Several lines of evidence point to a compromised proteostasis associated with a reduction of the Ubiquitin Proteasome System (UPS) activity in patients affected by Alzheimer's Disease (AD) and ...suggest that the amyloid β peptide (Aβ) is an important player in the game. Inspired also by many reports, underlining the presence of ubiquitin (Ub) in the amyloid plaques of AD brains, here we set out to test whether Ub may bind the Aβ peptide and have any effect on its clearance pathways. By using an integrated array of MALDI-TOF/UPLC-HRMS, fluorescence, NMR, SPR, Microscale Thermophoresis (MST) and molecular dynamics studies, we consistently demonstrated that Aβ40 binds Ub with a 1 : 1 stoichiometry and
K
d
in the high micromolar range. In particular, we show that the N-terminal domain of the Aβ peptide (through residues D1, E3 and R5) interacts with the C-terminal tail of Ub (involving residues K63 and E64), inducing the central region of Aβ (
14
HQKLVFFAEDVGSNK
28
) to adopt a mixed α-helix/β-turn structure. ELISA assays, carried out in neuroblastoma cell lysates, suggest that Aβ competitively binds Ub also in the presence of the entire pool of cytosolic Ub binding proteins. Ub-bound Aβ has a lower tendency to aggregate into amyloid-like fibrils and is more slowly degraded by the Insulin Degrading Enzyme (IDE). Finally, we observe that the water soluble fragment Aβ1-16 significantly inhibits Ub chain growth reactions. These results evidence how the non-covalent interaction between Aβ peptides and Ub may have relevant effects on the regulation of the upstream events of the UPS and pave the way to future
in vivo
studies addressing the role played by Aβ peptide in the malfunction of proteome maintenance occurring in AD.
Appetite for ubiquitin: a gushy travel companion in the intracellular journey of the amyloid β peptide.
We report an ATP-dependent ubiquitin conjugation with IDE which, in turn, promotes Ub-Ub linkages in tube tests. We propose a novel function for IDE as a non-canonical ubiquitin activating enzyme.
Purpose
Nearly, 40% of the causes of male infertility remain idiopathic. The only suggested treatment in idiopathic oligo- and/or asthenozoospermia in normogonadotropic patients is the FSH. In the ...current clinical practice, efficacy is exclusively assessable through semen analysis after 3 months of treatment. No molecular markers of treatment efficacy are appliable in clinical practice. The aim of the present work is to evaluate the combination of extracellular signal regulated kinase (ERK) 1 and 2 and prolactin inducible peptide (PIP) as potential markers of idiopathic infertility and FSH treatment efficacy.
Methods
Western blot and confocal microscopy were performed to analyze the modulation of PIP and ERK1/2 in idiopathic infertile patients (IIP) sperm cells. Taking advantage of mass spectrometry analysis, we identified these proteins unequivocally in sperm cells.
Results
We demonstrated a significant decrease of both PIP protein and of ERK1/2 levels in spermatozoa obtained from IIP in comparison to healthy fertile patients (HFP). Conversely, we reported a significant increase of these markers comparing infertile patients before and after 3 months of FSH treatment. Importantly, this correlated with an increase in total number of sperm and sperm motility after FSH treatment. Finally, we identified of PIP and ERK2 proteins in sperm samples by proteomic analysis.
Conclusions
The combined evaluation of ERK1/2 and PIP proteins might represent a useful molecular marker to tailor FSH treatment in the management of male normogonadotropic idiopathic infertility.
Abstract The present study addresses the question of evaluating, by combining both experimental and numerical approaches, the stress/strain distribution within a complete model of the entire lower ...bony chain. With this purpose an experimental model and a complete 3D finite element one were realised. A load of 700 N has been applied at the top of pelvis and the feet were rigidly fixed. Obtained results reveal interesting consequences deriving by taking into account the complete bony chain; it is possible to get information on load sharing between bones, location of high strain concentrations, and bone relative motion.
Introduction
Skeletal fragility with high risk of vertebral fractures is an emerging complication of acromegaly in close relationship with duration of active disease. The aim of this cross-sectional ...study was to evaluate the prevalence and determinants of vertebral fractures in males and females with a history of long-standing active acromegaly undergoing treatment with Pegvisomant.
Subjects and methods
Thirty-eight patients (25 females, 13 males) with acromegaly under Pegvisomant therapy were evaluated for vertebral fractures and bone mineral density at lumbar spine and femoral neck. Gonadal status, serum IGF1 levels and growth hormone receptor genotype were also assessed.
Results
Vertebral fractures were detected in 12 patients (31.6%). Fractured patients had longer duration of active disease (
p
= 0.01) with higher frequency of active acromegaly (
p
= 0.04), received higher dose of Pegvisomant (
p
= 0.008), and were more frequently hypogonadic (
p
= 0.02) as compared to patients who did not fracture. Stratifying the patients for gender, vertebral fractures were significantly associated with Pegvisomant dose (
p
= 0.02) and untreated hypogonadism (
p
= 0.02) in males and with activity of disease (
p
= 0.03), serum insulin-like growth factor-I values (
p
= 0.01) and
d3GHR
polymorphism (
p
= 0.005) in females. No significant association was found between vertebral fractures and bone mineral density at either skeletal site.
Conclusion
Vertebral fractures are a frequent complication of long-standing active acromegaly. When patients are treated with Pegvisomant, vertebral fractures may occur in close relationship with active acromegaly and coexistent untreated hypogonadism.
