Identificazione delle Lamine di tipo A come nuovi substrati della protein chinasi Akt/PKB

Akt (also called PKB) is a 63 kDa serine/threonine kinase involved in promotion of cell survival, proliferation a nd metabolic responses downstream the phosphoinositide-3-kinase (PI 3-kinase) signaling pathway. In resting cells, Akt is a predominantly cytosolic enzyme; however generation of PI 3-kinase lipid products recruits Akt to the plasma membrane, resulting in a conformational change which confers full enzymatic activity through the phosphorylation of the membrane-bound protein at two residues, Thr308, and Ser473. Activated Akt redistributes to cytoplasm and nucleus, where phosphorylation of specific substrates occurs. Both the presence and the activity of Akt in the nucleus have been described. An interesting mechanism that mediates nuclear translocation of Akt has been described in human mature T-cell leukemia: the product of TCL1 gene, Tcl1, interacts with the PH domain of phosphorylated Akt, thus driving Akt to the nucleus. In this context, Tcl1 may act as a direct transporter of Akt or may contribute to the formation of a complex that promotes the transport of active Akt to the nucleus, where it can phosphorylate nuclear substrates. A well described nuclear substrate if Foxo. IGF-1 triggers phosphorylation of Foxo by Akt inside the nucleus, where phospho-Foxo associates to 14.3.3 proteins that, in turn, promote its export to the cytoplasm where it is sequestered. Remarkably, Foxo phosphorylation by Akt has been shown to be a crucial event in Akt-dependent myogenesis. However, most Akt nuclear substrates have so far remained elusive, as well as nuclear Akt functions. This lack of information prompted us to undertake a search of substrates of Akt in the nucleus, by the combined use of 2D-separation/mass spectrometry and anti-Akt-phosphosubstrate antibody. This study presents evidence of A-type lamins as novel nuclear substrates of Akt. Lamins are type V intermediate filaments proteins found in the nucleus of higher eukaryotes where, together with lamin-binding proteins, they form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane. By coimmunoprecipitation, it is demonstrated here that endogenous lamin A and Akt interact, and that A-type lamins are phosphorylated by Akt both in vitro and in vivo. Moreover, by phosphoaminoacid analysis and mutagenesis, it is further demonstrated that Akt phosphorylates lamin A at Ser404, and, more importantly, that while lamin A/C phosphorylation is stable throughout the cell cycle, phosphorylation of the precursor prelamin A becomes detectable as cells enter the G2 phase, picking at G2/M. This study also shows that lamin phosphorylation by Akt creates a binding site for 14.3.3 adaptors which, in turn, promote prelamin A degradation. While this mechanism is in agreement with a general role of Akt in the regulation of a subset of its substrates, opposite to what has been described, degradation is not mediated through a ubiquitination and proteasomal mechanism but through a lysosomal pathway, as indicated by the reverting action of the lysosomal inhibitor cloroquine. Phosphorylation is a key event in the mitotic breakdown of the nuclear lamina. However, the kinases and the precise sites of phosphorylation are scarcely known. Therefore, these results represent an important breakthrough in this very significant but understudied area. The phosphorylation of the precursor protein prelamin A and its subsequent degradation at G2/M, when both the nuclear envelop and the nuclear lamina disassemble, can be view as part of a mechanism to dispose off the precursor that is not needed in this precise context. The recently reported finding that patients affected by Emery-Dreifuss muscular dystrophy carry a mutation at Arg 401, in the Akt phosphorylation motif, open new perspective that warrant further investigation in this very important field.

Retrograde cochlear implantation in postmeningitic basal turn ossification

Postmeningitic basal turn ossification is a challenge for successful cochlear implantation despite the availability of sophisticated implants and advanced drill-out procedures. A less complex concept consisting of a cochleostomy near the apex with retrograde array insertion is evaluated clinically and experimentally with emphasis on imaging of intracochlear array morphology.

