Carnitine is a pharmacological allosteric chaperone of the human lysosomal α-glucosidase

Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient’s enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.     

Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy

Aims

Activation of Ca²⁺/Calmodulin protein kinase II (CaMKII) is an important step in signaling of cardiac hypertrophy. The molecular mechanisms by which CaMKII integrates with other pathways in the heart are incompletely understood. We hypothesize that CaMKII association with extracellular regulated kinase (ERK), promotes cardiac hypertrophy through ERK nuclear localization.

Methods and Results

In H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction. Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses. To determine the role of CaMKII in cardiac hypertrophy in vivo, spontaneously hypertensive rats were subjected to intramyocardial injections of AntCaNtide or tat-CN17β. Left ventricular hypertrophy was evaluated weekly for 3 weeks by cardiac ultrasounds. We observed that the treatment with CaMKII inhibitors induced similar but significant reduction of cardiac size, left ventricular mass, and thickness of cardiac wall. The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart.

Conclusion

These results indicate that CaMKII and ERK interact to promote activation in hypertrophy; the inhibition of CaMKII-ERK interaction offers a novel therapeutic approach to limit cardiac hypertrophy.     

Site-specific replacement of the thymine methyl group by fluorine in thrombin binding aptamer significantly improves structural stability and anticoagulant activity

Here we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy, molecular modelling, differential scanning calorimetry and prothrombin time assay, on analogues of the thrombin binding aptamer (TBA) in which individual thymidines were replaced by 5-fluoro-2′-deoxyuridine residues. The whole of the data clearly indicate that all derivatives are able to fold in a G-quadruplex structure very similar to the ‘chair-like’ conformation typical of the TBA. However, only ODNs TBA-F4 and TBA-F13 have shown a remarkable improvement both in the melting temperature (ΔT_m ≈ +10) and in the anticoagulant activity in comparison with the original TBA. These findings are unusual, particularly considering previously reported studies in which modifications of T4 and T13 residues in TBA sequence have clearly proven to be always detrimental for the structural stability and biological activity of the aptamer. Our results strongly suggest the possibility to enhance TBA properties through tiny straightforward modifications.     

Zebularine partially reverses GST methylation in prostate cancer cells and restores sensitivity to the DNA minor groove binder brostallicin

Brostallicin is a DNA minor groove binder that shows enhanced antitumor activity in cells with high glutathione S-transferase (GST)/glutathione content. Prostate cancer cells present, almost invariably, methylation of the GSTP1 gene promoter and, as a consequence, low levels of GST-pi expression and activity. In these cells, brostallicin shows very little activity. We tested whether pretreatment of heavily GST-methylated prostate cancer cells with demethylating agents could enhance the activity of brostallicin. Human prostate cancer cells LNCaP and DU145 were used for these studies both in vitro and in vivo. The demethylating agent zebularine was used in combination with brostallicin. Methylation specific PCR and pyrosequencing were used to determine the level of GST methylation. Pretreatment with demethylating agents enhanced the in vitro activity of brostallicin in LNCaP cells. Zebularine, in particular, induced an enhancement of activity in vivo comparable to that obtained by transfecting the human GSTP1 gene in LNCaP cells in vitro. Molecular analysis performed on tumor xenografts in mice pretreated with zebularine failed to detect re-expression of GST-pi and demethylation of GSTP1. However, we found demethylation in the GSTM1 gene, with consequent re-expression of GST-mu at the mRNA level. These results indicate that zebularine, both in vitro and in vivo, enhances the activity of brostallicin and that this enhancement correlates with re-expression of GST-pi and GST-mu. These findings highlight the potential therapeutic value of combining demethylating agents and brostallicin in tumors with GST methylation that poorly respond to brostallicin.     

