Expression of the repressor element-1 silencing transcription factor (REST) is regulated by IGF-I and PKC in human neuroblastoma cells

The repressor element 1-silencing transcription factor (REST) was first identified as a protein that binds to a 21-bp DNA sequence element (known as repressor element 1 (RE1)) resulting in transcriptional repression of the neural-specific genes [Chong et al., 1995; Schoenherr and Anderson, 1995]. The original proposed role for REST was that of a factor responsible for restricting neuronal gene expression to the nervous system by silencing expression of these genes in non-neuronal cells. Although it was initially thought to repress neuronal genes in non-neuronal cells, the role of REST is complex and tissue dependent. In this study I investigated any role played by REST in the induction and patterning of differentiation of SH-SY5Y human neuroblastoma cells exposed to IGF-I. and phorbol 12- myristate 13-acetate (PMA) To down-regulate REST expression we developed an antisense (AS) strategy based on the use of phosphorothioate oligonucleotides (ODNs). In order to evaluate REST mRNA levels, we developed a real-time PCR technique and REST protein levels were evaluated by western blotting. Results showed that nuclear REST is increased in SH-SY5Y neuroblastoma cells cultured in SFM and exposed to IGF-I for 2-days and it then declines in 5-day-treated cells concomitant with a progressive neurite extension. Also the phorbol ester PMA was able to increase nuclear REST levels after 3-days treatment concomitant to neuronal differentiation of neuroblastoma cells, whereas, at later stages, it is down-regulated. Supporting these data, the exposure to PKC inhibitors (GF10923X and Gö6976) and PMA (16nM) reverted the effects observed with PMA alone. REST levels were related to morphological differentiation, expression of growth coneassociated protein 43 (GAP-43; a gene not regulated by REST) and of synapsin I and βIII tubulin (genes regulated by REST), proteins involved in the early stage of neuronal development. We observed that differentiation of SH-SY5Y cells by IGF-I and PMA was accompanied by a significant increase of these neuronal markers, an effect that was concomitant with REST decrease. In order to relate the decreased REST expression with a progressive neurite extension, I investigated any possible involvement of the ubiquitin–proteasome system (UPS), a multienzymatic pathway which degrades polyubiquinated soluble cytoplasmic proteins [Pickart and Cohen, 2004]. For this purpose, SH-SY5Y cells are concomitantly exposed to PMA and the proteasome inhibitor MG132. In SH-SY5Y exposed to PMA and MG 132, we observed an inverse pattern of expression of synapsin I and β- tubulin III, two neuronal differentiation markers regulated by REST. Their cytoplasmic levels are reduced when compared to cells exposed to PMA alone, as a consequence of the increase of REST expression by proteasome inhibitor. The majority of proteasome substrates identified to date are marked for degradation by polyubiquitinylation; however, exceptions to this principle, are well documented [Hoyt and Coffino, 2004]. Interestingly, REST degradation seems to be completely ubiquitin-independent. The expression pattern of REST could be consistent with the theory that, during early neuronal differentiation induced by IGF-I and PKC, it may help to repress the expression of several genes not yet required by the differentiation program and then it declines later. Interestingly, the observation that REST expression is progressively reduced in parallel with cell proliferation seems to indicate that the role of this transcription factor could also be related to cell survival or to counteract apotosis events [Lawinger et al., 2000] although, as shown by AS-ODN experiments, it does not seem to be directly involved in cell proliferation. Therefore, the decline of REST expression is a comparatively later event during maturation of neuroroblasts in vitro. Thus, we propose that REST is regulated by growth factors, like IGF-I, and PKC activators in a time-dependent manner: it is elevated during early steps of neural induction and could contribute to down-regulate genes not yet required by the differentiation program while it declines later for the acquisition of neural phenotypes, concomitantly with a progressive neurite extension. This later decline is regulated by the proteasome system activation in an ubiquitin-indipendent way and adds more evidences to the hypothesis that REST down-regulation contributes to differentiation and arrest of proliferation of neuroblastoma cells. Finally, the glycosylation pattern of the REST protein was analysed, moving from the observation that the molecular weight calculated on REST sequence is about 116 kDa but using western blotting this transcription factor appears to have distinct apparent molecular weight (see Table 1.1): this difference could be explained by post-translational modifications of the proteins, like glycosylation. In fact recently, several studies underlined the importance of O-glycosylation in modulating transcriptional silencing, protein phosphorylation, protein degradation by proteasome and protein–protein interactions [Julenius et al., 2005; Zachara and Hart, 2006]. Deglycosilating analysis showed that REST protein in SH-SY5Y and HEK293 cells is Oglycosylated and not N-glycosylated. Moreover, using several combination of deglycosilating enzymes it is possible to hypothesize the presence of Gal-β(1-3)-GalNAc residues on the endogenous REST, while β(1-4)-linked galactose residues may be present on recombinant REST protein expressed in HEK293 cells. However, the O-glycosylation process produces an immense multiplicity of chemical structures and monosaccharides must be sequentially hydrolyzed by a series of exoglycosidase. Further experiments are needed to characterize all the post-translational modification of the transcription factor REST.

