High-throughput determination of urinary hexosamines for diagnosis of mucopolysaccharidoses by capillary electrophoresis and high-performance liquid chromatography

Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur, making possible therapies less effective. This highlights the importance of early and accurate diagnosis. A high-throughput procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and HPLC has been performed and validated in subjects affected by various MPS including their mild and severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio, and Maroteaux-Lamy. Contrary to other analytical approaches, the present single analytical procedure, which is able to measure total abnormal amounts of urinary GAGs, high molecular mass, and related fragments, as well as specific hexosamines belonging to a group of GAGs, would be useful for possible application in their early diagnosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized with 2-aminobenzoic acid and separated by CE/UV in ∼10 min and reverse-phase (RP)-HPLC in fluorescence in ∼21 min. The total content of hexosamines was found to be indicative of abnormal urinary excretion of GAGs in patients compared to the controls, and the galactosamine/glucosamine ratio was observed to be related to specific MPS syndromes in regard to both their mild and severe forms. As a consequence, important correlations between analytical response and clinical diagnosis and the severity of the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be ascribed to the quantity of total GAGs, as high-molecular-mass polymers and fragments, accumulated in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method is suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment.     

Lycopene and preclinical carotid atherosclerosis

Evidence from epidemiological and clinical studies suggests a possible correlation between serum antioxidant levels and cardiovascular disease risk. High plasma concentrations of lycopene have been associated with reduced prevalence of cardiovascular disease. The aim of this study is to compare plasma concentrations of lycopene in subjects with or without ultrasonic evidence of asymptomatic carotid atherosclerosis. One hundred and twenty subjects underwent physical examination, ultrasonic measurement of common carotid artery intima-media thickness and serum profile analysis. Logistic regression methods and analysis of variance were used to determine whether differences existed between participants with or without evidence of carotid atherosclerosis. Of the 120 participants, 58 exhibited evidence of carotid atherosclerosis. Participants with ultrasonic evidence of carotid atherosclerosis exhibited significantly higher serum concentrations of total cholesterol, LDL-cholesterol and triglycerides. In contrast, participants with ultrasonic evidence of carotid atherosclerosis exhibited significantly lower plasma concentrations of lycopene. These data suggest that higher serum levels of lycopene may play a protective role versus cardiovascular diseases, in particular carotid atherosclerosis.     

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.     

The Role of Extracellular Medium Components and Specific Amino Acids in the Cytotoxic Response of Escherichia Coli and Chinese Hamster Ovary Cells to Hydrogen Peroxide

A concentration of H₂O₂ resulting in mode one killing of Escherichia coli is more toxic when exposure to the oxidant is performed in complete medium (K medium), as compared to a saline (M9 salts). Inorganic salts (MgSO₄ and CaCl₂), thiamine or glucose, when added separately, or combined, to M9 salts had no effect on the cytotoxic response to H₂O₂. In contrast, the lethality of the oxidant was highly dependent on the presence of the amino acids in the incubation medium. The addition of glucose further enhanced this response. Among the seventeen amino acids which are present in the complete amino acid mixture, only two, i.e. L-histidine and L-cystine, were found to increase the toxicity of H₂O₂. Again, glucose augmented this response.

The effect of these amino acids on the growth inhibitory action of hydrogen peroxide was also tested in Chinese Hamster Ovary cells. It was found that L-histidine was capable of increasing the toxicity of the oxidant whereas all the other amino acids did not affect the toxicity of the oxidant. Glucose only slightly augmented this effect of L-histidine.

DNA single strand breakage produced by H₂O₂, was increased by L-histidine and was not significantly modified by the other amino acids. DNA double strand breakage was also shown to occur in cells exposed to H₂O₂-L-histidine, and this effect was independent on the presence of glucose.

These results demonstrate that the cytotoxic response of bacterial and mammalian cells to challenge with H₂O₂ is highly dependent on the composition of the extracellular milieu. Particularly relevant seems to be the effect of L-histidine, which markedly sensitizes both types of cells to the insult elicited by the oxidant, and that of L-cystine, which increases the sensitivity of E. coli cells.     

The benzomorphan-based LP1 ligand is a suitable MOR/DOR agonist for chronic pain treatment

AimsPowerful analgesics relieve pain primarily through activating mu opioid receptor (MOR), but the long-term use of MOR agonists, such as morphine, is limited by the rapid development of tolerance. Recently, it has been observed that simultaneous stimulation of the delta opioid receptor (DOR) and MOR limits the incidence of tolerance induced by MOR agonists. 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide (LP1) is a centrally acting agent with antinociceptive activity comparable to morphine and is able to bind and activate MOR and DOR. The aim of this work was to evaluate and compare the induction of tolerance to antinociceptive effects from treatment with LP1 and morphine.Main methodsHere, we evaluated the pharmacological effects of LP1 administered at a dose of 4 mg/kg subcutaneously (s.c.) twice per day for 9 days to male Sprague–Dawley rats. In addition, the LP1 mechanism of action was assessed by measurement of LP1-induced [³⁵S]GTPγS binding to the MOR and DOR.Key findingsData obtained from the radiant heat tail flick test showed that LP1 maintained its antinociceptive profile until the ninth day, while tolerance to morphine (10 mg/kg s.c. twice per day) was observed on day 3. Moreover, LP1 significantly enhanced [³⁵S]GTPγS binding in the membranes of HEK293 cells expressing either the MOR or the DOR.SignificanceLP1 is a novel analgesic agent for chronic pain treatment, and its low tolerance-inducing capability may be correlated with its ability to bind both the MOR and DOR.     

Peroxynitrite-induced mitochondrial translocation of PKCalpha causes U937 cell survival

Our previous work has shown that non-toxic concentrations of peroxynitrite nevertheless commit U937 cells to mitochondrial permeability-transition (MPT)-dependent necrosis that is however prevented by a parallel survival signaling pathway involving cytosolic phospholipase A2 (cPLA2)-dependent arachidonic acid release and PKCalpha activation associated with the cytosolic translocation of Bad. The present study provides evidence of an early mitochondrial translocation of PKCalpha. Inhibition of the survival signaling at the level of either cPLA2, or PKC, was invariably associated with prevention of the mitochondrial localization of PKCalpha, with the mitochondrial translocation of Bad and Bax and with a very rapid lethal response. Collectively, the results presented in this study demonstrate that peroxynitrite, while committing U937 cells to necrosis, triggers a parallel signaling response leading to the cytosolic localization of two important members of the Bcl-2 family implicated in the onset of MPT.