Role of autophagy in experimental varicocele: a Review

Varicocele (VC) is an abnormal venous dilatation and/or tortuosity of the pampiniform plexus in the scrotum (1). VC is one of the main factors causing male infertility and affects about 15% of all healthy male adults and about35% of patients with primary infertility (2), so it isparticularly important to clarify the pathogenetic mechanism in the course of male infertility. There is no single pathogenetic factor that alone justifies the adverse effects of varicocele on the testes. The pathogenesis of disease is complex and multifactorial, with several proposed mechanisms acting in synergy. Indeed, blood stasis negatively affects spermatogenesis by causing an increase in scrotal temperature (usually below 2°C at body temperature), hypoxic damage (stagnant hypoxia), a reflux of renal and adrenal catabolites, and last a modification of the testicular hormonal balance

Clinical score for surgical treatment of acute scrotum in pediatric age

Acute scrotum is a set of signs and symptoms that should lead the patient to go to a first aid center. There are different causes of these signs and symptoms, but testicular torsion is identified as the main ones and can be suspected thanks to a careful history, physical examination and testicular ultrasound. It is important to quickly differentiate the various conditions associated with acute scrotum in order to optimize surgery times and preserve testicular vitality. Often the radiological study is essential to identify the causes of acute scrotum, in particular the testicular Doppler ultrasound is sensitive and specific for testicular torsion, infact it is considered the gold standard for diagnosis. However, the radiological study can delay surgical treatment and reduce testicular vitality. So TWIST score was proposed. It is based on the identification of signs and symptoms that can lead at clinical diagnosis of testicular torsion. Our narrative of literature review analyzes TWIST score validity and shows the utility of an adequate clinical examination obtaining a diagnosis of torsion cord, reducing the costs and the time of surgical treatment resulting in a higher likelihood of preserving testicular vitalit

The N-Terminal Domain of ERK1 Accounts for the Functional Differences with ERK2

The Extracellular Regulated Kinase 1 and 2 transduce a variety of extracellular stimuli regulating processes as diverse as proliferation, differentiation and synaptic plasticity. Once activated in the cytoplasm, ERK1 and ERK2 translocate into the nucleus and interact with nuclear substrates to induce specific programs of gene expression. ERK1/2 share 85% of aminoacid identity and all known functional domains and thence they have been considered functionally equivalent until recent studies found that the ablation of either ERK1 or ERK2 causes dramatically different phenotypes. To search a molecular justification of this dichotomy we investigated whether the different functions of ERK1 and 2 might depend on the properties of their cytoplasmic-nuclear trafficking. Since in the nucleus ERK1/2 is predominantly inactivated, the maintenance of a constant level of nuclear activity requires continuous shuttling of activated protein from the cytoplasm. For this reason, different nuclear-cytoplasmic trafficking of ERK1 and 2 would cause a differential signalling capability. We have characterised the trafficking of fluorescently tagged ERK1 and ERK2 by means of time-lapse imaging in living cells. Surprisingly, we found that ERK1 shuttles between the nucleus and cytoplasm at a much slower rate than ERK2. This difference is caused by a domain of ERK1 located at its N-terminus since the progressive deletion of these residues converted the shuttling features of ERK1 into those of ERK2. Conversely, the fusion of this ERK1 sequence at the N-terminus of ERK2 slowed down its shuttling to a similar value found for ERK1. Finally, computational, biochemical and cellular studies indicated that the reduced nuclear shuttling of ERK1 causes a strong reduction of its nuclear phosphorylation compared to ERK2, leading to a reduced capability of ERK1 to carry proliferative signals to the nucleus. This mechanism significantly contributes to the differential ability of ERK1 and 2 to generate an overall signalling output

Differential involvement of Ras-GRF1 and Ras-GRF2 in L-DOPA-induced dyskinesia

Recent findings have shown that pharmacogenetic manipulations of the Ras-ERK pathway provide a therapeutic means to tackle l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID). First, we investigated whether a prolonged l-DOPA treatment differentially affected ERK signaling in medium spiny neurons of the direct pathway (dMSNs) and in cholinergic aspiny interneurons (ChIs) and assessed the role of Ras-GRF1 in both subpopulations. Second, using viral-assisted technology, we probed Ras-GRF1 and Ras-GRF2 as potential targets in this pathway. We investigated how selective blockade of striatal Ras-GRF1 or Ras-GRF2 expression impacted on LID (induction, maintenance, and reversion) and its neurochemical correlates

Comparison of the nucleo-cytoplasmic shuttling of ERK1 and ERK2.

<p>A) After photobleaching of the nucleus of starved NIH 3T3 cells, the ERK1 fluorescence recovered more slowly than ERK2, indicating a slower turnover of ERK1 across the nuclear membrane. Calibration bar 10 µm. B) Time course of the recovery of the cells showed in A. The data point were fitted with a single exponential with time constant τ of 235 sec (ERK2) and 430 sec (ERK1). C) Summary data for the sampled cells. The turnover of ERK1 is slower both in starved cells and after stimulation with FGF. In ERK1 starved cells the turnover is 3.7 time slower (653 s) than ERK2 (178 s) and in the stimulated cells ERK1 turnover (266 s) is 3.1 time slower than ERK2 (84 s). The blue symbol shows the turnover for a GFP dimer. This molecule is smaller than ERK-GFP but it crosses the nuclear membrane more slowly than ERK1, indicating that also for ERK1 is operating a mechanism of facilitated diffusion. D) Scatter diagram showing the recovery and Concentration Index of all paired measurements. Lines join observations relative to the same cell before and after stimulation. The filled symbols are the averages of the two groups.</p

The N-terminal domain is responsible for the slow nucleo-cytoplasmic shuttling of ERK1.

