The role of the ATPase inhibitor factor 1 (IF1) in cancer cells adaptation to hypoxia and anoxia.

The physiological role of the mitochondrial ATP synthase complex is to generate ATP through oxidative phosphorylation. Indeed, the enzyme can reverse its activity and hydrolyze ATP under ischemic conditions, as shown in isolated mitochondria and in mammalian heart and liver. However, what occurs when cancer cells experience hypoxia or anoxia has not been well explored. In the present study, we investigated the bioenergetics of cancer cells under hypoxic/anoxic conditions with particular emphasis on ATP synthase, and the conditions driving it to work in reverse. In this context, we further examined the role exerted by its endogenous inhibitor factor, IF1, that it is overexpressed in cancer cells. Metabolic and bioenergetic analysis of cancer cells exposed to severe hypoxia (down to 0.1% O2) unexpectedly showed that Δψm is preserved independently of the presence of IF1 and that ATP synthase still phosphorylates ADP though at a much lower rate than in normoxia. However, when we induced an anoxia-mimicking condition by collapsing ΔµΗ+ with the FCCP uncoupler, the IF1-silenced clones only reversed the ATP synthase activity hydrolyzing ATP in order to reconstitute the electrochemical proton gradient. Notably, in cancer cells IF1 overexpression fully prevents ATP synthase hydrolytic activity activation under uncoupling conditions. Therefore, our results suggest that IF1 overexpression promotes cancer cells survival under temporary anoxic conditions by preserving cellular ATP despite mitochondria dysfunction.

Hypoxia decreases ROS level in human fibroblasts.

We have previously demonstrated that cells adapt to hypoxia using different metabolic reprogramming mechanisms depending on metabolism. We now investigate how the different adapting mechanisms affect reactive oxygen species (ROS) levels, and how ROS levels and cellular metabolism are linked. We show that when skin fibroblasts grew under short-term hypoxia (1% oxygen tension) ROS level markedly decreased (-50%) whatever substrate was available to the cells. Indeed, cellular ROS level linearly and directly decreased with oxygen tension. However, these relationships cannot explain the progressive ROS level decrease observed after prolonged cells hypoxia exposure. In glucose-enriched medium reduced mitochondrial mass and greater fragmentation are observed, both clear-cut indications of mitophagy suggesting that this is responsible for cellular ROS level decrease. Otherwise, in glucose-free medium exposure to prolonged hypoxia resulted in only minor mass reduction, but significantly enhanced expression of antioxidant enzymes. Interestingly, cellular ROS levels were lower in glucose-free compared to glucose-enriched medium under either normoxic or hypoxic conditions. Taken together, these findings reveal that in primary human fibroblasts hypoxia induces a decline in ROS and that different metabolism-dependent mechanisms contribute it, besides the major oxygen concentration decrease. In addition, the present data support the notion that metabolisms generating fewer ROS are associated with lower HIF-1α stabilization.

Diagnostic pitfalls in the preoperative 18F-FDG PET/CT evaluation of a case of giant malignant solitary fibrous tumor of the pleura.

Solitary fibrous tumor of the pleura (SFTP) is an uncommon entity, generally with an indolent behavior. Nevertheless, some malignant forms have been rarely reported. These, often have an aggressive biological behavior with pathological findings of invasiveness. The preoperative diagnosis and evaluation of the grade of malignancy are extremely challenging. Herein we report a case of a 64-year-old man who presented with a left giant intra-thoracic mass imaged with fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography ((18)F-FDG/PET-CT) and sampled via fine-needle aspiration biopsy (FNAB). Imaging and FNAB findings showed suspicion of a benign form of SFTP. Surgical radical resection of the giant mass was performed. The definitive histological diagnosis showed a malignant SFTP. Based on this report, we take the opportunity to briefly discuss the insidious pitfalls concerning the radiological and (18)F-FDG/PET-CT features as well as cyto/histological findings in the pre-operative diagnostic work-up examination of this rare entity.

Hyperoxia fully protects mitochondria of explanted livers.

