Increased rate of superoxide ion generation in Fanconi anemia erythrocytes

The rate of generation of superoxide ion, the concentration of Cu, Zn superoxide dismutase and the hematological parameters were measured in red blood cells obtained from Fanconi anemia patients and from healthy individuals. No significative difference in the superoxide dismutase concentration was found, while the rate of generation of the superoxide ion doubled in Fanconi anemia patients. The steady-state concentration of the superoxide ion was calculated from these data and was found to be 2.3 times higher in Fanconi anemia erythrocytes than in controls. The possible consequences with respect to the alterations in FA are discussed.     

Evaluation of the Antioxidant Properties of Propofol and its Nitrosoderivative. Comparison with Homologue Substituted Phenols

Propofol (2,6-diisopropylphenol), some substituted phenols (2,6-dimethylphenol and 2,6-ditertbutylphenol) and their 4-nitrosoderivatives have been compared for their scavenging ability towards 1,1-diphenyl-2-picrylhydrazyl and for their inhibitory action on lipid peroxidation. These products were also compared to the classical antioxidants butylated hydroxytoluene and butylated hydroxyanisole. When measuring the reactivity of the various phenolic derivatives with 1,1-diphenyl-2-picrylhydrazyl the following order of effectiveness was observed: butylated hydroxyanisole>propofol>2,6-dimethylphenol>2,6-di-tertbutylphenol?>?butylated hydroxytoluene. In cumene hydroperoxide-dependent microsomal lipid peroxidation, propofol acts as the most effective antioxidant, while butylated hydroxyanisole, 2,6-di-tertbutylphenol and butylated hydroxytoluene exhibit a rather similar effect, although lower than propofol. In the iron/ascorbate-dependent lipid peroxidation propofol, at concentrations higher than 10?μM, exhibits antioxidant properties comparable to those of butylated hydroxytoluene and butylated hydroxyanisole. 2,6-Dimethylphenol is scarcely effective in both lipoperoxidative systems. The antioxidant properties of the various molecules depend on their hydrophobic characteristics and on the steric and electronic effects of their substituents. However, the introduction of the nitroso group in the 4-position almost completely removes the antioxidant properties of the examined compounds. The nitrosation of the aromatic ring of antioxidant molecules and the consequent loss of antioxidant capacity can be considered a condition potentially occurring in vivo since nitric oxide and its derivatives are continuously formed in biological systems.     

Specific nitration at tyrosine 430 revealed by high resolution mass spectrometry as basis for redox regulation of bovine prostacyclin synthase

Treatment of bovine aortic microsomes containing active prostacyclin synthase (PGI(2) synthase) with increasing concentrations of peroxynitrite (PN) up to 250 microm of PN yielded specific staining of this enzyme on Western blots with antibodies against 3-nitrotyrosine (3-NT), whereas above 500 microm PN staining of additional proteins was also observed. Following treatment of aortic microsomes with 25 microm PN, PGI(2) synthase was about half-maximally nitrated and about half-inhibited. It was then isolated by gel electrophoresis and subjected to proteolytic digestion with several proteases. Digestion with thermolysin for 24 h provided a single specific peptide that was isolated by high performance liquid chromatography and identified as a tetrapeptide Leu-Lys-Asn-Tyr(3-nitro)-COOH corresponding to positions 427-430 of PGI(2) synthase. Its structure was established by precise mass determination using Fourier transform-ion cyclotron resonance-nanoelectrospray mass spectrometry and Edman microsequencing and ascertained by synthesis and mass spectrometric characterization of the authentic Tyr-nitrated peptide. Complete digestion by Pronase to 3-nitrotyrosine was obtained only after 72 h, suggesting that the nitrated Tyr-430 residue may be embedded in a tight fold around the heme binding site. These results provide evidence for the specific inhibition of PGI(2) synthase by nitration at Tyr-430 that may occur already at low levels of PN as a consequence of endothelial co-generation of nitric oxide and superoxide.     

