A mutation leading to super-assembly of twin-arginine translocase (Tat) protein complexes

The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAy^P2A complexes may have attached to the TatAyCy complex. EM and AFM analyses show that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9–16 nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500 nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10–12 nm comparable to the size of single TatAy^P2A complexes. The combined data indicate that the mutation leads to super-assembly of TatAy^P2A complexes and we propose that an individual TatAy^P2A complex assembles initially with a TatAy^P2ACy complex, after which further TatAy^P2A complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.     

Carotid Intima-Media Thickness Is Predicted by Combined Eotaxin Levels and Severity of Hepatic Steatosis at Ultrasonography in Obese Patients with Nonalcoholic Fatty Liver Disease

Background

Non-Alcoholic Fatty Liver Disease (NAFLD) is a distinct coronary artery disease (CAD) risk factor. The atherosclerotic process predisposing to CAD includes altered lipid profile and inflammatory processes. The available evidence suggests that increased circulating levels of eotaxin, an eosinophil chemoattractant cytokine implicated in allergic responses, are detected in the serum of patients with CAD. Relationships were sought between serum eotaxin on the one hand, and intima-media thickness—an early predictor of the atherosclerotic process, hepatic steatosis, arterial blood pressure values, as well as inflammation/immune markers and angiogenetic factors—on the other.

Methods

Eighty obese patients with NAFLD, diagnosed at ultrasonography, without evident cytolysis, formed our study population. Anthropometric measures, metabolic profile, serum concentrations of interleukin-1β, C-reactive protein, interleukin-6, fibrinogen, ferritin, TNF-α, spleen size, vascular endothelial growth factor, platelet-derived growth factor-BB and heat shock protein-70 were evaluated.

Results

Serum eotaxin concentrations were distinctly associated with TNF α, IL-6, IL-1β, VEGF and PDGF-BB levels but not with CRP, fibrinogen, heat shock protein-70 or spleen size. Among the metabolic and anthropometric parameters, a significant predictive power emerged when comparing eotaxin to insulin resistance, expressed as HOMA. NAFLD was distinctly associated with HOMA (P = 0.0005). Intima-media thickness was well predicted by both eotaxin levels and severity of NAFLD at ultrasonography, although no relation was detected between these last two variables.

Discussion and Conclusion

A role for insulin resistance in mediating the interplay between eotaxin and other inflammation/immune parameters could be evidenced in the induction/maintenance of atherosclerosis of obese patients with NAFLD.     

Clinical and Organizational Issues in the Management of Surviving Breast and Colorectal Cancer Patients: Attitudes and Feelings of Medical Oncologists

Background

The fast growing demand and the shortage of resources are pushing toward more efficient models of survivorship care delivery. The Associazione Italiana di Oncologia Medica (AIOM) established an interdisciplinary working group with the purpose of promoting organizational improvements at the national level. A survey aimed at assessing attitudes and feelings of oncologists was considered preliminary to further initiatives.

Methods

A 25-item questionnaire, sent to the mailing list of the Society, explored the following issues on the practice of breast and colorectal cancer patients'' follow up: 1) organization; 2) clinical features; 3) feelings about the different meanings of follow-up.

Results

Ninety-one oncologists of 160 institutions (57%) answered to the questionnaire. Although follow up is considered a distinct oncological activity in 68%, a fully shared organization between specialists is not common and communications with Primary Care Physicians are not structured in the majority of the cases. Fifty-five and 30% of the oncologists follow breast and colorectal cancer patients indefinitely. In case of discharge a survivorship care plan is delivered in only 9%. The majority of respondents do not hold a role of follow up in mortality reduction.

Conclusions

Although survivorship care represents a significant part of the oncologists'' workload, an “oncology-centered” model is largely adopted and established care pathways are still incomplete. Survivorship care needs to be put at the center of an educational policy and of a widespread organizational effort, directed at improving appropriateness and quality.     

Environmental conditions modulate the switch among different states of the hydrophobin Vmh2 from Pleurotus ostreatus

Fungal hydrophobins are amphipathic, highly surface-active, and self-assembling proteins. The class I hydrophobin Vmh2 from the basidiomycete fungus Pleurotus ostreatus seems to be the most hydrophobic hydrophobin characterized so far. Structural and functional properties of the protein as a function of the environmental conditions have been determined. At least three distinct phenomena can occur, being modulated by the environmental conditions: (1) when the pH increases or in the presence of Ca(2+) ions, an assembled state, β-sheet rich, is formed; (2) when the solvent polarity increases, the protein shows an increased tendency to reach hydrophobic/hydrophilic interfaces, with no detectable conformational change; and (3) when a reversible conformational change and reversible aggregation occur at high temperature. Modulation of the Vmh2 conformational/aggregation features by changing the environmental conditions can be very useful in view of the potential protein applications.     