Longterm peripheral baroreflex and chemoreflex function after bilateral eversion carotid endarterectomy

Introduction The “eversion” technique for carotid endarterectomy (e-CEA), that involves the transection of the internal carotid artery at the carotid bulb and its eversion over the atherosclerotic plaque, has been associated with an increased risk of postoperative hypertension possibly due to a direct iatrogenic damage to the carotid sinus fibers. The aim of this study is to assess the long-term effect of the e-CEA on arterial baroreflex and peripheral chemoreflex function in humans. Methods A retrospective review was conducted on a prospectively compiled computerized database of 3128 CEAs performed on 2617 patients at our Center between January 2001 and March 2006. During this period, a total of 292 patients who had bilateral carotid stenosis ≥70% at the time of the first admission underwent staged bilateral CEAs. Of these, 93 patients had staged bilateral e-CEAs, 126 staged bilateral s- CEAs and 73 had different procedures on each carotid. CEAs were performed with either the eversion or the standard technique with routine Dacron patching in all cases. The study inclusion criteria were bilateral CEA with the same technique on both sides and an uneventful postoperative course after both procedures. We decided to enroll patients submitted to bilateral e-CEA to eliminate the background noise from contralateral carotid sinus fibers. Exclusion criteria were: age >70 years, diabetes mellitus, chronic pulmonary disease, symptomatic ischemic cardiac disease or medical therapy with b-blockers, cardiac arrhythmia, permanent neurologic deficits or an abnormal preoperative cerebral CT scan, carotid restenosis and previous neck or chest surgery or irradiation. Young and aged-matched healthy subjects were also recruited as controls. Patients were assessed by the 4 standard cardiovascular reflex tests, including Lying-to-standing, Orthostatic hypotension, Deep breathing, and Valsalva Maneuver. Indirect autonomic parameters were assessed with a non-invasive approach based on spectral analysis of EKG RR interval, systolic arterial pressure, and respiration variability, performed with an ad hoc software. From the analysis of these parameters the software provides the estimates of spontaneous baroreflex sensitivity (BRS). The ventilatory response to hypoxia was assessed in patients and controls by means of classic rebreathing tests. Results A total of 29 patients (16 males, age 62.4±8.0 years) were enrolled. Overall, 13 patients had undergone bilateral e-CEA (44.8%) and 16 bilateral s-CEA (55.2%) with a mean interval between the procedures of 62±56 days. No patient showed signs or symptoms of autonomic dysfunction, including labile hypertension, tachycardia, palpitations, headache, inappropriate diaphoresis, pallor or flushing. The results of standard cardiovascular autonomic tests showed no evidence of autonomic dysfunction in any of the enrolled patients. At spectral analysis, a residual baroreflex performance was shown in both patient groups, though reduced, as expected, compared to young controls. Notably, baroreflex function was better maintained in e-CEA, compared to standard CEA. (BRS at rest: young controls 19.93 ± 2.45 msec/mmHg; age-matched controls 7.75 ± 1.24; e-CEA 13.85 ± 5.14; s-CEA 4.93 ± 1.15; ANOVA P=0.001; BRS at stand: young controls 7.83 ± 0.66; age-matched controls 3.71 ± 0.35; e-CEA 7.04 ± 1.99; s-CEA 3.57 ± 1.20; ANOVA P=0.001). In all subjects ventilation (VÝ E) and oximetry data fitted a linear regression model with r values > 0.8. Oneway analysis of variance showed a significantly higher slope both for ΔVE/ΔSaO2 in controls compared with both patient groups which were not different from each other (-1.37 ± 0.33 compared with -0.33±0.08 and -0.29 ±0.13 l/min/%SaO2, p<0.05, Fig.). Similar results were observed for and ΔVE/ΔPetO2 (-0.20 ± 0.1 versus -0.01 ± 0.0 and -0.07 ± 0.02 l/min/mmHg, p<0.05). A regression model using treatment, age, baseline FiCO2 and minimum SaO2 achieved showed only treatment as a significant factor in explaining the variance in minute ventilation (R2= 25%). Conclusions Overall, we demonstrated that bilateral e-CEA does not imply a carotid sinus denervation. As a result of some expected degree of iatrogenic damage, such performance was lower than that of controls. Interestingly though, baroreflex performance appeared better maintained in e-CEA than in s-CEA. This may be related to the changes in the elastic properties of the carotid sinus vascular wall, as the patch is more rigid than the endarterectomized carotid wall that remains in the e-CEA. These data confirmed the safety of CEA irrespective of the surgical technique and have relevant clinical implication in the assessment of the frequent hemodynamic disturbances associated with carotid angioplasty stenting.