Bmp2 and Bmp4 genetically interact to support multiple aspects of mouse development including functional heart development

Bone morphogenetic proteins (BMPs) have multiple roles during embryogenesis. Current data indicate that the dosage of BMPs is tightly regulated for normal development in mice. Since Bmp2 or Bmp4 homozygous mutant mice show early embryonic lethality, we generated compound heterozygous mice for Bmp2 and Bmp4 to explore the impact of lowered dosage of these BMP ligands. Genotyping pups bred between Bmp2 and Bmp4 heterozygous mice revealed that the ratio of adult compound heterozygous mice for Bmp2 and Bmp4 is much lower than expected. During embryogenesis, the compound heterozygous embryos showed several abnormalities, including defects in eye formation, body wall closure defects, and ventricular septal defects (VSD) in the heart. However, the ratio of the compound heterozygous embryos was the same as expected. Caesarean sections at E18.5 revealed that half of the compound heterozygotes died soon after birth, and the majority of the dead individuals exhibited VSD. Survivors were able to grow to adults, but their body weight was significantly lower than control littermates. They demonstrated progressive abnormalities in the heart, eventually showing a branched leaflet in atrioventricular valves. These results suggest that the dosage of both BMP2 and 4 is critical for functional heart formation during embryogenesis and after birth. genesis 47:374–384, 2009. © 2009 Wiley‐Liss, Inc.     

Polyketone polymer: a new support for direct enzyme immobilization

Polyketone polymer -[-CO-CH(2)-CH(2)-](n)-, obtained by copolymerization of ethene and carbon monoxide, is utilized for immobilization of three different enzymes, one peroxidase from horseradish (HRP) and two amine oxidases, from bovine serum (BSAO) and lentil seedlings (LSAO). The easy immobilization procedure is carried out in diluted buffer, at pH 7.0 and 3 degrees C, gently mixing the proteins with the polymer. No bifunctional reagents and spacer arms are required for the immobilization, which occurs exclusively via a large number of hydrogen bonds between the carbonyl groups of the polymer and the -NH groups of the polypeptidic chain. Experiments demonstrate a high linking capacity of polymer for BSAO and an extraordinary strong linkage for LSAO. Moreover, activity measurements demonstrate that immobilized LSAO totally retains the catalytic characteristics of the free enzyme, where only a limited increase of K(M) value is observed. Finally, the HRP-activated polymer is successfully used as active packed bed of an enzymatic reactor for continuous flow conversion and flow injection analysis of hydrogen peroxide containing solutions.     

Interaction of fluoxetine with phosphatidylcholine liposomes

Fluoxetine (Prozac) is one of the latest of a new generation of antidepressants, approved by FDA in 2002. The interactions of fluoxetine with multilamellar liposomes of pure phosphatidylcholine (PC) or containing cholesterol 10% molar were studied as a function of the lipid chain lengths, using differential scanning calorimetry and spin labelling EPR techniques. The DSC profiles of the gel-to-fluid state transition of liposomes of DMPC (C14:0) are broadened and shifted towards lower temperatures at increasing dopant concentrations and, with less than 10% fluoxetine, any detectable transition is destroyed. The broadened profiles and the lowered transition temperatures demonstrate that both the size and the packing of the cooperative units undergoing the transition are modified by fluoxetine, leading to a looser and more flexible bilayer. No phase separation was observed. The effects of fluoxetine on the thermotropic phase behaviour of DPPC (C16:0) and, even more, of DSPC (C18:0) are different from that of DMPC. In fact, in the former cases, two peaks appeared at increasing dopant concentrations, suggesting the occurrence of a phase separation phenomenon, which is a sign of a binding of fluoxetine in the phosphate region. In cholesterol containing membranes, fluoxetine, even at low concentrations, leads to a general corruption of the membrane, both in terms of packing and cooperativity, and the formation of any new phase is no longer observable. EPR spectra reflect the disordered motion of acyl chains in the bilayer. It was found that fluoxetine lowers the order of the lipid chains mainly in correspondence of the fifth carbon position of SASL, indicating a possible accumulation near the interfacial region.     

Scission of the lactyl ether bond of N-acetylmuramic acid by Escherichia coli "etherase"

