KIF5A de novo mutation associated with myoclonic seizures and neonatal onset progressive leukoencephalopathy.

The KIF5A gene (OMIM 602821) encodes a neuron-specific kinesin heavy chain involved in intracellular transport of mitochondria and other cargoes. KIF5A protein comprises the N terminal motor domain, the stalk domain and the C-terminal cargo binding domain. The binding between KIF5A and its cargoes is mediated by kinesin adaptor proteins such as TRAK1 and TRAK2. Numerous missense KIF5A mutations in the motor and stalk domains cause spastic paraplegia type 10 (SPG10, OMIM 604187). Conversely, the role of loss-of-function mutations, especially those affecting the cargo binding domain, is unclear. We describe a novel de novo KIF5A p.Ser974fs/c.2921delC mutation found by whole exome sequencing in a patient with a congenital severe disease characterized by myoclonic seizures and progressive leukoencephalopathy. Since this phenotype differs considerably from the KIF5A/SPG10 disease spectrum we propose that the KIF5A p.Ser974fs and possibly other mutations which lead to truncation of the C-terminal tail of the protein cause a novel disorder. We speculate that the unique effect of the C-terminal truncating KIF5A mutations may result from the previously described complex role of this protein domain in binding of the TRAK2 and possibly other kinesin adaptor protein(s).

Spondyloepimetaphyseal dysplasia with neurodegeneration associated with AIFM1 mutation - a novel phenotype of the mitochondrial disease.

In 1999, based on a single family, spondyloepimetaphyseal dysplasia (SEMD) with mental retardation (MR) was described as a novel syndrome with probably X-linked recessive inheritance and unknown molecular defect (MIM 300232). Our purpose was to search for the causative defect in the originally described family and in an independently ascertained second family. All patients had slowly progressive neurodegeneration with central and peripheral involvement and identical skeletal dysplasia. Whole exome sequencing performed in two subjects showed a single plausible candidate - the p.Asp237Gly variant in AIFM1 (chr. Xq26.1). The p.Asp237Gly segregated with disease as indicated by linkage analysis [maximum logarithm of odds score (LOD) score at theta 0 for the two families was 3.359]. This variant had not been previously reported and it was predicted to be pathogenic by Polyphen2, SIFT, MutationTaster and Mutation Assessor. AIFM1 encodes mitochondria associated apoptosis-inducing factor. The AIFM1 gene has been linked with COXPD6 encephalomyopathy (MIM 300816), Cowchock syndrome (MIM 310490) and X-linked deafness with neuropathy (DFNX5, MIM 300614), none of which are similar to SEMD-MR. Our results place SEMD as the third instance of a skeletal phenotype associated with a mitochondrial disease (the others being EVEN-PLUS syndrome caused by mutations of HSPA9 and CODAS syndrome due to LONP1 mutations).

Next-generation sequencing for diagnosis of thoracic aortic aneurysms and dissections: diagnostic yield, novel mutations and genotype phenotype correlations.

BACKGROUND: Thoracic aortic aneurysms and dissections (TAAD) are silent but possibly lethal condition with up to 40 % of cases being hereditary. Genetic background is heterogeneous. Recently next-generation sequencing enabled efficient and cost-effective examination of gene panels. Aim of the study was to define the diagnostic yield of NGS in the 51 TAAD patients and to look for genotype-phenotype correlations within families of the patients with TAAD. METHODS: 51 unrelated TAAD patients were examined by either whole exome sequencing or TruSight One sequencing panel. We analyzed rare variants in 10 established thoracic aortic aneurysms-associated genes. Whenever possible, we looked for co-segregation in the families. Kaplan-Meier survival curve was constructed to compare the event-free survival depending on genotype. Aortic events were defined as acute aortic dissection or first planned aortic surgery. RESULTS AND DISCUSSION: In 21 TAAD patients we found 22 rare variants, 6 (27.3 %) of these were previously reported, and 16 (73.7 %) were novel. Based on segregation data, functional analysis and software estimations we assumed that three of novel variants were causative, nine likely causative. Remaining four were classified as of unknown significance (2) and likely benign (2). In all, 9 (17.6 %) of 51 probands had a positive result when considering variants classified as causative only and 18 (35.3 %) if likely causative were also included. Genotype-positive probands (n = 18) showed shorter mean event free survival (41 years, CI 35-46) than reference group, i.e. those (n = 29) without any plausible variant identified (51 years, CI 45-57, p = 0.0083). This effect was also found when the 'genotype-positive' group was restricted to probands with 'likely causative' variants (p = 0.0092) which further supports pathogenicity of these variants. The mean event free survival was particularly low (37 years, CI 27-47) among the probands with defects in the TGF beta signaling (p = 0.0033 vs. the reference group). CONCLUSIONS: This study broadens the spectrum of genetic background of thoracic aneurysms and dissections and supports its potential role as a prognostic factor in the patients with the disease.