RGD motif of lipoprotein T, involved in adhesion of Mycoplasma conjunctivae to lamb synovial tissue cells

Lipoprotein T (LppT), a membrane-located 105-kDa lipoprotein of Mycoplasma conjunctivae, the etiological agent of infectious keratoconjunctivitis (IKC) of domestic sheep and wild Caprinae, was characterized. LppT was shown to promote cell attachment to LSM 192 primary lamb joint synovial cells. Adhesion of M. conjunctivae to LSM 192 cells is inhibited by antibodies directed against LppT. The RGD (Arg-Gly-Asp) motif of LppT was found to be a specific site for binding of M. conjunctivae to these eukaryotic host cells. Recombinant LppT fixed to polymethylmethacrylate slides binds LSM 192 cells, whereas LppT lacking the RGD site is deprived of binding capacity to LSM 192, and LppT containing RGE rather than RGD shows reduced binding. Synthetic nonapeptides derived from LppT containing RGD competitively inhibit binding of LSM 192 cells to LppT-coated slides, whereas nonapeptides containing RAD rather than RGD do not inhibit. RGD-containing, LppT-derived nonapeptides are able to directly inhibit binding of M. conjunctivae to LSM 192 cells by competitive inhibition, whereas the analogous nonapeptide containing RAD rather than RGD or the fibronectin-derived RGD hexapeptide has no inhibitory effect. These results reveal LppT as the first candidate of a RGD lectin in Mycoplasma species that is assumed to bind to beta integrins.

Problem of Tragedy and Tragic Consciousness in Russia at the turn of the 19th Century

