FAD Synthesis and Degradation in the Nucleus Create a Local Flavin Cofactor Pool

FAD is a redox cofactor ensuring the activity of many flavoenzymes mainly located in mitochondria but also relevant for nuclear redox activities. The last enzyme in the metabolic pathway producing FAD is FAD synthase (EC 2.7.7.2), a protein known to be localized both in cytosol and in mitochondria. FAD degradation to riboflavin occurs via still poorly characterized enzymes, possibly belonging to the NUDIX hydrolase family. By confocal microscopy and immunoblotting experiments, we demonstrate here the existence of FAD synthase in the nucleus of different experimental rat models. HPLC experiments demonstrated that isolated rat liver nuclei contain ∼300 pmol of FAD·mg⁻¹ protein, which was mainly protein-bound FAD. A mean FAD synthesis rate of 18.1 pmol·min⁻¹·mg⁻¹ protein was estimated by both HPLC and continuous coupled enzymatic spectrophotometric assays. Rat liver nuclei were also shown to be endowed with a FAD pyrophosphatase that hydrolyzes FAD with an optimum at alkaline pH and is significantly inhibited by adenylate-containing nucleotides. The coordinate activity of these FAD forming and degrading enzymes provides a potential mechanism by which a dynamic pool of flavin cofactor is created in the nucleus. These data, which significantly add to the biochemical comprehension of flavin metabolism and its subcellular compartmentation, may also provide the basis for a more detailed comprehension of the role of flavin homeostasis in biologically and clinically relevant epigenetic events.     

Utilization of nitrate abolishes the “Custers effect” in Dekkera bruxellensis and determines a different pattern of fermentation products

Nitrate is one of the most abundant nitrogen sources in nature. Several yeast species have been shown to be able to assimilate nitrate and nitrite, but the metabolic pathway has been studied in very few of them. Dekkera bruxellensis can use nitrate as sole nitrogen source and this metabolic characteristic can render D. bruxellensis able to overcome S. cerevisiae populations in industrial bioethanol fermentations. In order to better characterize how nitrate utilization affects carbon metabolism and the yields of the fermentation products, we investigated this trait in defined media under well-controlled aerobic and anaerobic conditions. Our experiments showed that in D. bruxellensis, utilization of nitrate determines a different pattern of fermentation products. Acetic acid, instead of ethanol, became in fact the main product of glucose metabolism under aerobic conditions. We have also demonstrated that under anaerobic conditions, nitrate assimilation abolishes the “Custers effect”, in this way improving its fermentative metabolism. This can offer a new strategy, besides aeration, to sustain growth and ethanol production for the employment of this yeast in industrial processes.     

Neuropsychopathological comorbidities in learning disorders

Background

Learning Disorders (LD) are complex diseases that affect about 2-10% of the school-age population. We performed neuropsychological and psychopathological evaluation, in order to investigate comorbidity in children with LD.

Methods

Our sample consisted of 448 patients from 7 to 16 years of age with a diagnosis of LD, divided in two subgroups: Specific Learning Disorders (SLD), including reading, writing, mathematics disorders, and Learning Disorders Not Otherwise Specified (LD NOS).

Results

Comorbidity with neuropsychopathologies was found in 62.2% of the total sample. In the LSD subgroup, ADHD was present in 33%, Anxiety Disorder in 28.8%, Developmental Coordination Disorder in 17.8%, Language Disorder in 11% and Mood Disorder in 9.4% of patients. In LD NOS subgroup, Language Disorder was present in 28.6%, Developmental Coordination Disorder in 27.5%, ADHD in 25.4%, Anxiety Disorder in 16.4%, Mood Disorder in 2.1% of patients. A statistically significant presence was respectively found for Language and Developmental Coordination Disorder comorbidity in LD NOS and for ADHD, mood and anxiety disorder comorbidity in SLD subgroup.

Conclusions

The different findings emerging in this study suggested to promote further investigations to better define the difference between SLD and LD NOS, in order to improve specific interventions to reduce the long range consequences.

     

Frequency of Discrimination,Harassment, and Violence in Lesbian,Gay Men,and Bisexual in Italy

Background

This cross-sectional study assessed the frequency of discrimination, harassment, and violence and the associated factors among a random sample of 1000 lesbian, gay men, and bisexual women and men recruited from randomly selected public venues in Italy.

