Impairment of enzymatic antioxidant defenses is associated with bilirubin-induced neuronal cell death in the cerebellum of Ugt1 KO mice

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration.Unconjugated bilirubin (UCB) is the metabolic product of heme degradation in mammals. The enzymes heme oxygenase 1 and 2 (HO-1, HO-2) catalyze the degradation of heme into biliverdin, which is subsequently reduced to bilirubin by biliverdin reductase. In the liver, UCB is conjugated to glucuronic acid by UDP-glucuronosil transferase (UGT1a1), rendering it water soluble and excretable in the bile. In neonates, the late induction of Ugt1a1 gene expression may result in a limited capacity to conjugate bilirubin. The imbalance between bilirubin production and its elimination result in neonatal hyperbilirubinemia. Moderate jaundice is present in a high proportion of babies and is considered beneficial because of the antioxidant and cytoprotective properties of UCB.¹ However, excessive hyperbilirubinemia in newborns may produce acute bilirubin encephalopathy (BE), bilirubin-induced neurological disorders (BINDs) and eventually death by kernicterus.² BIND is characterized by a wide array of neurological deficits, including irreversible abnormalities in motor, sensitive and cognitive functions, because of UCB accumulation in the cerebellum, hippocampus and basal ganglia. Infants affected by null mutations in the Ugt1 gene develop Crigler–Najjar syndrome type I (CNSI).³ CNSI patients present high UCB plasma levels and are particularly exposed to BE and BIND if untreated. Current therapy initially consists in intensive phototherapy (PT) but liver transplantation is required later in life because of a reduction in PT efficiency.^{4, 5}Despite extensive investigations in animal models and in vitro tissue culture cells, the basic mechanisms of hyperbilirubinemia neurotoxicity have not been fully clarified yet.^{6, 7} UCB affects a large number of cellular functions and neurological damage appears to be the result of their concerted disruption rather than misregulation of a single pathway. The reported observations range from energy metabolism,⁸ cell proliferation,⁹ DNA and protein synthesis,¹⁰ receptor functionality,¹¹ to neurotransmitter uptake and release.¹² Our group and others reported that UCB is associated with an increased oxidative stress condition in cell culture.^{13, 14} In vitro studies showed that exposure to UCB decreases neuronal and glia viability.^{15, 16} In primary cultures, it has been observed that UCB permeabilizes mitochondrial membranes, resulting in mitochondrial swelling, release of cytochrome c into the cytosol, caspase-3 activation, Bax translocation and cell death by apoptosis.^{14, 17} Bilirubin also decreases NGF signaling to AKT and ERKs, interfering with prosurvival signaling pathways.¹⁸ However, most of the studies leading to potential mechanisms of bilirubin neurotoxicity were performed on monotypic cultures with artificial dosing of bilirubin, lacking the complexity of the interactions between different cell types, as it occurs in vivo.The aim of this study was to get a deeper unbiased insight into the molecular processes underlying bilirubin-induced neurodegeneration in vivo by using a mouse model of neonatal hyperbilirubinemia that shows early lethality because of bilirubin-induced neurological damage.¹⁹ Previous experiments conducted by our lab showed that the cerebellum of the Ugt1a^−/− mouse is the most vulnerable region of the brain to bilirubin toxicity.^{19, 20} Cerebellar susceptibility to bilirubin resulted in important alterations of its architecture, being the external germinal layer (EGL) and the Purkinje cell layer (PCL) the most affected regions, associated with an increase of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells.^{19, 20}In this study, we performed a differential proteomic analysis of the cerebellum from Ugt1 mutant and wild-type (WT) mice followed by validation of candidate genes both at protein and RNA levels. Our data point to an impairment of enzymatic antioxidant processes as important intracellular mechanisms involved in the onset of bilirubin-induced neurotoxicity in vivo.     

Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1

Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.     