This project intertwines philosophical and historico-literary themes, taking as its starting point the concept of tragic consciousness inherent in the epoch of classicism. The research work makes use of ontological categories in order to describe the underlying principles of the image of the world which was created in philosophical and scientific theories of the 17th century as well as in contemporary drama. Using these categories brought Mr. Vilk to the conclusion that the classical picture of the world implied a certain dualism; not the Manichaean division between light and darkness but the discrimination between nature and absolute being, i.e. God. Mr. Vilk begins with an examination of the philosophical essence of French classical theatre of the XVII and XVIII centuries. The history of French classical tragedy can be divided into three periods: from the mid 17th to early 19th centuries when it triumphed all over France and exerted a powerful influence over almost all European countries; followed by the period of its rejection by the Romantics, who declared classicism to be "artificial and rational"; and finally our own century which has taken a more moderate line. Nevertheless, French classical tragedy has never fully recovered its status. Instead, it is ancient tragedy and the works of Shakespeare that are regarded to be the most adequate embodiment of the tragic. Consequently they still provoke a great number of new interpretations ranging from specialised literary criticism to more philosophical rumination. An important feature of classical tragedy is a system of rules and unities which reveals a hidden ontological structure of the world. The ontological picture of the dramatic world can be described in categories worked out by medieval philosophy - being, essence and existence. The first category is to be understood as a tendency toward permanency and stability (within eternity) connected with this or that fragment of dramatic reality. The second implies a certain set of permanent elements that make up the reality. And the third - existence - should be understood as "an act of being", as a realisation of permanently renewed processes of life. All of these categories can be found in every artistic reality but the accents put on one or another and their interrelations create different ontological perspectives. Mr. Vilk plots the movement of thought, expressed in both philosophical and scientific discourses, away from Aristotle's essential forms, and towards a prioritising of existence, and shows how new forms of literature and drama structured the world according to these evolving requirements. At the same time the world created in classical tragedy fully preserves another ontological paradigm - being - as a fundamental permanence. As far as the tragic hero's motivations are concerned this paradigm is revealed in the dedication of his whole self to some cause, and his oath of fidelity, attitudes which shape his behaviour. It may be the idea of the State, or personal honour, or something borrowed from the emotional sphere, passionate love. Mr. Vilk views the conflicting ambivalence of existence and being, duty as responsibility and duty as fidelity, as underlying the main conflict of classical tragedy of the 17th century. Having plotted the movement of the being/existence duality through its manifestations in 17th century tragedy, Mr. Vilk moves to the 18th century, when tragedy took a philosophical turn. A dualistic view of the world became supplanted by the Enlightenment idea of a natural law, rooted in nature. The main point of tragedy now was to reveal that such conflicts as might take place had an anti-rational nature, that they arose as the result of a kind of superstition caused by social reasons. These themes Mr. Vilk now pursues through Russian dramatists of the 18th and early 19th centuries. He begins with Sumarakov, whose philosophical thought has a religious bias. According to Sumarakov, the dualism of the divineness and naturalness of man is on the one hand an eternal paradox, and on the other, a moral challenge for humans to try to unite the two opposites. His early tragedies are not concerned with social evils or the triumph of natural feelings and human reason, but rather the tragic disharmony in the nature of man and the world. Mr Vilk turns next to the work of Kniazhnin. He is particularly keen to rescue his reputation from the judgements of critics who accuse him of being imitative, and in order to do so, analyses in detail the tragedy "Dido", in which Kniazhnin makes an attempt to revive the image of great heroes and city-founders. Aeneas represents the idea of the "being" of Troy, his destiny is the re-establishment of the city (the future Rome). The moral aspect behind this idea is faithfulness, he devotes himself to Gods. Dido is also the creator of a city, endowed with "natural powers" and abilities, but her creation is lacking internal stability grounded in "being". The unity of the two motives is only achieved through Dido's sacrifice of herself and her city to Aeneus. Mr Vilk's next subject is Kheraskov, whose peculiarity lies in the influence of free-mason mysticism on his work. This section deals with one of the most important philosophical assumptions contained in contemporary free-mason literature of the time - the idea of the trinitarian hierarchy inherent in man and the world: body - soul - spirit, and nature - law - grace. Finally, Mr. Vilk assess the work of Ozerov, the last major Russian tragedian. The tragedies which earned him fame, "Oedipus in Athens", "Fingal" and "Dmitri Donskoi", present a compromise between the Enlightenment's emphasis on harmony and ontological tragic conflict. But it is in "Polixene" that a real meeting of the Russian tradition with the age-old history of the genre takes place. The male and female characters of "Polixene" distinctly express the elements of "being" and "existence". Each of the participants of the conflict possesses some dominant characteristic personifying a certain indispensable part of the moral world, a certain "virtue". But their independent efforts are unable to overcome the ontological gap separating them. The end of the tragedy - Polixene's sacrificial self-immolation - paradoxically combines the glorification of each party involved in the conflict, and their condemnation. The final part of Mr. Vilk's research deals with the influence of "Polixene" upon subsequent dramatic art. In this respect Katenin's "Andromacha", inspired by "Polixene", is important to mention. In "Andromacha" a decisive divergence from the principles of the philosophical tragedy of Russian classicism and the ontology of classicism occurs: a new character appears as an independent personality, directed by his private interest. It was Katenin who was to become the intermediary between Pushkin and classical tragedy.

ANKLE IMPEDANCE AND ANKLE ANGLES DURING STEP TURN AND STRAIGHT WALK: IMPLICATIONS FOR THE DESIGN OF A STEERABLE ANKLE-FOOT PROSTHETIC ROBOT