Amniocentesis for the detection of congenital toxoplasmosis: results from the nationwide Austrian prenatal screening program.

Prenatal diagnosis of congenital toxoplasmosis (CT) influences therapeutical management in pregnant women and their offspring. In Austria, a nationwide serological healthcare program to identify potential maternal toxoplasma infections during pregnancy exists. We assessed the clinical use of amniocentesis for toxoplasma-specific polymerase chain reaction (PCR) on amniotic fluid to detect CT. Data on serology, amniocentesis, PCR, complications, treatment, and paediatric clinical outcome were collected retrospectively among the birth cohort 1992-2008. There were 1386 women with amniocentesis, but only in 707 cases (51%) was acute maternal infection confirmed serologically. A high proportion (49%) of amniocenteses with negative PCR results in women with chronic infection or seronegativity were performed without clinical justification for the women or their foetuses. The positive and negative predictive values of PCR were 94.4% and 99.3%, respectively. Thirty-nine foetuses with CT, including four deaths, were reported. The five PCR-negative but infected infants were identified by the serological and clinical follow-up program. Thirty percent of amniocenteses were performed in the third trimester, and gestational age or treatment did not influence PCR sensitivity. Amniocentesis is indicated in women with acute maternal infection, and facilitated targeted therapies in pregnant women and their offspring. In women with late toxoplasma infection, negative amniotic fluid PCR made treatment of infants unnecessary. Serological and clinical follow-up of infants is important to confirm the infection status of the infant. Recommendations, based on our 17-year experience, to improve the current diagnostic strategies and to reduce unnecessary amniocentesis, are given.

Type of delivery onset has a significant impact on post-natal mortality in preterm infants of less than 30 weeks' gestation.

AIM: Type of delivery onset is not currently evaluated for its predictive impact. This study explored whether the type of preterm delivery onset was an antenatal predictor for post-natal mortality in preterm infants <30 weeks' gestation and should be included in antenatal counselling. METHODS: This retrospective cohort study included 1117 preterm infants <30 weeks' gestation born between 1999 and 2008 in a tertiary perinatal referral centre. Study patients were classified into spontaneous or iatrogenic preterm deliveries. Spontaneous deliveries included deliveries after preterm premature rupture of membranes (PPROM) and preterm labour. The study outcome was infant mortality before discharge from hospital. RESULTS: We included 499 patients born after PPROM (44.7%) and 247 born after preterm labour (22.1%). Iatrogenic preterm birth was noted in 282 patients (25.2%) and 89 patients fulfilled both criteria for spontaneous and iatrogenic preterm delivery (8.0%). Babies born after iatrogenic preterm delivery in gestational weeks 25-29 had significantly higher mortality rates. Logistic regression revealed that type of preterm delivery onset was an independent antenatal predictor for post-natal mortality. CONCLUSION: Type of preterm delivery onset had a significant impact on post-natal mortality in preterm infants <30 weeks' gestation, with a higher mortality rate after iatrogenic preterm delivery.

Long-term effects of maternal immune activation on depression-like behavior in the mouse.

Depression is a debilitating mental disease affecting a large population worldwide, the pathophysiological mechanisms of which remain incompletely understood. Prenatal infection and associated activation of the maternal immune system (MIA) are prominently related to an increased risk for the development of several psychiatric disorders including schizophrenia and autism in the offsprings. However, the role of MIA in the etiology of depression and its neurobiological basis are insufficiently investigated. Here we induced MIA in mice by challenge with polyinosinic:polycytidylic phosphate salt-a synthetic analog of double-stranded RNA, which enhances maternal levels of the cytokine interleukin-6 (IL-6)-and demonstrate a depression-like behavioral phenotype in adult offsprings. Adult offsprings additionally show deficits in cognition and hippocampal long-term potentiation (LTP) accompanied by disturbed proliferation of newborn cells in the dentate gyrus and compromised neuronal maturation and survival. The behavioral, neurogenic and functional deficiencies observed are associated with reduced hippocampal expression of vascular endothelial growth factor (VEGF)A-VEGFR2. IL-6-STAT3-dependent aberrant VEGFA-VEGFR2 signaling is proposed as neurobiological mechanism mediating the effects of MIA on the developing fetal brain and ensuing consequences in adulthood.

Blood transfusions using 27 gauge PICC lines: a retrospective clinical study on safety and feasibility.