Prediction of Cardiorespiratory Fitness by the Six-Minute Step Test and Its Association with Muscle Strength and Power in Sedentary Obese and Lean Young Women: A Cross-Sectional Study

Impaired cardiorespiratory fitness (CRF) is a hallmark characteristic in obese and lean sedentary young women. Peak oxygen consumption (VO_2peak) prediction from the six-minute step test (6MST) has not been established for sedentary females. It is recognized that lower-limb muscle strength and power play a key role during functional activities. The aim of this study was to investigate cardiorespiratory responses during the 6MST and CPX and to develop a predictive equation to estimate VO_2peak in both lean and obese subjects. Additionally we aim to investigate how muscle function impacts functional performance. Lean (LN = 13) and obese (OB = 18) women, aged 20–45, underwent a CPX, two 6MSTs, and isokinetic and isometric knee extensor strength and power evaluations. Regression analysis assessed the ability to predict VO_2peak from the 6MST, age and body mass index (BMI). CPX and 6MST main outcomes were compared between LN and OB and correlated with strength and power variables. CRF, functional capacity, and muscle strength and power were lower in the OB compared to LN (<0.05). During the 6MST, LN and OB reached ~90% of predicted maximal heart rate and ~80% of the VO_2peak obtained during CPX. BMI, age and number of step cycles (NSC) explained 83% of the total variance in VO_2peak. Moderate to strong correlations between VO_2peak at CPX and VO_2peak at 6MST (r = 0.86), VO_2peak at CPX and NSC (r = 0.80), as well as between VO_2peak, NSC and muscle strength and power variables were found (p<0.05). These findings indicate the 6MST, BMI and age accurately predict VO_2peak in both lean and obese young sedentary women. Muscle strength and power were related to measures of aerobic and functional performance.     

Morphological and physiological modifications of Cistus salvifolius L. winter leaves in response to the rise in winter temperatures

The global climate is predicted to change in the next century; for the Mediterranean Basin, an increase in air temperature more than 4°C and a higher frequency of extreme climatic events such as drought and heat waves are expected. In this work, the response of Cistus salvifolius L. to the rise in winter temperature has been studied. Plants acclimated to winter conditions [outdoor (OUT)] were moved into a greenhouse [indoor (IND)] at higher temperature and eco-physiological behaviour was analysed on leaves after 15 days from plant transferring (IND_15d) and on leaves developed IND. IND leaves were characterized by reduced thickness, higher specific leaf area, higher CO₂ mesophyll conductance and photosynthetic rate, and lower respiratory rate than leaves grown OUT upon current winter conditions. In IND_15d leaves, no improvement of photochemical activity was found. When IND leaves were subjected to a rapid increase in air temperature, the CO₂ fixation was not limited indicating a high thermotolerance of photosynthetic machinery. The results for IND leaves indicate the occurrence of a strategy that merging changes in leaf structure as well as in photosynthetic regulation allow C. salvifolius to maintain an elevated carbon gain in response to temperature increase.     

Response to biotic and oxidative stress in Arabidopsis thaliana: analysis of variably phosphorylated proteins

Protein phosphorylation plays a pivotal role in the regulation of many cellular events; increasing evidences indicate that this post-translational modification is involved in plant response to various abiotic and biotic stresses. Since phosphorylated proteins may be present at low abundance, enrichment methods are generally required for their analysis. We here describe the quantitative changes of phosphoproteins present in Arabidopsis thaliana leaves after challenging with elicitors or treatments mimicking biotic stresses, which stimulate basal resistance responses, or oxidative stress. Phosphoproteins from elicited and control plants were enriched by means of metal oxide affinity chromatography and resolved by 2D electrophoresis. A comparison of the resulting proteomic maps highlighted phosphoproteins showing quantitative variations induced by elicitor treatment; these components were identified by MALDI-TOF peptide mass fingerprinting and/or nanoLC-ESI-LIT-MS/MS experiments. In total, 97 differential spots, representing 75 unique candidate phosphoproteins, were characterized. They are representative of different protein functional groups, such as energy and carbon metabolism, response to oxidative and abiotic stresses, defense, protein synthesis, RNA processing and cell signaling. Ascertained protein phosphorylation found a positive confirmation in available Arabidopsis phosphoproteome database. The role of each identified phosphoprotein is here discussed in relation to plant defense mechanisms. Our results suggest a partial overlapping of the responses to different treatments, as well as a communication with key cellular functions by imposed stresses.     

