Cellular and functional analysis of four mutations located in the mitochondrial ATPase6 gene

The smallest rotary motor of living cells, F0F1‐ATP synthase, couples proton flow—generated by the OXPHOS system—from the intermembrane space back to the matrix with the conversion of ADP to ATP. While all mutations affecting the multisubunit complexes of the OXPHOS system probably impact on the cell's output of ATP, only mutations in complex V can be considered to affect this output directly. So far, most of the F0F1‐ATP synthase variations have been detected in the mitochondrial ATPase6 gene. In this study, the four most frequent mutations in the ATPase6 gene, namely L156R, L217R, L156P, and L217P, are studied for the first time together, both in primary cells and in cybrid clones. Arginine (“R”) mutations were associated with a much more severe phenotype than Proline (“P”) mutations, in terms of both biochemical activity and growth capacity. Also, a threshold effect in both “R” mutations appeared at 50% mutation load. Different mechanisms seemed to emerge for the two “R” mutations: the F1 seemed loosely bound to the membrane in the L156R mutant, whereas the L217R mutant induced low activity of complex V, possibly the result of a reduced rate of proton flow through the A6 channel. J. Cell. Biochem. 106: 878–886, 2009. © 2009 Wiley‐Liss, Inc.     

SING: Subgraph search In Non-homogeneous Graphs

Background  

Finding the subgraphs of a graph database that are isomorphic to a given query graph has practical applications in several fields, from cheminformatics to image understanding. Since subgraph isomorphism is a computationally hard problem, indexing techniques have been intensively exploited to speed up the process. Such systems filter out those graphs which cannot contain the query, and apply a subgraph isomorphism algorithm to each residual candidate graph. The applicability of such systems is limited to databases of small graphs, because their filtering power degrades on large graphs.     

Involvement of the mitochondrial compartment in human NCL fibroblasts

Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.     

Novel large-range mitochondrial DNA deletions and fatal multisystemic disorder with prominent hepatopathy

Hepatic involvement in mitochondrial cytopathies rarely manifests in adulthood, but is a common feature in children. Multiple OXPHOS enzyme defects in children with liver involvement are often associated with dramatically reduced amounts of mtDNA. We investigated two novel large scale deletions in two infants with a multisystem disorder and prominent hepatopathy. Amount of mtDNA deletions and protein content were measured in different post-mortem tissues. The highest levels of deleted mtDNA were in liver, kidney, pancreas of both patients. Moreover, mtDNA deletions were detected in cultured skin fibroblasts in both patients and in blood of one during life. Biochemical analysis showed impairment of mainly complex I enzyme activity. Patients manifesting multisystem disorders in childhood may harbour rare mtDNA deletions in multiple tissues. For these patients, less invasive blood specimens or cultured fibroblasts can be used for molecular diagnosis. Our data further expand the array of deletions in the mitochondrial genomes in association with liver failure. Thus analysis of mtDNA should be considered in the diagnosis of childhood-onset hepatopathies.     

The utility of microsatellite DNA markers for the evaluation of area-wide integrated pest management using SIT for the fruit fly, Bactrocera dorsalis (Hendel), control programs in Thailand

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a key pest that causes reduction of the crop yield within the international fruit market. Fruit flies have been suppressed by two Area-Wide Integrated Pest Management programs in Thailand using Sterile Insect Technique (AW-IPM-SIT) since the late 1980s and the early 2000s. The projects’ planning and evaluation usually rely on information from pest status, distribution, and fruit infestation. However, the collected data sometimes does not provide enough detail to answer management queries and public concerns, such as the long term sterilization efficacy of the released fruit fly, skepticism about insect migration or gene flow across the buffer zone, and the re-colonisation possibility of the fruit fly population within the core area. Established microsatellite DNA markers were used to generate population genetic data for the analysis of the fruit fly sampling from several control areas, and non-target areas, as well as the mass-rearing facility. The results suggested limited gene flow (m < 0.100) across the buffer zones between the flies in the control areas and flies captured outside. In addition, no genetic admixture was revealed from the mass-reared colony flies from the flies within the control area, which supports the effectiveness of SIT. The control pests were suppressed to low density and showed weak bottleneck footprints although they still acquired a high degree of genetic variation. Potential pest resurgence from fragmented micro-habitats in mixed fruit orchards rather than pest incursion across the buffer zone has been proposed. Therefore, a suitable pest control effort, such as the SIT program, should concentrate on the hidden refuges within the target area.     

