Primary cutaneous interdigitating dendritic cell sarcoma (IDCS): Report of a new case and literature review.

Interdigitating dendritic cell sarcoma is a very rare entity in the spectrum of histiocytic and dendritic cell neoplasms that mostly occurs in lymph nodes, generally presenting as solitary lymphadenopathy, but may affect every organ. Among extra nodal sites, cutaneous interdigitating dendritic cell sarcoma is exceedingly rare; to date, only 9 cases have been described in English literature. The mean age at diagnosis was 60 years, with a male-female ratio of 1,5 to 1; clinically, two different modalities of skin presentation have been reported: solitary, represented by a single red-brownish nodular lesion, or diffuse, characterized by multiple nodular lesions in one or more body districts. The extreme rarity of this sarcoma and its morphological similarity to other poorly differentiated tumors may lead to a delay in diagnosis; in particular, cutaneous localization may be difficult to differentiate from follicular dendritic cell sarcoma, Langerhans cell sarcoma, poorly differentiated squamous cell carcinoma and more generally sarcomatoid carcinoma, atypical fibroxanthoma, malignant melanoma and several sarcomas. Immunohistochemistry plays an important role in identifying this rare entity and formulating a correct histological diagnosis, fundamental requirement for choosing the best therapeutic approach. We report herein a further case of an 81-year-old Caucasian woman who presented to the Dermatology Department to remove an asymptomatic skin papule in the left temporal region, clinically diagnosed as dermatofibroma. The overall pathological and immunohistochemical features supported the diagnosis of a malignant dendritic cell tumor, consistent of interdigitating dendritic cell sarcoma.

A new methodological approach for in vitro determination of the role of DNA methylation on transcription factor binding using AlphaScreen® analysis: Focus on CREB1 binding at hBDNF promoter IV.

BACKGROUND: DNA methylation plays a relevant role in the regulation of gene transcription, but currently the exact quantification of transcription factors binding to methylated DNA is not being determined. The binding of the transcription factor cAMP response element-binding protein-1 to its cognate CpG containing motif is known to be impaired upon methylation. It thus represents a paradigmatic system to experimentally verify the validity of a new in vitro method to measure the role of methylation on DNA/transcription factors binding. METHOD: An AlphaScreen® assay was developed to quantitatively measure the contribution of DNA CpG methylation on the interaction with transcription factors. The method was validated measuring the variation in affinity of cAMP response element-binding protein-1 and its recognition motif in human Brain-derived neurotrophic factor gene exon IV promoter as a function of CpG methylation. RESULTS: For the first time, a quantitative direct correlation between DNA methylation and transcription factors binding is reported showing a dramatic reduction in binding affinity between fully methylated and non-methylated DNA. COMPARISON WITH EXISTING METHODS: This methodology allows to directly measure DNA/transcription factors binding ability as a function of DNA methylation levels thus improving not quantitative methods available today. Moreover, it allows to work with purified proteins and oligonucleotides without need of chromatin. CONCLUSIONS: The present methodology is suggested as a new analytical tool for the quantitative determination of the effect of CpG methylation on the interaction of gene promoters with transcription factors regulating gene expression, a key epigenetic mechanism implicated in many physiological and pathological conditions.

Dissociation kinetics of hemocyanin from Octopus vulgaris.

The native form of hemocyanin (Hc) from Octopus vulgaris can be completely dissociated, at alkaline pH and in the presence of EDTA, from 49S decamers to 11S monomers. The kinetics of this process was studied, using a Bio-Logic stopped flow system, by following the time dependence of the 450-nm light intensity, scattered at 90 degrees, in the 7.9-8.8 pH range. All experimental traces were best fitted by a sum of three exponential decay functions. We then tried to best fit these decay functions with a series of kinetic models, the best of them resulting in one whose dissociation of decamers to monomers takes place in three consecutive and irreversible steps, with a highly cooperative step concerning dissociation of octamers to dimers, which appears to be the only intermediate species. This model was preferred over several others, not only for the best norm value but also for the best accordance between each calculated and experimental kinetic parameter (rate constants and amplitudes). Although other more complex models may be considered, our best fit model represents the simplest one, which is able to describe the observed dissociation kinetics.

Metastasis of breast carcinoma to intracranial meningioma.