The role of light quality of photoperiodic lighting on photosynthesis,flowering and metabolic profiling in Ranunculus asiaticus L.

Photoperiodic light quality affects flowering of long day plants, by influencing the phytochrome photoequilibria (PPE) at plant level; however, the most effective light spectrum to promote flowering is still unknown for most of the flower crops. We evaluated the influence of light spectrum of three light sources, with different induced PPE, on photosynthesis, metabolic profiling, plant growth and flowering in two hybrids of Ranunculus asiaticus L., MBO (early flowering) and MDR (medium earliness). Three photoperiodic treatments were compared to natural day length (NL): white fluorescent light (PPE 0.84), light emitting diodes (LEDs) with red:far red (R:FR) light at 3:1 ratio (PPE, 0.84) and LEDs with R:FR light at 1:3 ratio (PPE 0.63). Under natural light, net photosynthesis was higher in MDR than in MBO, while photochemistry was similar in the hybrids. Compared to NL, photoperiodic treatments did not affect net photosynthesis, while they promoted the quantum yield of PSII and reduced the non-photochemical quenching. Under NL, plant growth was greater in MBO, while flowering started earlier in MDR and flowers characteristics were similar in the hybrids. Despite the greater sensitivity of MDR plants in terms of metabolism, photoperiodic lighting improved plant growth and reduced the flowering time only in MBO, with a stronger effect under R:FR 3:1 light. MDR plants were characterized by higher soluble sugars, polyphenols, photosynthetic pigments and proteins, while MBO plants by higher starch and amino acid content. The morphological effects of photoperiodic light quality and the hybrid-specific response should be taken into account to optimize lighting protocols in commercial farms.     

Prevalence of systemic diseases in patients undergoing minor oral surgeries

It is of interest to evaluate the prevalence of systemic disorders in patients undergoing minor oral surgeries at a dental hospital. This will help to take necessary precautions prior to oral surgeries. We used the digital case records of 1288 patients who underwent minor oral surgeries in a hospital. Demographic details and systemic diseases of the patients were recorded from digital case records. Data shows that 103 patients (7.9%) of the total number of patients undergoing minor oral surgeries had systemic diseases with 3.8% of patients diagnosed with diabetes. Statistically significant associations were found between type of minor oral surgery and the type of systemic disease (p<0.001); age of patients and type of minor oral surgery (p<0.001); age and type of systemic diseases (p<0.001) and gender of patient and type of minor oral surgery (p = 0.005). Thus, data shows the prevalence of systemic diseases in patients undergoing minor oral surgeries was 7.9%.     

Characterization of a <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strain Able to Produce Tyramine and Partial Cloning of a Putative Tyrosine Decarboxylase Gene

The aim of this article was to analyze the ability of wine Lactobacillus plantarum strains to form tyramine. Preliminary identification of L. plantarum strains was performed by amplification of the recA gene. Primers pREV and PlanF, ParaF and PentF were used respectively as reverse and forward primers in the polymerase chain reaction tests as previously reported. Furthermore, the gene encoding for the tyrosine decarboxylase (TDC) was partially cloned from one strain identified as L. plantarum. The strain was further analyzed by 16S rDNA sequence and confirmed as belonging to L. plantarum species. The tyrosine decarboxylase activity was investigated and tyramine was determined by the high-performance liquid chromatography method. Moreover, a negative effect of sugars such as glucose and fructose and L-malic acid on tyrosine decarboxylase activity was observed. The results suggest that, occasionally, L. plantarum is able to produce tyramine in wine and this ability is apparently confined only to L. plantarum strains harboring the tdc gene.