Muscular laminopathies: role of prelamin A in early steps of muscle differentiation

Lamin A is a nuclear envelope constituent involved in a group of human disorders, collectively referred to as laminopathies, which include Emery-Dreifuss muscular dystrophy. Because increasing evidence suggests a role of lamin A precursor in nuclear functions, we investigated the processing of prelamin A along muscle differentiation. Both protein levels and cellular localization of prelamin A appears to be modulated during C2C12 mouse myoblasts activation. Similar changes also occur in the expression of two lamin A-binding proteins: emerin and LAP2α. Furthermore prelamin A forms a complex with LAP2α in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affects LAP2α and PCNA amount and increases caveolin 3 mRNA and protein levels, whilst accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor inhibits caveolin 3 expression. These data provide evidence for a critical role of lamin A precursor in the early steps of muscle cell differentiation. In fact the post-translational processing of prelamin A affects caveolin 3 expression and influences the myoblast differentiation process. Thus, altered lamin A processing could affect myoblast differentiation and/or muscle regeneration and might contribute to the myopathic phenotype.     

Safety and efficacy of lactoferrin versus ferrous sulphate in curing iron deficiency and iron deficiency anaemia in hereditary thrombophilia pregnant women: an interventional study

Objective Evaluate the safety and efficacy of bovine lactoferrin (bLf) versus the ferrous sulphate standard intervention in curing iron deficiency (ID) and ID anaemia (IDA) in pregnant women affected by hereditary thrombophilia (HT). Design Interventional study. Setting Secondary-level hospital for complicated pregnancies in Rome, Italy. Population 295 HT pregnant women (≥18 years) suffering from ID/IDA. Methods Women were enrolled in Arm A or B in accordance with their personal choice. In Arm A, 156 women received oral administration of 100 mg of bLf twice a day; in Arm B, 139 women received 520 mg of ferrous sulphate once a day. Therapies lasted until delivery. Main outcome measures Red blood cells, haemoglobin, total serum iron, serum ferritin (haematological parameters) were assayed before and every 30 days during therapy until delivery. Serum IL-6, key factor in inflammatory and iron homeostasis disorders, was detected at enrolment and after therapy at delivery. Possible maternal, foetal, and neonatal adverse effects were assessed. Results Haematological parameters were significantly higher in Arm A than in Arm B pregnant women (P ≤ 0.0001). Serum IL-6 significantly decreased in bLf-treated women and increased in ferrous sulphate-treated women. BLf did not exert any adverse effect. Adverse effects in 16.5 % of ferrous sulphate-treated women were recorded. Arm A women experienced no miscarriage compared to five miscarriages in Arm B women. Conclusions Differently from ferrous sulphate, bLf is safe and effective in curing ID/IDA associated with a consistent decrease of serum IL-6. The absence of miscarriage among bLf-treated women provided an unexpected benefit. Trial registration: ClinicalTrials.gov Identifier NCT01221844.     

Lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases

Conflicting data are reported on pro- or anti-inflammatory activity of bovine lactoferrin (bLf) in different cell models as phagocytes or epithelial cell lines infected by bacteria. Here we evaluated the bLf effect on epithelial models mimicking two human pathologies characterized by inflammation and infection with specific bacterial species. Primary bronchial epithelium from a cystic fibrosis (CF) patient and differentiated intestinal epithelial cells were infected with Pseudomonas aeruginosa LESB58 isolated from a CF patient and Adherent-Invasive Escherichia coli LF82 isolated from a Crohn’s disease patient. Surprisingly, bLf significantly reduced the intracellular bacterial survival, but differently modulated the inflammatory response. These data lead us to hypothesize that bLf differentially acts depending on the epithelial model and infecting pathogen. To verify this hypothesis, we explored whether bLf could modulate ferroportin (Fpn), the only known cellular iron exporter from cells, that, by lowering the intracellular iron level, determines a non permissive environment for intracellular pathogens. Here, for the first time, we describe the bLf ability to up-regulate Fpn protein in infected epithelial models. Our data suggest that the mechanism underlying the bLf modulating activity on inflammatory response in epithelial cells is complex and the bLf involvement in modulating cellular iron homeostasis should be taken into account.