The ubiquitous bacterial cell wall sugar N-acetylmuramic acid (MurNAc) carries a unique D-lactyl ether substituent at the C3 position. Recently, we proposed an etherase capable of cleaving this lactyl ether to be part of the novel bacterial MurNAc dissimilation pathway (Dahl, U., Jaeger, T., Nguyen, B. T., Sattler, J. M., Mayer, C. (2004) J. Bacteriol. 186, 2385-2392). Here, we report the identification of the first known MurNAc etherase. The encoding gene murQ is located at 55 min on the Escherichia coli chromosome adjacent to murP, the MurNAc-specific phosphotransferase system. A murQ deletion mutant could not grow on MurNAc as the sole source of carbon and energy but could be complemented by expressing murQ from a plasmid. The mutant had no obvious phenotype when grown on different carbon sources but accumulated MurNAc 6-phosphate at millimolar concentrations from externally supplied MurNAc. Purified MurQ-His6 fusion protein and extracts of cells expressing murQ both catalyze the cleavage of MurNAc 6-phosphate, with GlcNAc 6-phosphate and D-lactate being the primary products. The 18O label from enriched water is incorporated into the sugar molecule, showing that the C3-O bond is cleaved and reformed by the enzyme. Moreover, an intermediate was detected and identified as an unsaturated sugar molecule. Based on this observation, we suggested a lyase-type mechanism (beta-elimination/hydration) for the cleavage of the lactyl ether bond of MurNAc 6-phosphate. Close homologs of murQ were found on the chromosome of several bacteria, and amino acid sequence similarity with the N-terminal domain of human glucokinase-regulatory protein (GckR or GKRP) was recognized.     

Respiratory responses of three Antarctic ascidians and a sea pen to increased sediment concentrations

Glacial retreat and subglacial bedrock erosion are consequences of rapid regional warming on the West Antarctic Peninsula. Sedimentation of fine-grained eroded particles can impact the physiology of filter-feeding benthic organisms. We investigated the effect of increasing concentrations of sediment on the oxygen consumption of suspension feeding species, the ascidians Molgula pedunculata, Cnemidocarpa verrucosa, Ascidia challengeri, and the pennatulid Malacobelemnon daytoni in Potter Cove (South Shetland Islands, Antarctica). In A. challengeri and C. verrucosa, oxygen consumption increased gradually up to a critical sediment concentration (C _crit) where species oxygen consumption was maximal (O _max in mg O₂?g^?1dm?day^?1) and further addition of sediments decreased respiration. C _crit was 200?mg?L^?1 for A. challengeri (O _max of 0.651?±?0.238) and between 100 and 200?mg?L^?1 for C. verrucosa (O _max of 0.898?±?0.582). Oxygen consumption of M. pedunculata increased significantly even at low sediment concentrations (15–50?mg sediment?L^?1). Contrary to the ascidians, sediment exposure did not affect oxygen consumption of the sea pen. The tiered response to sedimentation in the four species corroborates recent field observations that detected a reduction in the abundance of the sensitive ascidian M. pedunculata from areas strongly affected by glacial sediment discharge, whereas sea pens are increasing in abundance. Our investigation relates consequences (population shifts in filter-feeder communities) to causes (glacial retreat) and is of importance for modelling of climate change effects in Antarctic shallow coastal areas.     

Spina bifida cystica and severe congenital bilateral talipes equinovarus in one twin of a monoamniotic pair: a case report

Background

Spina bifida and congenital talipes equinovarus (CTEV) are common congenital malformations which may occur together and increase morbidity. Monozygous twins are particularly at risk of these malformations and discordance in one type of malformation is typical. The occurrence of both spina bifida and CTEV in one twin of a monozygotic pair is rare.

Case presentation

A 22 year-old Cameroonian primigravida at 36 weeks of a twin gestation was received in our district hospital at the expulsive phase of labour on a background of sub-optimal antenatal care. A caesarean section indicated for cephalo-pelvic disproportion was performed and life monoamniotic male twins were extracted. The first twin was normal. The second twin had spina bifida cystica and severe bilateral CTEV. Routine postnatal care was ensured and at day 2 of life, the affected twin was evacuated to a tertiary hospital for proper management. He was later on reported dead from complications of hydrocephalus.

Conclusions

Spina bifida cystica with severe bilateral CTEV in one twin of a monoamniotic pair illustrates the complexity in the interplay of causal factors of these malformations even among monozygotic twins who are assumed to share similar genetic and environmental features. The occurrence and poor outcome of the malformations was probably potentiated by poor antenatal care. With postnatal diagnoses, a better outcome was difficult to secure even with prompt referral. Early prenatal diagnoses and appropriate counseling of parents are cardinal.