<p>A) At the N-terminus, ERK1 wild type (wt) contains 20 residues not present in ERK2. We produced fusion with GFP of three different deletions of ERK1, as indicated in the diagram (mouse sequence is exemplified). B) The time constant of the nucleo-cytoplasmic shuttling of ERK1 fusion proteins is strongly affected by the different deletions of the N-terminus. In this and the next figure, the number of cells is indicated over each column.</p

ERK1-GFP translocates in the nucleus of NIH-3T3 cells after stimulation.

<p>A) Cells transfected with ERK1-GFP in control conditions and after treatment with 80 ng/ml of FGF4 (calibration bar 20 µm). B) Time course of the normalized Concentration Index of cells stimulated with FGF4 (n = 18). The vertical bars are the standard error of the mean at the given point and are representative of the experimental variability of the entire data set. C) ERK-GFP fusion proteins are phosphorylated following serum stimulation, as demonstrated by western blot with a phospho-specific ERK antibody (upper panel). The fusion proteins have the correct molecular weight also when assayed with an anti-GFP antibody.</p

Computational estimate of the functional consequences of the different shuttling rates of ERK1 and 2.

<p>A) Cells transfected with ERK1-GFP and ERK2-GFP were treated with FGF4 for 15 min to allow complete nuclear translocation and then with the ERK blocker U0126. The inactivation of the ERK pathway caused the immediate loss of nuclear accumulation of ERK-GFP unmasking the action of nuclear dephosphorylation. Calibration bar 20 µm. B) By fitting the time course of the decay of nuclear ERK2-GFP (empty red diamonds) with the model output (continuous red line), we could estimate the dephosphorylation rate. This estimate was included in the model together with the shuttling rates measured for ERK1 to predict the outcome of this experiment practiced on the cells transfected with ERK1-GFP. The model prediction (green line) describes with great accuracy the experimental points (filled green symbols). C) Reaction scheme of the model. It has been considered an equilibrium among 4 different states regulated by first order kinetics: pERK and ERK indicates the concentration of the phosphorylated and not-phosphorylated pools in the cytoplasm (Cyt) or in the nucleus (Nuc); the rate constants α and β are the time constant of recovery of FRAP experiments; γ is the phosphorylation rate; γ' and δ' are the dephosphorylation rates, respectively, in the cytoplasm and in the nucleus. D) The model was used to compute the phosphorylation in the nucleus as a function of the shuttling speed. The empty symbols represent the result of a single simulation run and the filled symbols are the averages of each group. The phosphorylation level has been normalized to ERK2, therefore the computation shows that the total level of phosphorylation of ERK1 is only about half of ERK2. The numbers under each set of data points are the time constants (in seconds) of nuclear shuttling in the starved (above) and stimulated conditions (below).</p

Alignment of the amino acid sequences of rat ERK1 and ERK2.

<p>The N-terminus is shown with a larger font. The 20 aa present only in ERK1 are displayed in bold.</p

Functional consequences of N-terminus mutations of ERK1 and ERK2.

<p>A) In vitro assay testing the capacity of the indicated ERK to be activated by MEK and to phosphorylate the downstream target MBP. The upper lanes show the purified protein revealed by the anti-GFP antibody. The fusion proteins showed correct molecular weights (ERK1, E2>E1 71 kD; ERK2, E1>E2 69 kD). The lower panel indicates that each protein is able to phosphorylate MBP. The experiment was performed in triplicate with similar results. B) Representative examples of the cell colonies transfected with the indicated constructs. C) Immunoblot anti GFP shows that all the colonies expressed the GFP tagged proteins; the anti-HA and anti-Myc staining show that the mutated forms of Ras (respectively: H-Ras G12V and H-Ras Q61L), were expressed in the colonies with constitutive activation of the pathway, but they were absent in the wild type background. D) Quantification of the effect on proliferation of the various expressed fusion proteins. The symbols indicates the number of colonies counted after transfection with the specified vector, normalized to the colonies counted after transfection with the empty vector. The empty symbols represent the result of a single experiment and the filled symbols are the averages of each group. Expression of constitutively active Ras (H-Ras G12V and H-Ras Q61L) causes a large increase in proliferation that is inhibited by co-expressing ERK1 but not by co-expressing ERK2. The mutant of ERK1 characterized by fast shuttling (E1Δ39 indicated as E1>E2) did not prevent H-Ras G12V and Q61L-induced proliferation behaving similarly to ERK2. In contrast the slow mutant of ERK2 (Δ39E2 indicated as E2>E1) inhibited proliferation. Statistical significativity was essayed by <i>t</i>-test.</p