The optimization of protocols for proteome difference gel electrophoresis (DiGE) analysis of preneoplastic skin

Difference gel electrophoresis (DiGE) allows the reliable comparison of proteome differences between two or three samples within a single gel, by way of a CyDye fluorescent labeling system. This facilitates identification of protein differences avoiding the difficulties associated with gel-to-gel variation. A drawback of this approach is the necessity for high-purity protein samples, since contaminants can interfere with the labeling process, affecting subsequent analysis. Thus far, DiGE has been applied to the study of various sample types derived from relatively simple starting materials such as serum, cell lines, or primary cells. Herein, we describe optimization of protein extraction and purification from a complex tissue (the murine ear) of which a major component is skin, which is compatible with the CyDye labeling system and DiGE. Protein samples obtained by this method from preneoplastic, transgenic tissue have been effectively compared to normal tissue samples to reveal bona fide differences, verifiable by Western blotting. In total, 41 protein differences (21 up- and 20 down-regulated in the pathological samples) were identified by mass spectrometry (MS). This method can therefore form a guide for those wishing to perform DiGE on complex tissues, and is especially useful for samples with relatively insoluble components such as skin.     

Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells

We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ-producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100(-/-) L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100(-/-) leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100(-/-) exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.     

GAPDH mediates nitrosylation of nuclear proteins

S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS--neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)--are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO-GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.     

Biosynthesis of the xanthophyll plectaniaxanthin as a stress response in the red yeast Dioszegia (Tremellales, Heterobasidiomycetes, Fungi)

Carotenoid biosynthesis was examined in a phylloplane yeast identified by ITS, 18S and 28S rDNA analysis as a Dioszegia sp. close to D. takashimae. In well-aerated flask or fermentor cultures, this strain produced essentially a single pigment confirmed as the xanthophyll plectaniaxanthin by NMR analysis, at concentrations of 103-175 microgg(-1) biomass dry weight. Detailed studies showed increases in plectaniaxanthin concentrations in the presence of 5 mM hydrogen peroxide (1.8-fold), 50 and 100 microM duroquinone (3.1- and 3.7-fold, respectively), and 2% ethanol (4.9-fold). Whereas oxidative stress is known to enhance the biosynthesis of torularhodin or astaxanthin in other red yeasts where they are associated with an antioxidant function, this is the first report implicating plectaniaxanthin in a similar role. At reduced aeration, biosynthesis of plectaniaxanthin was suppressed and its putative precursor gamma-carotene accumulated. The carotenoid cyclase inhibitor nicotine (5-20 mM) inhibited plectaniaxanthin formation, with lycopene accumulating stoichiometrically. Hydroxy groups at C-1' and C-2' therefore seem to be introduced late in plectaniaxanthin biosynthesis, following cyclization of the beta-ionone ring.     

Levothyroxine monotherapy cannot guarantee euthyroidism in all athyreotic patients

Context

Levothyroxine monotherapy is the treatment of choice for hypothyroid patients because peripheral T4 to T3 conversion is believed to account for the overall tissue requirement for thyroid hormones. However, there are indirect evidences that this may not be the case in all patients.

Objective

To evaluate in a large series of athyreotic patients whether levothyroxine monotherapy can normalize serum thyroid hormones and thyroid-pituitary feedback.

Design

Retrospective study.

Setting

Academic hospital.

Patients

1,811 athyreotic patients with normal TSH levels under levothyroxine monotherapy and 3,875 euthyroid controls.

Measurements

TSH, FT4 and FT3 concentrations by immunoassays.

Results

FT4 levels were significantly higher and FT3 levels were significantly lower (p<0.001 in both cases) in levothyroxine-treated athyreotic patients than in matched euthyroid controls. Among the levothyroxine-treated patients 15.2% had lower serum FT3 and 7.2% had higher serum FT4 compared to euthyroid controls. A wide range of FT3/FT4 ratios indicated a major heterogeneity in the peripheral T3 production capacity in different individuals. The correlation between thyroid hormones and serum TSH levels indicated an abnormal feedback mechanism in levothyroxine-treated patients.

Conclusions

Athyreotic patients have a highly heterogeneous T3 production capacity from orally administered levothyroxine. More than 20% of these patients, despite normal TSH levels, do not maintain FT3 or FT4 values in the reference range, reflecting the inadequacy of peripheral deiodination to compensate for the absent T3 secretion. The long-term effects of chronic tissue exposure to abnormal T3/T4 ratio are unknown but a sensitive marker of target organ response to thyroid hormones (serum TSH) suggests that this condition causes an abnormal pituitary response. A more physiological treatment than levothyroxine monotherapy may be required in some hypothyroid patients.     

Characterization of diet induced aortic atherosclerosis in Syrian F1B hamsters

We characterized atherosclerotic lesions in Syrian F₁B hamsters fed a diet high in saturated fat and cholesterol. Total cholesterol, non-high-density lipoprotein cholesterol, and triglycerides were significantly higher for treated animals than for low fat controls. After 4, 12, 18, 26, 32 and 44 weeks on either diet, the vasculature was fixed in situ and the aortic arch prepared for light and electron microscopy and immunohistochemistry. Fatty streak lesions comprised of foam cells were noted at 4 weeks along the inner curvature of the aortic arch. Fibromuscular lesions became evident at 26 weeks with excess connective tissue and a thickened media. Lesion size increased as foam cells accumulated in the subendothelial space and collagen was deposited in the upper media beneath an intact internal elastic lamina. By 44 weeks an advanced lesion had developed that consisted of a smooth muscle and extracellular matrix cap with an intact endothelium over a lipid rich core. The core consisted of foam cells, extracellular lipid, necrotic debris, cholesterol clefts, calcium deposits, and extracellular proteins. Oxidized LDL was only detected in the treated hamsters and localized to foam cells in early lesions, spread to extracellular matrix in fibrofatty lesions, and further involved medial smooth muscle cells in advanced lesions. Cyclooxygenases-1 and -2 were observed at low levels in both groups; however, cyclooxygenase-2 was noticeably upregulated in the early lesions of treated animals. Atherosclerotic lesions similar to each major stage of pathology in humans developed at a predictable site in the hamster aorta in a relatively short period.     

Dalmatian: spotting the difference in cohesin protectors

The cohesin complex prevents separation of chromosomes following their duplication until the appropriate time during cell division. In vertebrates, establishment and maintenance of cohesin‐dependent linkages depend on two distinct proteins, sororin and shugoshin. New findings published in The EMBO Journal show that in Drosophila, the function of both of these cohesin regulators is carried out by a single hybrid protein, Dalmatian.     

Isolation and characterization of 23 polymorphic microsatellite loci for a West Indian iguana (Cyclura pinguis) from the British Virgin Islands

Twenty-three polymorphic microsatellite markers were identified and characterized for Cyclura pinguis, a critically endangered species of lizard (Sauria: Iguanidae) native to Anegada Island in the British Virgin Islands. We examined variation at these loci for 39 C. pinguis, finding up to five alleles per locus and an average expected heterozygosity of 0.55. Allele frequency estimates for these microsatellite loci will be used to characterize genetic diversity of captive and wild C. pinguis populations and to estimate relatedness among adult iguanas at the San Diego Zoo that form the nucleus of a captive breeding programme for this critically endangered species.