     

Developmental Change of Sialidase Neu4 Expression in Murine Brain and Its Involvement in the Regulation of Neuronal Cell Differentiation

Sialidase Neu4 is reported to be dominantly expressed in the mouse brain, but its functional significance is not fully understood. We previously demonstrated that sialidase Neu3, also rich in mouse brain, is up-regulated during neuronal differentiation with involvement in acceleration of neurite formation. To elucidate physiological functions of Neu4, as well as Neu3, we determined expression during mouse brain development by quantitative RT-PCR. Expression was relatively low in the embryonic stage and then rapidly increased at 3–14 days after birth, whereas Neu3 demonstrated high levels in the embryonic stage and down-regulation after birth. Murine Neu4 was found to possess two isoforms differing in expression levels, developmental pattern, and enzymatic character. Distinct from the human isoforms, the murine forms, to a different extent, both catalyzed the removal of sialic acid from gangliosides as well as glycoproteins, and one isoform seemed to act on polysialylated NCAM efficiently, despite the low activity toward ordinary substrates. In situ hybridization demonstrated Neu4 mRNA to be present mainly in the hippocampus in which NCAM is rich and decreases after birth. During retinoic acid-induced differentiation, Neu4 expression was down-regulated in Neuro2a cells. Overexpression of Neu4 resulted in suppression of neurite formation, and its knockdown showed the acceleration. Thin layer chromatography of the glycolipids from Neu4-transfected cells showed ganglioside compositions to be only slightly affected, although lectin blot analysis revealed increased binding to Ricinus communis agglutinin (RCA) lectin of a ∼95-kDa glycoprotein, which decreased with cell differentiation. These results suggest that mouse Neu4 plays an important regulatory role in neurite formation, possibly through desialylation of glycoproteins.Sialidases catalyze the removal of sialic acid from non-reducing ends of glycoproteins and glycolipids. In mammals, four types of sialidases have so far been cloned, classified according to their subcellular localization and enzymatic properties (abbreviated to Neu1, Neu2, Neu3, and Neu4) (1–3). Studies have provided strong evidence that these sialidases play crucial roles in various physiological functions such as cell differentiation, cell growth, and malignant transformation. Among these sialidases, Neu4 is unique in its tissue expression pattern and enzymatic properties. In the mouse, it is dominantly expressed in brain, but its sialidase activity is very weak compared with other mouse sialidases (4). In contrast, human NEU4 is expressed not only in brain, but also in liver, kidney, and colon (5–7). We have demonstrated that NEU4 has two isoforms, differing in the N-terminal 12-amino acid residues that act as a mitochondrial-targeting sequence (7). Except for the subcellular localization, enzymatic properties are very similar. The short form of NEU4 (NEU4S) suppresses malignancy in colon cancer cells, mainly through desialylation of some glycoproteins, whereas the long form of NEU4 (NEU4L) may be involved in apoptosis with hydrolysis of ganglioside GD3 in mitochondria (8). Recently, Neu4 knockout mice (Neu4^−/−) were generated for pathological analysis (9). Neu4^−/− grew normally with a normal lifespan and proved fertile, but vacuolization of the lung and spleen was observed with a lysosomal storage phenotype, and the GM1/GD1a ratio was decreased in the brain. The observations on Neu4^−/− are very interesting, but there is some ambiguity in the available previous reports, because, as mentioned above, mouse Neu4 has been reported to have weak sialidase activity in vitro, and its expression is restricted in brain. To clarify this ambiguity and further understand the physiological functions of Neu4, we examined expression in the mouse brain and observed a possible involvement in neural differentiation in connection with another sialidase, Neu3, which greatly increases during differentiation of neuroblastoma cells (10, 11) and causes acceleration of neurite formation (10–13).In the GenBank^TM data base, nucleotide sequences of mouse Neu4 have been submitted as {"type":"entrez-nucleotide","attrs":{"text":"AY258421","term_id":"32402573","term_text":"AY258421"}}AY258421 and {"type":"entrez-nucleotide","attrs":{"text":"AK034236","term_id":"26329804","term_text":"AK034236"}}AK034236. The former contains a complete coding sequence of 1506 bp, with two ATGs at positions 1 and 70, and {"type":"entrez-nucleotide","attrs":{"text":"AK034236","term_id":"26329804","term_text":"AK034236"}}AK034236 encodes only the second ATG (4). The gene from {"type":"entrez-nucleotide","attrs":{"text":"AY258421","term_id":"32402573","term_text":"AY258421"}}AY258421 has been reported to encode Neu4, showing weak sialidase activity, but there is no information on whether the gene based on {"type":"entrez-nucleotide","attrs":{"text":"AK034236","term_id":"26329804","term_text":"AK034236"}}AK034236 encodes Neu4 with sialidase activity toward natural substrates. We have now extended our studies to the existence of different mouse Neu4 isoforms, focusing on their significance in neuronal cells by measuring expression levels during cell differentiation. We present, here, evidence that two murine Neu4 isoforms contribute to neurite formation.