A patient who had been treated for bilateral breast carcinoma subsequently developed a metastatic breast lesion in a meningioma. Although it is not uncommon for more than one tumor to occur in the same patient, metastases from one tumor into another tumor are rare (''tumor to tumor'' phenomenon). Meningiomas are the most common primary, intracranial tumors to harbor metastases, the majority of which arise from breast and lung carcinomas. Patients with a history of breast cancer and a solitary, intracranial mass with radiological features consistent of meningioma should be evaluated surgically. The lesion may be a primary cerebral malignancy, a metastatic lesion, or a meningioma with or without a metastatic lesion.

Characterisation of a specific phycocyanin-hydrolysing protease purified from Spirulina platensis.

A novel protease has been identified, purified and partially characterised from complete medium grown Spirulina platensis, which could be responsible for the selective proteolysis of phycobiliproteins. It is an 80 kDa homodimeric enzyme; its N-terminal sequence is not related to any known protease sequence. It hydrolyses native phycocyanins in both crude extracts and reconstructed systems with purified Allo- or C-phycocyanin. It is inactive on several native proteins, including ribulose-1,5-bisphosphate carboxylase. The two phycocyanins are degraded at different velocities since C-phycocyanin is the better substrate, in agreement with the earlier observations on the progress of the phycobilisome disassembly. Specificity for synthetic substrates and inhibitors strongly suggests its assignment to the serine-protease family. The enzyme, however, is insensitive to the commercially available protein inhibitors of trypsin-like proteases.

GPR55 and its interaction with membrane lipids: comparison with other endocannabinoid-binding receptors.

A number of integral membrane G protein-coupled receptors (GPCRs) share common structural features (including palmytoilated aminoacid residues and consensus sequences specific for interaction with cholesterol) that allow them to interact with lipid rafts, membrane cholesterol-rich microdomains able to regulate GPCR signalling and functions. Among GPCRs, type-1 and type-2 cannabinoid receptors, the molecular targets of endocannabinoids (eCBs), control many physiological and pathological processes through the activation of several signal transduction pathways. Recently, the orphan GPR55 receptor has been proved to be activated by many eCBs, thus leading to the hypothesis that it might be the "type-3" cannabinoid receptor. While the biological activity of eCBs and the influence of membrane lipids on their functions are rather well established, information regarding GPR55 is still scarce and often controversial. Based on this background, here we shall review current data about GPR55 pharmacology and signalling, highlighting its involvement in several pathophysiological conditions. We shall also outline the structural features that allow GPR55 to interact with cholesterol and to associate with lipid rafts; how the latter lipid microdomains impact the biological activity of GPR55 is also addressed, as well as their potential for the discovery of new therapeutics useful for the treatment of those human diseases that might be associated with alterations of GPR55 activity.

Molecular modeling and simulation of membrane lipid-mediated effects on GPCRs.

Functioning of G protein-coupled receptors (GPCRs) is tightly linked to the membrane environment, but a molecular level understanding of the modulation of GPCR by membrane lipids is not available. However, specific receptor-lipid interactions as well as unspecific effects mediated by the bulk properties of the membrane (thickness, curvature, etc.) have been proposed to be key regulators of GPCR modulation. In this review, we examine computational efforts made towards modeling and simulation of (i) the complex behavior of membrane lipids, (ii) membrane lipid-GPCR interactions as well as membrane lipid-mediated effects on GPCRs and (iii) GPCR oligomerization in a native-like membrane environment. We propose that, from the perspective of computational modeling, all three of these components need to be addressed in order to achieve a deeper understanding of GPCR functioning. Presently, we are able to simulate numerous lipid properties applying advanced computational techniques, although some barriers, such as the time-length of these simulations, need to be overcome. Implementing three-dimensional structures of GPCRs in such validated membrane systems can give novel insights in membrane-dependent receptor modulation and formation of higher order receptor complexes. Finally, more realistic GPCR-membrane models would provide a very useful tool in studying receptor behavior and its modulation by small drug-like ligands, a relevant issue for drug discovery.

The endogenous cannabinoid system in the gut of patients with inflammatory bowel disease.