Identification of the N-Linked Glycosylation Sites of the Transcription Factor Rest and Effect of Glycosylation on DNA Binding and Transcriptional Activity

REST is a zinc-finger transcription factor implicated in several processes such as maintenance of embryonic stem cell pluripotency and regulation of mitotic fidelity in non-neuronal cells [Chong et al., 1995]. The gene encodes for a 116-kDa protein that acts as a molecular platform for co-repressors recruitment and promotes modifications of DNA and histones [Ballas, 2005]. REST showed different apparent molecular weights, consistent with the possible presence of post-translational modifications [Lee et al., 2000]. Among these the most common is glycosylation, the covalent attachment of carbohydrates during or after protein synthesis [Apweiler et al., 1999] My thesis has ascertained, for the first time, the presence of glycan chians in the transcription factor REST. Through enzymatic deglycosylation and MS, oligosaccharide composition of glycan chains was evaluated: a complex mixture of glycans, composed of N-acetylgalactosamine, galactose and mannose, was observed thus confirming the presence of O- and N-linked glycan chains. Glycosylation site mapping was done using a 18O-labeling method and MS/MS and twelve potential N-glycosylation sites were identified. The most probable glycosylation target residues were mutated through site-directed mutagenesis and REST mutants were expressed in different cell lines. Variations in the protein molecular weight and mutant REST ability to bind the RE-1 sequence were analyzed. Gene reporter assays showed that, altogether, removal of N-linked glycan chains causes loss of transcriptional repressor function, except for mutant N59 which showed a slight residual repressor activity in presence of IGF-I. Taken togheter these results demonstrate the presence of complex glycan chians in the transcription factor REST: I have depicted their composition, started defining their position on the protein backbone and identified their possible role in the transcription factor functioning. Considering the crucial role of glycosylation and transcription factors activity in the aetiology of many diseases, any further knowledge could find important and interesting pharmacological application.

Kinematics and statics of cable-driven parallel robots by interval-analysis-based methods

In the past two decades the work of a growing portion of researchers in robotics focused on a particular group of machines, belonging to the family of parallel manipulators: the cable robots. Although these robots share several theoretical elements with the better known parallel robots, they still present completely (or partly) unsolved issues. In particular, the study of their kinematic, already a difficult subject for conventional parallel manipulators, is further complicated by the non-linear nature of cables, which can exert only efforts of pure traction. The work presented in this thesis therefore focuses on the study of the kinematics of these robots and on the development of numerical techniques able to address some of the problems related to it. Most of the work is focused on the development of an interval-analysis based procedure for the solution of the direct geometric problem of a generic cable manipulator. This technique, as well as allowing for a rapid solution of the problem, also guarantees the results obtained against rounding and elimination errors and can take into account any uncertainties in the model of the problem. The developed code has been tested with the help of a small manipulator whose realization is described in this dissertation together with the auxiliary work done during its design and simulation phases.