Methods

A face-to-face interview sought information about: socio-demographics, frequency of discrimination, verbal harassment, and physical and sexual violence because of their sexual orientation, and their fear of suffering each types of victimization.

Results

In the whole sample, 28.3% and 11.9% self-reported at least one episode of victimization because of the sexual orientation in their lifetime and in the last year. Those unmarried, compared to the others, and with a college degree or higher, compared to less educated respondents, were more likely to have experienced an episode of victimization in their lifetime. Lesbians, compared to bisexual, had almost twice the odds of experiencing an episode of victimization. The most commonly reported experiences across the lifetime were verbal harassment, discrimination, and physical or sexual violence. Among those who had experienced one episode of victimization in their lifetime, 42.1% self-reported one episode in the last year. Perceived fear of suffering violence because of their sexual orientation, measured on a 10-point Likert scale with a higher score indicative of greater fear, ranges from 5.7 for verbal harassment to 6.4 for discrimination. Participants were more likely to have fear of suffering victimization because of their sexual orientation if they were female (compared to male), lesbian and gay men (compared to bisexual women and men), unmarried (compared to the others), and if they have already suffered an episode of victimization (compared to those who have not suffered an episode).

Conclusions

The study provides important insights into the violence experiences of lesbian, gay men, and bisexual women and men and the results may serve for improving policy initiatives to reduce such episodes.     

Evidence for the presence of photorespiration in desiccation-sensitive leaves of the C4 'resurrection' plant Sporobolus stapfianus during dehydration stress

The possible role of photorespiration as a general stress protectionmechanism, and in C₄ plant metabolism, is controversial. Inparticular, the potential involvement of photorespiration inthe acquisition of desiccation tolerance in ‘resurrection’plants is unknown. An investigation was carried out into whetherphotorespiration is present in leaves of the C₄ resurrectionplant Sporobolus stapfianus Gandoger (Poaceae) and whether itfunctions as a mechanism of stress resistance in the desiccation-tolerantyounger leaves (YL) of this plant. It is shown that the enzymesinvolved in the photorespiratory pathway maintain their activityuntil 88% relative water content (RWC) in both YL and desiccation-sensitiveolder leaves (OL). In subsequent stages of dehydration stress,the enzymatic activity declined similarly in both YL and OL.The content of the phorespiratory metabolite, serine, and ethanolamine,a direct product of serine decarboxylation, is higher in theearly stages of dehydration (88% RWC) in OL, suggesting a transientlyenhanced photorespiratory activity in these leaves. This wasconfirmed by simultaneous gas exchange and fluorescence measurements,showing suppression of the electron transport rate in OL exposedto non-photorespiratory conditions (2% O₂) at 85% RWC. It isconcluded that a higher photorespiratory electron transportoccurs in desiccation-sensitive OL, and it is therefore proposedthat the capacity to scavenge excess electrons through photorespirationdoes not contribute to protect leaves of the desiccation-tolerantYL of S. stapfianus during the stress. Key words: Ethanolamine, glycine, photorespiratory enzymes, photosynthesis, poikilohydric plant, serine Received 5 June 2007; Revised 3 September 2007 Accepted 17 September 2007     

Epithelial lining fluid free IGF-I-to-PAPP-A ratio is associated with bronchopulmonary dysplasia in preterm infants

Preterm newborns developing retinopathy of prematurity (ROP) and bronchopulmonary dysplasia (BPD) show persistently low levels of insulin-like growth factor-I (IGF-I) in sera. They also present higher free IGF-I concentrations in epithelial lining fluids (ELFs) and lung tissues. Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase that dissociates three binding proteins from the active form of IGF-I, namely free IGF-I. The present study analyzes the ELF concentrations of free IGF-I, PAPP-A, and their ratios in preterm newborns developing or not BPD, defined as O(2) dependence at 36 wk postmenstrual age. Bronchoalveolar lavage fluids of 41 infants (34 without and 7 with BPD) were analyzed on the 2nd and 4th day after birth. Infants developing BPD showed increased ELF free IGF-I and decreased PAPP-A concentrations on both days 2 and 4 compared with newborns without BPD. A nonsignificant trend between these 2 days was observed for free IGF-I (increasing) and PAPP-A (decreasing). On the same days, the free IGF-I-to-PAPP-A ratio was always significantly higher in patients developing BPD. These differences were more significant than those of IGF-I or PAPP-A when individually evaluated. A multivariate analysis confirmed the significance for free IGF-I on day 4, whereas the ratio was confirmed on both days 2 and 4. The same ratio was significantly correlated with some indexes of disease severity, such as hours of oxygen administration, days of hospitalization, and ROP severity scores. Finally, the ratio between ELF free IGF-I and PAPP-A appears to be a useful marker for lung injury of premature newborns.     