Activation of cannabinoid receptors (CBs) by endocannabinoids impacts on a number of gastrointestinal functions. Recent data indicate that CB1 agonists improve 2,4-dinitrobenzene sulfonic acid-induced colitis in mice, thus suggesting a role for the endocannabinoid agonist anandamide (AEA) in protecting the gut against inflammation. We here examined the gut endocannabinoid system in inflammatory bowel disease (IBD) patients, and investigated the ex vivo and in vitro effects of the non-hydrolysable AEA analog methanandamide (MAEA) on the mucosal proinflammatory response. The content of AEA, but not of 2-arachidonoyl-glycerol and N-palmitoylethanolamine, was significantly lower in inflamed than uninflamed IBD mucosa, and this was paralleled by lower activity of the AEA-synthesizing enzyme N-acyl-phosphatidylethanolamine-specific phospholipase D and higher activity of the AEA-degrading enzyme fatty acid amide hydrolase. MAEA significantly downregulated interferon-γ and tumor necrosis factor-α secretion by both organ culture biopsies and lamina propria mononuclear cells. Although these results are promising, further studies are needed to determine the role of cannabinoid pathways in gut inflammation.

Interaction of endocannabinoid receptors with biological membranes.

Cellular signaling is regulated by several biochemical reactions, whose dynamics depends on changes in the fluxes of specific ligands through the containment barriers that are the biological membranes. The regulation of this complex dynamic equilibrium is mainly due to the activity of border proteins, that must be able to interact simultaneously with the lipid bilayer and the extracellular milieu. Endocannabinoid receptors, that include type-1 and type-2 cannabinoid receptors, the transient vanilloid potential receptors and the peroxisome proliferator-activated receptors, represent one of the most intriguing examples of "border" proteins. They have also been identified as important drug discovery targets with potential therapeutic applications, from antiemesis, appetite enhancement, analgesia, glaucoma treatment, and immune suppression. However, as yet the molecular details of endocannabinoid receptor regulation remain elusive. In this review we summarize the most relevant aspects of the structural/functional characterization of these receptors, with a focus on the active role played by biological membranes (in particular lipid rafts) in the modulation of their accessibility and mode of ligand binding. Based on available evidence, we propose that endocannabinoid receptors can be regulated by the rate of interlayer exchange and lateral diffusion of endocannabinoid/cholesterol complexes within lipid bilayers, thus suggesting innovative approaches for the therapeutic exploitation of the membrane component of endocannabinoid signaling.

Lipid-mediated dimerization of beta2-adrenergic receptor reveals important clues for cannabinoid receptors.

The high-resolution crystal structure of an engineered human beta2-adrenergic receptor has recently been resolved, suggesting a molecular mechanism by which cholesterol may mediate receptor dimerization. Here, we present a critical examination of new structural and functional insights derived from unprecedented preliminary homology modeling of cannabinoid receptors, obtained using the crystal structure of beta2-adrenergic receptor as a template. The structural comparison between the two cannabinoid receptor subtypes and the beta2-adrenergic receptor may be of particular interest, by providing important clues for the elucidation of the structural determinants involved in cholesterol binding. In addition, the implications of G protein coupled receptor dimerization, as well as the role of cholesterol in this process, are briefly discussed.

Evidence for the intracellular accumulation of anandamide in adiposomes.

Anandamide is a lipid messenger that carries out a wide variety of biological functions. It has been suggested that anandamide accumulation involves binding to a saturable cellular component. To identify the structure(s) involved in this process, we analyzed the intracellular distribution of both biotinylated and radiolabeled anandamide, providing direct evidence that lipid droplets, also known as adiposomes, constitute a dynamic reservoir for the sequestration of anandamide. In addition, confocal microscopy and biochemical studies revealed that the anandamide-hydrolase is also spatially associated with lipid droplets, and that cells with a larger adiposome compartment have an enhanced catabolism of anandamide. Overall, these findings suggest that adiposomes may have a critical role in accumulating anandamide, possibly by connecting plasma membrane to internal organelles along the metabolic route of this endocannabinoid.

["Proximal type" epithelioid sarcoma of the vulva: differential diagnosis with other extrarenal rhabdoid tumors]. / Sarcoma epitelioide di tipo prossimale della vulva: diagnosi differenziale con altri tumori rabdoidi extrarenali.

Proximal type epithelioid sarcoma is a rare neoplasia in which morphological findings are characterized by nodular proliferation of epithelioid cells with focal rhabdoid features. It shares some histological features with other neoplasias and this gives an account of several differential diagnosis with other extrarenal rhabdoid tumors. Immunohistochemical and ultrastructural analysis are important in defining this entity: vimentin, cytokeratin, EMA and often CD34 expression of tumoral cells, moreover ultrastructurally evidence of large paranuclear whorls of intermediate filaments, are requested for diagnosis. A correct diagnostic framing is necessary because of the aggressive clinical behaviour of this tumor, that has a tendency to early spreading. We describe a case of vulvar proximal type epithelioid sarcoma in a 34 years old woman.