Liver ischemia-reperfusion injury is still an open problem in many clinical circumstances, including surgery and transplantation. This study investigates how mitochondrial structure, mass and oxidative phosphorylation change and may be preserved during a brief period of ischemia followed by a long period of reperfusion, an experimental model that mimics the condition to which a liver is exposed during transplantation. Livers were explanted from rats and exposed for 24 h to three different oxygen availability conditions at 4 °C. Mitochondrial mass, respiration, oxidative phosphorylation (OXPHOS), and levels of OXPHOS complexes were all significantly altered in livers stored under the currently used preservation condition of normoxia. Remarkably, liver perfusion with hyperoxic solutions fully preserved mitochondrial morphology and function, suggesting that perfusion of the graft with hyperoxic solution should be considered in human transplantation.

Is supercomplex organization of the respiratory chain required for optimal electron transfer activity?

The supra-molecular assembly of the main respiratory chain enzymatic complexes in the form of "super-complexes" has been proved by structural and functional experimental evidence. This evidence strongly contrasts the previously accepted Random Diffusion Model stating that the complexes are functionally connected by lateral diffusion of small redox molecules (i.e. Coenzyme Q and cytochrome c). This review critically examines the available evidence and provides an analysis of the functional consequences of the intermolecular association of the respiratory complexes pointing out the role of Coenzyme Q and of cytochrome c as channeled or as freely diffusing intermediates in the electron transfer activity of their partner enzymes.

Thymolipoma of the anterior mediastinum: videothoracoscopic removal using a bilateral approach.

Thymolipoma, a rare benign neoplasm of the anterior mediastinum, is often asymptomatic and as a result it can become quite large before it is diagnosed. CT is the most accurate diagnostic technique to identify the adipose tissue, but it often cannot make a differential diagnosis differentiating it from other anterior mediastinal masses; transthoracic biopsy also reveals the presence of fatty tissue, but a definitive diagnosis can only be achieved by means of surgical excision, which is also curative. We describe the case of a young woman who presented with a fatty neoplasm of the anterior mediastinum. The mass was removed using single-stage bilateral sequential videothoracoscopy. The histopathological diagnosis was thymolipoma.

[Calcified pseudotumor of the pleura: description of a case]. / Pseudotumore fibroso calcifico della pleura: descrizione di un caso.

We describe a calcifying fibrous pseudotumour of pleura in a 46-year-old female, smoker. The patient presented with a well-delimited pleural mass, 3-cm across, located at the base of the right lung and attached to the lung with a short pedicle. Seven years after surgical excision of the mass, the patient is alive and well. Microscopically, the lesion was mostly composed of dense collagenous tissue, with sparse benign spindle cells, a rich inflammatory infiltrate and scattered calcifications, sometimes laminated. Immunohistochemically, spindle cells were positive for vimentin and negative for smooth muscle actin, desmin, S100 protein, CD34, CD99 and Bcl2. Calcifying fibrous pseudotumour is rare in the pleura. Pertinent data from the literature and problems in differential diagnosis are briefly discussed.

Preoperative localization of indeterminate pulmonary nodules before videothoracoscopic resection.

BACKGROUND: The management of a solitary pulmonary nodule is the subject of debate and minimally invasive diagnostic approaches have low sensitivity for small peripheral nodules. We discuss the role of video-assisted thoracoscopic surgery (VATS) in the management of solitary pulmonary nodules (SPNs) < or = 1 cm performed with a preoperative computed tomography-guided wire localization. METHODS: Thirty-five selected patients underwent VATS resection for SPN, with localization by guide wire before surgery. RESULTS: Seven patients, after VATS exploration, underwent thoracotomy because of pleuropulmonary adhesions, depth or dimensions. Histological diagnosis was obtained in all procedures; there was no postoperative morbidity or morbidity. CONCLUSION: Preoperative computed tomography hook-wire localization is a suitable strategy for peripheral nodules < or = 1 cm in diameter.

[Clear-cell tumor of the lung: description of a case 1 mm in diameter ("micro-sugar tumor")]. / Tumore a cellule chiare del polmone: descrizione di un caso di 1 mm di diametro ("micro-sugar tumour").