Glutaredoxin 1 is a major player in copper metabolism in neuroblastoma cells

Background

Glutaredoxin 1 (Grx1), a small protein belonging to the thioredoxin family, is involved in redox-regulation since it catalyzes the reduction of protein disulfides and that of mixed disulfides. It was reported to modulate active copper extrusion from cells, by affecting the function of the pumps ATP7A and B. These are components of the network of protein chaperones involved in the control of the homeostasis of copper, an essential, though harmful, metal. However, the effect of Grx1 on copper levels, copper chaperones and copper-elicited cell toxicity was never investigated.

Methods

In order to investigate the effect of Grx1 on copper metabolism, we constitutively overexpressed Grx1 in human neuroblastoma SH-SY5Y cells (SH-Grx1 cells) and assessed a number of copper-related parameters.

Results

SH-Grx1 cells show a basal intracellular copper level higher than control cells, accumulate more copper upon CuSO₄ treatment, but are more resistant to copper-induced toxicity. Grx1 shows copper-binding properties and copper overload produces a decrease of Grx1 enzyme activity in SH-Grx1 cells. Finally, Grx1 overexpression decreases copper accumulation in mitochondria upon copper overload and modulates the expression of copper transporter 1 (Ctr1).

Conclusion

Altogether, these data demonstrate that Grx1 is a major player in copper metabolism in neuronal cells.     

Diversity of Prokaryotic Community at a Shallow Marine Hydrothermal Site Elucidated by Illumina Sequencing Technology

To investigate the prokaryotic community structure and composition in an active hydrothermal site, named Black Point, off Panarea Island (Eolian Islands, Italy), we examined sediment and fluid samples, differing in temperature, by a massive parallel sequencing (Illumina) technique targeting the V3 region of the 16S rRNA gene. The used technique enabled us to detect a greater prokaryotic diversity than that until now observed and to reveal also microorganisms occurring at very low abundance (≤0.01 %). Most of sequences were assigned to Bacteria while Archaea were a minor component of the microbial community in both low- and high-temperature samples. Proteobacteria (mainly consisting of Alpha-, Gamma-, and Epsilonproteobacteria) dominated among all samples followed by Actinobacteria and Bacteroidetes. Analyzed DNA obtained from samples taken at different temperatures indicated the presence of members of different dominant genera. The main differences were observed between sediment samples where Rhodovulum and Thiohalospira prevailed at high temperature, while Thalassomonas and Sulfurimonas at low temperature. Chlorobium, Acinetobacter, Sulfurimonas, and Brevundimonas were abundant in both low- and high-temperature fluid samples. Euryarchaeota dominated the archaeal community in all samples. Classes of Euryarchaeota embracing hyperthermophilic members (Thermococci and Thermoplasmata) and of Crenarchaeota (Thermoprotei) were more abundant in high-temperature samples. A great number of sequences referred to Bacteria and Archaea still remained unaffiliated, indicating that Black Point site represents a rich source of so-far uncharted prokaryotic diversity.     

Identification of H ferritin-dependent and independent genes in K562 differentiating cells by targeted gene silencing and expression profiling

Ferritin is best known as the key molecule in intracellular iron storage, and is involved in several metabolic processes such as cell proliferation, differentiation and neoplastic transformation. We have recently demonstrated that the shRNA silencing of the ferritin heavy subunit (FHC) in a melanoma cell line is accompanied by a consistent modification of gene expression pattern leading to a reduced potential in terms of proliferation, invasiveness, and adhesion ability of the silenced cells.