Cacospongionolide and scalaradial, two marine sesterterpenoids as potent apoptosis-inducing factors in human carcinoma cell lines

Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects of scalaradial and cacospongionolide, two sesterterpenoids from Cacospongia scalaris and Fasciospongia cavernosa marine sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot. The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116 (human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation. Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-κB subunits, nuclear level. In conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising compounds to inhibit cancer cell proliferation.     

Differential protein expression in tears of patients with primary open angle and pseudoexfoliative glaucoma

Primary open angle (POAG) and pseudoexfoliative glaucoma (PXG) are the most common primary and secondary forms of glaucoma, respectively. Even though the patho-physiology, aqueous humor composition, risk factors, clinical features, therapy and drug induced ocular surface changes in POAG and PXG have been widely studied, to date information concerning tear protein characterization is lacking. Tears are a source of nourishment for ocular surface tissues and a vehicle to remove local waste products, metabolized drugs and inflammatory mediators produced in several ophthalmic diseases. In glaucoma, the proteomic definition of tears may provide insights concerning patho-physiology of the disease and ocular surface modifications induced by topical therapy. Our study aimed at characterizing protein patterns in tears of patients with medically controlled POAG and PXG. A comparative tears proteomic analysis by label-free LC-MS(E) highlighted differences in the expression of several proteins in the two glaucoma sub-types and control subjects, highlighting inflammation pathways expressed in both diseases. Results were independently reconfirmed by SDS-PAGE and linear MALDI-TOF MS, validating altered levels of Lysozyme C, Lipocalin-1, Protein S100, Immunoglobulins and Prolactin Inducible Protein. Moreover, we found a differential pattern of phosphorylated Cystatin-S that distinguishes the two pathologies. The most relevant results suggest that in both pathologies there may be active inflammation pathways related to the disease and/or induced by therapy. We show, for the first time, tear protein patterns expressed under controlled intraocular pressure conditions in POAG and PXG subjects. These findings could help in the understanding of molecular machinery underlying these ophthalmologic diseases, resulting in early diagnosis and more specific therapy.     

Cutting edge: Autocrine TGF-beta sustains default tolerogenesis by IDO-competent dendritic cells

CD8(-) and CD8(+) dendritic cells (DCs) are distinct subsets of mouse splenic accessory cells with opposite but flexible programs of Ag presentation, leading to immunogenic and tolerogenic responses, respectively. In this study, we show that the default tolerogenic function of CD8(+) DCs relies on autocrine TGF-beta, which sustains the activation of IDO in response to environmental stimuli. CD8(-) DCs do not produce TGF-beta, yet externally added TGF-beta induces IDO and turns those cells from immunogenic into tolerogenic cells. The acquisition of a suppressive phenotype by CD8(-) DCs correlates with activation of the PI3K/Akt and noncanonical NF-kappaB pathways. These data are the first to link TGF-beta signaling with IDO in controlling spontaneous tolerogenesis by DCs.     

Autocrine regulation of IL-21 production in human T lymphocytes

IL-21 has pathologic function in immune-inflammatory diseases. IL-21 mediates its functions through a heterodimeric receptor, composed of a specific subunit, termed IL-21R, and the common gamma-chain. IL-21 is mostly produced by CD4(+) T cells, but molecular mechanisms that regulate IL-21 synthesis are not fully understood. The fact that CD4(+) T cells express high levels of IL-21R and are capable of functionally responding to IL-21 raises the possibility that IL-21 may regulate its own production. We here show that IL-21 enhances IL-21 RNA and protein expression in human peripheral blood CD3(+) T cells in a dose- and time-dependent fashion. Additionally, both IL-7 and IL-15, but not IL-4, induce IL-21, thus suggesting that common gamma-chain signals are not sufficient to promote IL-21 synthesis. Analysis of molecular mechanisms underlying IL-21 induction reveals that IL-21 activates Stat3 and enhances its recruitment to IL-21 gene promoter. Pharmacologic inhibition and knockdown of Stat3 by small interference RNA largely prevent IL-21 induction in IL-21-treated cells. Consistently, IL-21 is inducible in T cells by IL-6, another cytokine that activates Stat3. Finally, we show that IL-21 positively regulates its own expression in human intestinal CD3(+) lamina propria lymphocytes, and blockade of endogenous IL-21 in cultures of CD3(+) lamina propria lymphocytes isolated from patients with Crohn's disease, a chronic inflammatory bowel disease characterized by high IL-21, down-regulates Stat3 activation and IL-21 expression. These data suggest the existence of a positive autocrine loop that could help to amplify and stabilize IL-21-driven, T cell-mediated responses.