The interaction of the porphyrin derivative H
TCPPSpm4, having spermine pendants in the four meso positions, with the G-quadruplex (GQ) structure formed by the DNA aptamer TGGGAG has been ...investigated by means of UV, electronic circular dichroism and PAGE studies. The results reported here demonstrate that the porphyrin derivative is capable of stabilizing or destabilizing the higher-ordered structures of parallel GQs, depending on the method used to reach their relative stoichiometry (titration vs. single addition). Noteworthily, when two equivalents of H
TCPPSpm4 were mixed directly with one equivalent of the (TGGGAG)
GQ to reach a 2 : 1 H
TCPPSpm4 : GQ ratio T
higher than 80 °C was also observed confirming the presence of higher-ordered GQ structures.
The chemical composition of the cervical mucus (CM), its physical characteristics and the volume of secretion change cyclically throughout the menstrual cycle. The aim of this study was to identify ...the constitutive protein composition of CM of fertile women and the changes in the CM proteome throughout the menstrual cycle. Five fertile women who had a term delivery within 1 year before the study were enrolled. Proteomic analysis was performed using an Ultimate 3000 Nano/Micro-HPLC apparatus equipped with an FLM-3000-Flow manager module and coupled with an LTQ Orbitrap XL hybrid mass spectrometer; bioinformatic software was used for functional and quantitative analysis. 59, 81 and 43 proteins (mean) were respectively identified in the pre-ovulatory, ovulatory and post-ovulatory samples. 38 common proteins were identified. 42, 38 and 17 exclusive proteins were respectively identified in pre-ovulatory, ovulatory and post-ovulatory CM. The main part of CM constituents has a catalytic activity, which is mainly related to hydrolase activity. The label-free quantitative analysis of the common proteins revealed a significant reduction in the protein abundance index for antileukoproteinase, after the ovulation, and a peak of haptoglobin at ovulation. This is the first application of high-resolution MS-based proteomics for the identification of protein constituents of CM. This approach may contribute to the identification of putative biomarkers of the female reproductive tract.
Mature microRNAs are short non-coding RNA sequences which upon incorporation into the RISC ribonucleoprotein complex, play a crucial role in regulation of gene expression. However, miRNAs can exist ...within the cell also as free molecules fulfilling their biological activity. Therefore, it is emerging that in addition to sequence even the structure adopted by mature miRNAs might play an important role to reach the target. Indeed, we analysed by several spectroscopic techniques the secondary structures of two artificial miRNAs selected by computational tool (miR-Synth) as best candidates to silence c-MET and EGFR genes and of two endogenous miRNAs (miR-15a and miR-15b) having the same seed region, but different biological activity. Our results demonstrate that both endogenous and artificial miRNAs can arrange in several 3D-structures which affect their activity and selectivity toward the targets.
Summary
Introduction
Growth hormone deficiency is considered the most important factor determining skeletal fragility in hypopituitary patients. Osteoblasts and chondrocytes express growth hormone ...(GH) receptor. Two GH receptor isoforms (GHRi) have been identified: they differ for the presence/absence of a protein fragment encoded by exon 3 of GHR gene. Consequently, three genotypes were identified: carriers of both the full‐length proteins (flfl‐GHR), carriers of one full‐length protein and one deleted protein (fld3‐GHR) and carriers of both deleted proteins (d3d3‐GHR). This polymorphism confers a higher sensitivity to endogenous GH and to recombinant human GH (rhGH); its effect on bone metabolism and skeletal fragility is unknown. The aim of this article was to investigate the role of GHRi in predicting skeletal fragility in adult‐onset GHD (AO‐GHD) patients.
Subjects and methods
A cross‐sectional study was conducted to investigate the association between the d3‐GHR isoform and the prevalence of morphometric vertebral fractures (VFs) in AO‐GHD. Ninety‐three AO‐GHD were enrolled. Forty‐nine patients carried flfl‐GHRi (52·7%), and 44 patients (47·3%) carried at least one allele of the d3‐GHR isoform. Thirty‐two VFs were documented. Fifty‐seven patients underwent rhGH replacement therapy.
Results
Median age was significantly higher in fractured patients as compared to nonfractured ones; d3‐carrier patients showed a lower VF risk as compared to flfl‐GHRi (OR: 0·37, 95% IC: 0·24–0·55, P < 0·0001). This finding was also confirmed in AO‐GHD undergoing rhGH replacement therapy.
Conclusion
This study suggests that d3‐GHR may protect AO‐GHD particularly when treated with rhGH from the risk of VFs.
Periodontal disease is characterized by inflammation and bone loss. The balance between inflammatory mediators and their counter-regulatory molecules may be fundamental for determining the outcome of ...immune pathology of periodontal disease. Cytokines play crucial roles in the maintenance of tissue homeostasis, a process which requires a delicate balance between anabolic and catabolic activities. In particular, two families of growth factors-such as transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF) are thought to play important roles in modulating the proliferation and/or migration of structural cells involved in inflammation and regulation of immune responses. The aim of this work was to analyze gingival samples and periodontal tissue specimens collected from thirty-eight patients with chronic periodontal disease and from forty healthy individuals, in order to detect the expression and distribution of TGF-β1 and VEGF between the two groups. TGF-β1 and VEGF expression levels were detected using immunohistochemical analysis and computer-assisted morphometric analysis. The findings presented here suggest that biomarker such as TGF-β1 and VEGF have an important regulating role in the orchestration of the immune response, which in turn influence the outcome of disease establishment and evolution.
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