During locomotion, turning is a common and recurring event which is largely neglected in the current state-of-the-art ankle-foot prostheses, forcing amputees to use different steering mechanisms for turning, compared to non-amputees. A better understanding of the complexities surrounding lower limb prostheses will lead to increased health and well-being of amputees. The aim of this research is to develop a steerable ankle-foot prosthesis that mimics the human ankle mechanical properties. Experiments were developed to estimate the mechanical impedance of the ankle and the ankles angles during straight walk and step turn. Next, this information was used in the design of a prototype, powered steerable ankle-foot prosthesis with two controllable degrees of freedom. One of the possible approaches in design of the prosthetic robots is to use the human joints’ parameters, especially their impedance. A series of experiments were conducted to estimate the stochastic mechanical impedance of the human ankle when muscles were fully relaxed and co-contracting antagonistically. A rehabilitation robot for the ankle, Anklebot, was employed to provide torque perturbations to the ankle. The experiments were performed in two different configurations, one with relaxed muscles, and one with 10% of maximum voluntary contraction (MVC). Surface electromyography (sEMG) was used to monitor muscle activation levels and these sEMG signals were displayed to subjects who attempted to maintain them constant. Time histories of ankle torques and angles in the lateral/medial (LM) directions, inversion-eversion (IE), and dorsiflexionplantarflexion (DP) were recorded. Linear time-invariant transfer functions between the measured torques and angles were estimated providing an estimate of ankle mechanical impedance. High coherence was observed over a frequency range up to 30 Hz. The main effect of muscle activation was to increase the magnitude of ankle mechanical impedance in all degrees of freedom of the ankle. Another experiment compared the three-dimensional angles of the ankle during step turn and straight walking. These angles were measured to be used for developing the control strategy of the ankle-foot prosthesis. An infrared camera system was used to track the trajectories and angles of the foot and leg. The combined phases of heel strike and loading response, mid stance, and terminal stance and pre-swing were determined and used to measure the average angles at each combined phase. The Range of motion (ROM) in IE increased during turning while ML rotation decreased and DP changed the least. During the turning step, ankle displacement in DP started with similar angles to straight walk and progressively showed less plantarflexion. In IE, the ankle showed increased inversion leaning the body toward the inside of the turn. ML rotation initiated with an increased medial rotation during the step turn relative to the straight walk transitioning to increased lateral rotation at the toe off. A prototype ankle-foot prosthesis capable of controlling both DP and IE using a cable driven mechanism was developed and assessed as part of a feasibility study. The design is capable of reproducing the angles required for straight walk and step turn; generates 712N of lifting force in plantarflexion, and shows passive stiffness comparable to a nonload bearing ankle impedance. To evaluate the performance of the ankle-foot prosthesis, a circular treadmill was developed to mimic human gait during steering. Preliminary results show that the device can appropriately simulate human gait with loading and unloading the ankle joint during the gait in circular paths.

Transforming growth factor-beta2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF expression

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

Comparison of the receptor FGFRL1 from sea urchins and humans illustrates evolution of a zinc binding motif in the intracellular domain

BACKGROUND: FGFRL1, the gene for the fifth member of the fibroblast growth factor receptor (FGFR) family, is found in all vertebrates from fish to man and in the cephalochordate amphioxus. Since it does not occur in more distantly related invertebrates such as insects and nematodes, we have speculated that FGFRL1 might have evolved just before branching of the vertebrate lineage from the other invertebrates (Beyeler and Trueb, 2006). RESULTS: We identified the gene for FGFRL1 also in the sea urchin Strongylocentrotus purpuratus and cloned its mRNA. The deduced amino acid sequence shares 62% sequence similarity with the human protein and shows conservation of all disulfides and N-linked carbohydrate attachment sites. Similar to the human protein, the S. purpuratus protein contains a histidine-rich motif at the C-terminus, but this motif is much shorter than the human counterpart. To analyze the function of the novel motif, recombinant fusion proteins were prepared in a bacterial expression system. The human fusion protein bound to nickel and zinc affinity columns, whereas the sea urchin protein barely interacted with such columns. Direct determination of metal ions by atomic absorption revealed 2.6 mole zinc/mole protein for human FGFRL1 and 1.7 mole zinc/mole protein for sea urchin FGFRL1. CONCLUSION: The FGFRL1 gene has evolved much earlier than previously assumed. A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.