miR135a administration ameliorates brain ischemic damage by preventing TRPM7 activation during brain ischemia.

BACKGROUND: miRNA-based strategies have recently emerged as a promising therapeutic approach in several neurodegenerative diseases. Unregulated cation influx is implicated in several cellular mechanisms underlying neural cell death during ischemia. The brain constitutively active isoform of transient receptor potential melastatin 7 (TRPM7) represents a glutamate excitotoxicity-independent pathway that significantly contributes to the pathological Ca2+ overload during ischemia. AIMS: In the light of these premises, inhibition of TRPM7 may be a reasonable strategy to reduce ischemic injury. Since TRPM7 is a putative target of miRNA135a, the aim of the present paper was to evaluate the role played by miRNA135a in cerebral ischemia. Therefore, the specific objectives of the present paper were: (1) to evaluate miR135a expression in temporoparietal cortex of ischemic rats; (2) to investigate the effect of the intracerebroventricular (icv) infusion of miR135a on ischemic damage and neurological functions; and (3) to verify whether miR135a effects may be mediated by an alteration of TRPM7 expression. METHODS: miR135a expression was evaluated by RT- PCR and FISH assay in temporoparietal cortex of ischemic rats. Ischemic volume and neurological functions were determined in rats subjected to transient middle cerebral artery occlusion (tMCAo) after miR135a intracerebroventricular perfusion. Target analysis was performed by Western blot. RESULTS: Our results demonstrated that, in brain cortex, 72 h after ischemia, miR135a expression increased, while TRPM7 expression was parallelly downregulated. Interestingly, miR135a icv perfusion strongly ameliorated the ischemic damage and improved neurological functions, and downregulated TRPM7 protein levels. CONCLUSIONS: The early prevention of TRPM7 activation is protective during brain ischemia.

Polychlorinated biphenyls, organochlorine pesticides, and polycyclic aromatic hydrocarbons in wild, farmed, and frozen marine seafood marketed in Campania, Italy.

Polychlorinated biphenyls (PCBs), organochlorine pesticides, and polycyclic aromatic hydrocarbons (PAHs) were detected in fresh-catch, farmed, and frozen marine fish marketed in Campania, Italy. Additionally, polychlorobiphenyl congeners were found: six were non-dioxin-like (NDL-PCB) (IUPAC no. 28, 52, 101, 138, 153, and 180), and one was dioxin-like (DL-PCB) (IUPAC no. 118). In all, 93% of fresh-catch, 100% of aquaculture, and 74% of the frozen specimens contained PCBs at concentrations varying from 0.12 to 35.11 ng/g, wet weight; NDL-PCBs ranged between 0.12 and 32.44 ng/g. Penta-, hexa-, and heptachlorobiphenyls were predominant. Regarding organochlorine pesticides, hexachlorobenzene was detected in 35% of fresh catch, 36% of farmed, and 46% of the frozen fish specimens, in a range between < 0.01 and 3.29 ng/g. Contents of the dichlorodiphenyltrichloroethane isomer amounted to 0.12 to 11.00 ng/g. Finally, PAHs were detected in 100% of the specimens. Benzo[a]pyrene was detected in 66% of the aquaculture, 35% of the fresh catch, and 24% of the frozen species, at concentrations varying from 0.03 to 9.18 ng/g. On the basis of annual fish consumption, an average daily intake of NDL-PCBs of 6.02 ng/kg of body weight was estimated. Calculated daily hexachlorobenzene and total dichlorodiphenyltrichloroethane intakes were, respectively, 0.11 and 0.90 ng/kg of body weight per day. The contribution of fish to the daily consumption of the noncarcinogenic PAHs can be considered low; for benzo[a]pyrene, the estimated daily intake is considerably lower than the doses considered carcinogenic for experimental animals by the European Union Scientific Committee on Food.

Occurrence of polychlorobiphenyls in buffalo mozzarella cheese from Campania (Italy).

Buffalo milk and mozzarella cheese produced in the Caserta and Salerno areas in Campania region have been investigated on the presence and the levels of polychlorobiphenyls (PCBs). Seven congeners, six non dioxin-like (NDL-PCBs nos. 28, 52, 101, 138, 153 and 180) and one dioxin-like (DL-PCB n. 118), were detected. PCBs were found at detectable levels in the 83% of the buffalo milk and in the 100% of the mozzarella cheese samples from Caserta; in those from Salerno the prevalence of contamination was 77% for milk and 73% for mozzarellas, respectively. The NDL-PCB content of mozzarellas collected in Caserta was significantly higher than that found in those from Salerno. The more diffuse congeners were PCB 28, 138 and 153 both in milk and in mozzarella cheese; PCB 118 contributed to the total PCB content for the 7% in milk and 2-3% in mozzarella cheese. On the basis of the Italian annual average consumption the contribution of mozzarella to the daily dietary intake of NDL-PCB can vary between 0.41 and 21.33 ng kg(-1) bw, median value of 3.66 ng kg(-1) bw. The levels of contamination in milk and dairies analyzed are similar or quite lower than those found in other European countries.

GTP-cyclohydrolase I gene mutations in patients with autosomal dominant and recessive GTP-CH1 deficiency: identification and functional characterization of four novel mutations.

GTP-cyclohydrolase I (GTP-CH1, EC 3.5.4.16) is encoded by the GCH1 gene. Mutations in the GCH1 gene cause both dopa-responsive dystonia (McKusick 128230) and recessive GTP-CH1 deficiency (McKusick 600225). The exact molecular mechanism resulting in decreased GTP-CH1 activity in the patients is still obscure. We report the clinical features and molecular and functional study of the GCH1 gene in eight Italian patients affected by dominant and recessive GTP-CH1 deficiency. All the studied patients had mutations in the GCH1 gene. Three missense mutations (V205G, K224R, P199A), a frameshift mutation (Delta G693), and a splice-site mutation (ivs5 + 1g > c) were found. Except for K224R these are all novel mutations. To analyse the defect caused by the novel mutations, an in vivo functional assay in a Saccharomyces cerevisiae strain lacking the endogenous gene encoding GTP-CH1 ( FOL2 ) was performed. Complementation analysis showed that the Delta G693 and V205G mutations abolish the enzymatic function, while the P199A mutation causes a conditional defect. In conclusion, the clinical phenotypes displayed by our patients confirm the wide clinical spectrum of the disease and further support the lack of correlation between a given mutation and a clinical phenotype. Complementation analysis in yeast is a useful tool for confirming the pathogenetic effect of GCH1 mutations.