BACKGROUND: Blood transfusions are required by most extremely low birth weight (ELBW) infants, but sometimes an adequate peripheral venous access cannot be achieved. Under these circumstances, we used 27 Gauge (G) peripherally inserted central catheter (PICC) lines that are routinely inserted on the second day of life. Due to their narrow lumen, hemolysis of transfused erythrocytes was a major concern. We therefore performed a retrospective study in ELBW infants to analyze the incidence, safety and feasibility of PRBC transfusions via 27 G PICC lines. METHODS: ELBW infants admitted from 08/2011-07/2012 were screened for packed red blood cell (PRBC) transfusions. Those applied via 27 G PICC lines were identified. For analysis of transfusion safety (hemolysis), hemoglobin and potassium levels as well as cardiovascular variables (invasive mean arterial blood pressure and heart rate) were evaluated before and after transfusion. For analysis of transfusion feasibility, catheter removal after transfusion and the reason for removal were recorded. RESULTS: A total of 648 transfusions were applied in 110 ELBW infants. 27 infants (24%) received no transfusion. In 12/83 (14.5%) infants who received PRBCs, transfusions were applied using a 27 G PICC line (38/648, 5.9%). Patients who received PRBCs via the PICC line were smaller at birth (582 g [range 380-752 g] vs. 710 g [430-972 g]; 23+6 [23+1-27+6] vs. 26+0 [23+1-31+4]) and required a higher number of PRBC transfusions (n=13 vs. n=5) overall. Transfusion analysis showed an appropriate increase of blood hemoglobin levels and stable potassium levels as well as cardiovascular parameters. 4/38 of PICC lines were removed within 24 h after transfusion, one due to occlusion (15 h after transfusion). CONCLUSIONS: We conclude that PRBC transfusions via 27 G PICC lines were feasible and performed without signs of hemolysis in ELBW infants. Our findings may help clinicians in the management of ELBW infants requiring transfusions if a peripheral venous access is not achievable.

Safety of blood transfusions using 27 gauge neonatal PICC lines: an in vitro study on hemolysis.

Blood transfusions are required by the majority of extremely premature infants. Packed red blood cells (PRBCs) are usually applied via simple peripheral cannulas. In situations where no peripheral venous access is achievable, 27 Gauge (G) neonatal PICC lines - that are ideally exclusively dedicated to application of parenteral nutrition - may represent a useful alternative access for PRBC transfusions. However, transfusion via small scaled catheters may damage PRBCs and lead to hemolysis. We here evaluate whether transfusion of irradiated PRBCs via 27 G PICC lines leads to hemolysis in vitro.Experimental transfusions of gamma-irradiated PRBCs were performed at increasing velocities (2.5, 3.7, 5 ml/h; full force manual push approximating 30 ml/h) via 27 G PICC lines of 20 and 30 cm length. Parameters of hemolysis (lactate dehydrogenase, potassium and free hemoglobin) were measured from the supernatants of transfused PRBCs and the percentage of hemolysis was calculated.Potassium and lactate dehydrogenase after transfusion at increasing velocities did not differ significantly from negative controls. Free hemoglobin levels showed a small but significant increase at the slowest transfusion speed (2.5 ml/h) using the 30 cm 27 G PICC line, with a relative hemolysis of only 0.13%. A manual push (approximating 30 ml/h) showed no significant changes of parameters from baseline.We conclude that transfusion of gamma-irradiated PRBCs using a 27 G neonatal PICC line does not cause clinically relevant hemolysis in vitro. Clinical studies are needed to confirm the feasibility and safety of the approach in vivo.

Preterm neonates display altered plasmacytoid dendritic cell function and morphology.

Bacterial and viral infections cause high rates of morbidity and mortality in premature newborns. In the setting of viral infection, pDCs play a key role as strong producers of IFN-α upon TLR9 activation. We analyzed pDC frequency, phenotype, morphology, and function in CB of preterm and term newborns in comparison with adults. Whereas all age groups show similar pDC numbers, BDCA-2, CD123, and TLR9 levels, the expression of BDCA-4 and capacity to produce IFN-α upon TLR9 challenge were decreased significantly in preterm neonates. Furthermore, we show by means of electron microscopy that pDCs from preterm newborns exhibit a distinct, "immature" morphology. Taken together, these findings suggest decreased functionality of pDCs in the premature newborn. The reduced capacity to produce IFN-α is likely to render such infants more susceptible to viral infections.

Impact of low-grade intraventricular hemorrhage on long-term neurodevelopmental outcome in preterm infants.