Hemocyanin subunit organization of the gastropod Rapana thomasiana.

RtH1 and RtH2, the two hemocyanin isoforms of the prosobranch gastropod Rapana thomasiana, have been purified by anion-exchange chromatography and studied by SDS-PAGE and immunoelectrophoresis. Both subunit types are built up of eight functional units (FUs). Under reducing conditions subunit RtH2 splits into two fragments, RtH2-a-f and RtH2-gh, suggesting the presence of a disulfide bridge between FU2-f and FU2-g. By proteolytic cleavage of the subunits into three-, two-, and single-FU fragments, purification of fragments by HPLC, N-terminal sequencing of the peptides, and crossed-line immunoelectrophoresis, FUs-a-h of RtH2 and FU-a, FU-d, FU-e, and FU-f of RtH1 were identified and correlated to the eight-FUs pattern of immunoelectrophoresis. FU-a, FU-e, and FU-f of RtH1 and RtH2 are very closely related immunologically. RtH1 and RtH2 both correspond immunologically to KLH2, one of the two hemocyanin isoforms of the prosobranch gastropod Megathura crenulata.

Low-resolution structure of the proteolytic fragments of the Rapana venosa hemocyanin in solution.

Rapana venosa hemocyanin (Hc) is a giant oxygen-binding protein consisting of different subunits assembled in a hollow cylinder. The polypeptide chain of each subunit is believed to be folded in several oxygen-binding functional units of molecular mass 50 kDa, each containing a binuclear copper active site. Limited proteolysis with alpha-chymotrypsin of native R. venosa hemocyanin allows the separation of three functional proteolytic fragments with molecular masses of approximately 150, 100, and 50 kDa. The functional fragments, purified by combining gel filtration chromatography and ion-exchange FPLC, were analyzed by means of small-angle X-ray scattering (SAXS). The gyration radius of the 50-kDa Rapana Hc fraction (2.4 nm) agrees well with that calculated on the basis of the dimensions determined by X-ray crystallography for one functional unit of Octopus Hc (2.1 nm). Independent shape determination of the 50- and 100-kDa proteolytic fragments yields consistent low-resolution models. Simultaneous fitting of the SAXS data from these fragments provides a higher-resolution model of the 100-kDa species made of two functional units tilted with respect to each other. The model of the 150-kDa proteolytic fragment consistent with the SAXS data displays a linear chain-like aggregation of the 50-kDa functional units. These observations provide valuable information for the reconstruction of the three-dimensional structure of the minimal functional subunit of gastropod hemocyanin in solution. Furthermore, the spatial relationships among the different functional units within the subunit will help in elucidation of the overall quaternary structure of the oligomeric native protein.

Subunits composition and allosteric control in Carcinus aestuarii hemocyanin.

Carcinus aestuarii hemocyanin (Hc) exists in two aggregation forms at pH 7.5 and 20 mM Ca2+: 24S accounting for 90% of total hemocyanin and 16S accounting for 10%. Removal of metal cations by EDTA at neutral pH causes the complete dissociation of 24S hemocyanin into two different 16S. At pH 9.2, 24S hemocyanin dissociates into a pH stable 16S and a 5S component. The 5S component consists of three monomeric fractions named CaeSS1 (10%), CaeSS2 (50%) and CaeSS3 (40%); the latter fraction consisting of two isoforms. The fractions CaeSS1, CaeSS2 and CaeSS3 have been studied as far as their reassociation properties to form hexamers are concerned. We investigated the oxygen-binding properties of the native form (24S), the mixture of the two 16S forms, the pH-stable 16S alone and of purified subunit fractions to define the role of each species on the expression of the allosteric behaviour of the 24S aggregate. The analysis of O2-binding data reveals that 24S-Hc can be well described by the modified Monod Wyman and Changeaux-model (nested MWC-model), while the half-molecules (16S) bind oxygen according to the simple MWC-model. The two hexameric 16S within the dodecameric 24S hemocyanin can be regarded as nested allosteric units. They behave as being functionally coupled in the T-states (tT and rT). In the R-states (tR and rR) the two half-molecules seem to be functionally uncoupled since they have the same values of oxygen binding constants as deduced for isolated 16S hexamers.