encoded by the
FAH1 gene. A yeast strain that lacks the
FAH1 gene was resistant to syringomycin E, and failed to complement BW7. These results demonstrate that BW7 carries a mutation in the
FAH1 gene, and that the lack of α-hydroxylated very long chain fatty acids in yeast sphingolipids confers resistance to syringomycin E.
PDF