Clear cell ("sugar") tumour of the lung is a rare neoplasm, generally presenting as a discrete nodule on the chest X-ray. We report a case of clear cell tumour of the lung in a 64-year-old woman. The tumour at presentation was 1 mm in diameter.

T cell activation by coxsackievirus B4 antigens in type 1 diabetes mellitus: evidence for selective TCR Vbeta usage without superantigenic activity.

Numerous clinical and epidemiological studies link enteroviruses such as the Coxsackie virus group with the autoimmune disease type 1 diabetes mellitus (DM). In addition, there are reports that patients with type 1 DM are characterized by skewing of TCR Vbeta chain selection among peripheral blood and intraislet T lymphocytes. To examine these issues, we analyzed TCR Vbeta chain-specific up-regulation of the early T cell activation marker, CD69, on CD4 T cells after incubation with Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the Vbeta chains 2, 7, and 8 were the most frequently activated by CVB4. Up-regulation of CD69 by different TCR families was significantly more frequent in new onset type 1 DM patients (p = 0.04), 100% of whom (n = 8) showed activation of CD4 T cells bearing Vbeta8, compared with 50% of control subjects (n = 8; p = 0.04). T cell proliferation after incubation with CVB4 Ags required live, nonfixed APCs, suggesting that the selective expansion of CD4 T cells with particular Vbeta chains resulted from conventional antigen processing and presentation rather than superantigen activity. Heteroduplex analysis of TCR Vbeta chain usage after CVB4 stimulation indicated a relatively polyclonal, rather than oligo- or monoclonal response to viral Ags. These results provide evidence that new-onset patients with type 1 DM and healthy controls are primed against CVB4, and that CD4 T cell responses to the virus have a selective TCR Vbeta chain usage which is driven by viral Ags rather than a superantigen.

Lymphocyte dysmetabolism: an immunocytochemical comparative approach in IDDM and control subjects.

We have investigated by immuno-electron microscopy the presence of phosphotyrosine in cells as a whole and in different cell districts (nucleus, cytoplasm, plasma membrane, and mitochondria) in peripheral blood lymphocytes of IDDM (insulin-dependent diabetes mellitus) patients and age-matched controls. Immuno-gold particle density was highest in mitochondria and decreased in cytoplasm, nucleus and plasma membrane. The time dependence of phosphotyrosine labelling after cell isolation was very strong in all subcellular populations, with a fall in immunogold staining after 30 min. Staining levels at zero time were similar in controls and IDDM patients; the loss of phosphotyrosine labelling was much stronger in controls, except in the plasma membrane. Plasma membrane NADH oxidoreductase activity, studied using cytosolic NADH as substrate and assayed with DCIP as acceptor, was significantly increased in IDDM patients, suggesting a response to a deficient mitochondrial energetic activity. The fact that NADH oxidoreductase is a growth factor related to tyrosine phosphorylation pathways raises intriguing questions on the cellular derangement occurring in peripheral lymphocytes in IDDM, although the relationships among the immunocytochemical and biochemical changes is still obscure.

Decreased Pasteur effect in platelets of aged individuals.

We have investigated the mitochondrial energy state in human platelets of young (19-30 years old) and aged individuals (65-87 years old) exploiting the Pasteur effect, i.e. stimulation of lactate production by incubation of the purified platelets with the mitochondrial respiratory chain inhibitor, antimycin A. This assay allows the determination of mitochondrial function with respect to glycolysis, and the ratio of mitochondrial adenosine triphosphate (ATP) to glycolytic ATP. A significant increase of basal, non-stimulated lactate production and decrease of the stimulation by antimycin A were observed in the older individuals, suggesting that the impairment of oxidative phosphorylation detectable in post-mitotic tissues of aged individuals can be observed also in easily collectable blood cells.

T-Cell reactivity to the P2C nonstructural protein of a diabetogenic strain of coxsackievirus B4.