Protective Effects of Anthocyanins from Blackberry in a Rat Model of Acute Lung Inflammation

Anthocyanins are a group of naturally occuring phenolic compounds related to the coloring of plants, flowers and fruits. These pigments are important as quality indicators, as chemotaxonomic markers and for their antioxidant activities. Here, we have investigated the therapeutic efficacy of anthocyanins contained in blackberry extract (cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents) in an experimental model of lung inflammation induced by carrageenan in rats. Injection of carrageenan into the pleural cavity elicited an acute inflammatory response characterized by fluid accumulation which contained a large number of neutrophils as well as an infiltration of polymorphonuclear leukocytes in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx) and prostaglandin E₂ (PGE₂). All parameters of inflammation were attenuated in a dose-dependent manner by anthocyanins (10, 30 mg kg_-1 30 min before carrageenan). Furthermore, carrageenan induced an upregulation of the adhesion molecule ICAM-1, nitrotyrosine and poly (ADP-ribose) synthetase (PARS) as determined by immunohistochemical analysis of lung tissues. The degree of staining was lowered by anthocyanins treatment. Thus, the anthocyanins contained in the blackberry extract exert multiple protective effects in carrageenan-induced pleurisy.     

Genomic alterations in human umbilical cord–derived mesenchymal stromal cells call for stringent quality control before any possible therapeutic approach

Background aimsThe umbilical cord (UC) is a promising source of mesenchymal stromal cells (MSCs). UC-MSCs display very similar in vitro characteristics to bone marrow–MSCs and could represent a valuable alternative for cell-based therapies. However, it is still unclear whether UC-MSCs are prone or not to the acquisition of genomic imbalances during in vitro expansion.MethodsWith the use of array-comparative genomic hybridization, we compared copy number variations of early (P2–P3) and late (>P5) passages of in vitro–expanded UC-MSCs.ResultsIn two of 11 long-term UC-MSCs cultures, we observed the appearance of clones carrying genomic imbalances, which generated genetic mosaicism at intermediate passages. Although still able to reach the senescence phase, the cells carrying the genomic imbalance acquired a proliferative advantage, as demonstrated by the increase in frequency during long-term culture.ConclusionsAltogether, our results suggest that UC-MSC–based clinical protocols should be designed with caution; their clinical use should be preceded by array-comparative genomic hybridization screening for the acquisition of genomic imbalances during in vitro expansion.     

Genome‐wide linkage analysis for human longevity: Genetics of Healthy Aging Study

Clear evidence exists for heritability of human longevity, and much interest is focused on identifying genes associated with longer lives. To identify such longevity alleles, we performed the largest genome‐wide linkage scan thus far reported. Linkage analyses included 2118 nonagenarian Caucasian sibling pairs that have been enrolled in 15 study centers of 11 European countries as part of the Genetics of Healthy Aging (GEHA) project. In the joint linkage analyses, we observed four regions that show linkage with longevity; chromosome 14q11.2 (LOD = 3.47), chromosome 17q12‐q22 (LOD = 2.95), chromosome 19p13.3‐p13.11 (LOD = 3.76), and chromosome 19q13.11‐q13.32 (LOD = 3.57). To fine map these regions linked to longevity, we performed association analysis using GWAS data in a subgroup of 1228 unrelated nonagenarian and 1907 geographically matched controls. Using a fixed‐effect meta‐analysis approach, rs4420638 at the TOMM40/APOE/APOC1 gene locus showed significant association with longevity (P‐value = 9.6 × 10^?8). By combined modeling of linkage and association, we showed that association of longevity with APOEε4 and APOEε2 alleles explain the linkage at 19q13.11‐q13.32 with P‐value = 0.02 and P‐value = 1.0 × 10^?5, respectively. In the largest linkage scan thus far performed for human familial longevity, we confirm that the APOE locus is a longevity gene and that additional longevity loci may be identified at 14q11.2, 17q12‐q22, and 19p13.3‐p13.11. As the latter linkage results are not explained by common variants, we suggest that rare variants play an important role in human familial longevity.     

Design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide

Protein-inspired biomaterials have gained great interest as an alternative to synthetic polymers, in particular, for their potential use as biomedical devices. The potential inspiring models are mainly proteins able to confer mechanical properties to tissues and organs, such as elasticity (elastin, resilin, spider silk) and strength (collagen, silk). The proper combination of repetitive sequences, each of them derived from different proteins, represents a useful tool for obtaining biomaterials with tailored mechanical properties and biological functions. In this report we describe the design, the production, and the preliminary characterization of a chimeric polypeptide, based on sequences derived from the highly resilient proteins resilin and elastin and from collagen-like sequences. The results show that the obtained chimeric recombinant material exhibits promising self-assembling properties. Young's modulus of the fibers was determined by AFM image analysis and lies in the range of 0.1-3 MPa in agreement with the expectations for elastin-like and resilin-like materials.