Enteroviruses are proposed as initiating factors in the etiology of Type 1 diabetes mellitus (Type 1 DM). Molecular mimicry between the autoantigen glutamic acid decarboxylase 65 (GAD65) and the coxsackievirus B4 (CVB4) nonstructural protein P2C is frequently cited as a mechanism by which this virus triggers the disease, but little is known about the immunogenicity of this viral protein in humans, mainly due to the problem of obtaining highly pure preparations of P2C. We generated large amounts of highly pure, soluble P2C protein, coupled to the fusion partner maltose binding protein (MBP-P2C) using the PMAL-c2 bacterial expression plasmid and a two-step purification system comprising amylose resin and ion exchange. Using purified viral protein we show that specific T-cell responses against P2C are detected in the blood of healthy donors and Type 1 DM patients. Proliferation responses to P2C were detected only in subjects also demonstrating T-cell proliferation to CVB4 Vero cell lysates. However, in additional cases T-cell responses to P2C were detectable through the release of interferon-gamma or interleukin-4 in individuals who did not make proliferative responses. Taken together, our data show that the P2C nonstructural protein of CVB4 is targeted by T cells during the antiviral immune response and may trigger the production of T helper 1 and T helper 2 cytokines. The availability of pure, immunogenic P2C should allow the putative role of antiviral responses in the development of autoimmune diabetes to be investigated.

Tyrosine phosphorylation in type-1 diabetes by immunogold detection: an in vitro human aortic endothelial cell (HAEC) study in the presence of diabetic low density lipoproteins (LDL).

An immunomorphometric study of tyrosine phosphorylation was performed by the immunogold technique on cultured human aortic endothelial cells (HAEC) with a view to demonstrating their impaired signal transduction status, induced in vitro by incubation with low-density lipoproteins from the plasma of Type-1 diabetic patients. The results seem to sustain the hypothesis that extranuclear bioenergetic derangement induced by low-density lipoproteins from Type-1 diabetic patients may be associated with an up-regulation of the nuclear energetic machinery aimed at maintaining intracellular metabolic equilibrium. Our data demonstrate that phosphorylated tyrosine is a useful marker to monitor this metabolic condition.

Mitochondrial DNA in platelets from aged subjects.

This study aimed to assess platelets as a possible model for screening the accumulation of mitochondrial DNA mutations, particularly during normal ageing. For this purpose we isolated platelets from young and old donors selected by lack of systemic and haematological diseases. We studied the accumulation of a particular deletion (4977-bp deletion) that usually accumulates in an age-related manner in different post-mitotic tissues, such as brain, heart and skeletal muscle, and in some non-post-mitotic tissues (skin, liver). Using different primers, we failed to detect this particular species of deletion in platelets both from young and old individuals. However, we cannot exclude the presence of other species of deletions or point mutations affecting the mitochondrial DNA in platelets during the aging process.

Arterial injury of the axilla: an unusual case after blunt trauma of the shoulder.

The lesion of an axillary artery after a shoulder dislocation without bone fractures is very rare. The most common lesion is the subtotal transection of the vessel. Vascular injuries may be associated with nervous lesions whose incidence ranges from 27 to 44%. The nervous lesions are highly invalidating. The symptoms which recur most frequently are motor and sensitive deficiencies and a distal ischemia which, in some cases, may not be noted owing to the extensive collateral network. Whenever these symptoms are covered or absent, as in this case report, an angiography of the upper limb becomes essential for correct diagnosis and treatment.

[New diagnostic and therapeutic prospects in peripheral nodules of the lung: surgical video thoracoscopy]. / Nuove prospettive diagnostiche e terapeutiche dei noduli periferici del polmone: la videotoracoscopia operativa.

The new findings and results of laparoscopic surgery lead us to apply this technique in thoracic surgery. Thoracoscopy is an old diagnostic procedure, but advanced imaging techniques, improved optics and new classes of instrument have paved the way for many diagnostic and therapeutic approaches, before not feasible. Now it is possible to treat spontaneous pneumothorax, removal of mediastinal and peripheral tumors, with minimally invasive surgery. It is our belief that thoracoscopic surgery will be a valuable tool in the near future for a variety of chest disorders.