PURPOSE: Despite a decreasing incidence, intraventricular hemorrhage (IVH) remains a point of major concern in neonatology due to its association to adverse neurodevelopmental outcome (NDO). Aim of this study was to compare outcome of preterm infants with different grades of IVH born below 32 weeks of gestational age (GA) with outcome of controls without IVH and to especially evaluate the influence of low grade IVH on NDO. METHODS: Four hundred seventy-one preterm infants with a GA below 32 weeks were admitted to our neonatal intensive care unit between 1994 and 2005 and included into analysis. RESULTS: IVH patients showed significantly lower mean psychomotor and mental developmental indices and a significantly higher percentage of cerebral palsy and visual impairment. Results of IVH patients born below 28 weeks of GA were significantly worse than results of IVH patients born at or above 28 weeks of GA. In all parameters, an increase of abnormal results with increasing grade of IVH could be observed; even patients with low-grade IVH (grades I and II) showed higher percentages of impairment compared to controls without any IVH. CONCLUSION: Even low-grade IVH has an significant impact on neurodevelopmental outcome of preterm patients and gestational age influences the impact of intraventricular hemorrhage on neurodevelopmental outcome.

Is it possible to make a reliable prognosis within the first hour of life for very low birth weight infants delivered after preterm premature rupture of membranes?

BACKGROUND: One third of all preterm births are due to preterm premature rupture of membranes (pPROM). An accurate prognostic evaluation after admission to the neonatal intensive care unit is necessary. OBJECTIVE: The aim of this study was to identify prognostic factors within the first hour of life for mortality, short-term pulmonary morbidity, chronic lung disease (CLD) and severe cerebral morbidity in very low birth weight (VLBW) infants after pPROM. METHODS: This retrospective study included 300 infants with pPROM who fit the study criteria and were derived from a cohort of 1,435 VLBW infants. A total of 17 obstetric and neonatal factors were evaluated by univariate and multivariate analysis. RESULTS: Gestational age at birth and 5-min Apgar score correlated significantly with all 4 outcomes. The results of the first blood gas analysis correlated with 3 outcomes and the first mean arterial pressure with 2 outcomes. Anhydramnios and a lower number of courses of antenatal steroids correlated with higher mortality, and preterm labor correlated with CLD. The multivariate analysis revealed gestational age, 5-min Apgar score, the results of the first blood gas analysis, the first mean arterial pressure and anhydramnios to be significant predictors. The positive predictive value ranged from 20 to 81%, and the negative predictive value ranged from 79 to 92%. CONCLUSION: Gestational age at birth and parameters reflecting postnatal adaptation were the most precise factors for assessment of the prognosis of VLBW infants after pPROM within the first hour of life. Apart from anhydramnios, obstetric factors did not predict neonatal outcome. At 1 h of age, our models of perinatal risk factors were more effective in predicting a favorable outcome than an adverse outcome.

Neonatal colonization of mice with Lactobacillus plantarum producing the aeroallergen Bet v 1 biases towards Th1 and T-regulatory responses upon systemic sensitization.

BACKGROUND: The use of recombinant lactic acid bacteria (LAB) as vehicles for mucosal delivery of recombinant allergens is an attractive concept for antigen-defined allergy prevention/treatment. Interventions with LAB are of increasing interest early in life when immune programming is initiated. Here, we investigated the effect of neonatal colonization with a recombinant LAB producing the major birch pollen allergen Bet v 1 in a murine model of type I allergy. METHODS: We constructed a recombinant Lactobacillus (L.) plantarum NCIMB8826 strain constitutively producing Bet v 1 to be used for natural mother-to-offspring mono-colonization of germ-free BALB/c mice. Allergen-specific immunomodulatory effects of the colonization on humoral and cellular immune responses were investigated prior and after sensitization to Bet v 1. RESULTS: Mono-colonization with the Bet v 1 producing L. plantarum induced a Th1-biased immune response at the cellular level, evident in IFN-γ production of splenocytes upon stimulation with Bet v 1. After sensitization with Bet v 1 these mice displayed suppressed IL-4 and IL-5 production in spleen and mesenteric lymph node cell cultures as well as decreased allergen-specific antibody responses (IgG1, IgG2a, and IgE) in sera. This suppression was associated with a significant up-regulation of the regulatory marker Foxp3 at the mRNA level in the spleen cells. CONCLUSION: Intervention at birth with a live recombinant L. plantarum producing a clinically relevant allergen reduces experimental allergy and might therefore become an effective strategy for early intervention against the onset of allergic diseases.

Perinatal outcome of preterm infants <1500 g after IVF pregnancies compared with natural conception.

OBJECTIVE: In vitro fertilisation (IVF) pregnancies are at increased risk for adverse perinatal outcome including very low birth weight infants. The purpose of this study was to find out whether the perinatal outcome of preterm infants <1500 g after IVF is different from those in naturally conceived pregnancies. PATIENTS AND METHODS: This retrospective cohort study included preterm infants <1500 g born between 1999 and 2007 in a tertiary perinatal referral centre. All analyses were made separately for singletons and multiples, divided into infants <1000 g and 1000-1499 g. The primary study outcomes were infant mortality, short term pulmonary morbidity and cerebral morbidity. The secondary study outcomes were small for gestational age, Apgar score at 5 min, the results of the first venous blood gas analysis of the preterm infant, and the first mean arterial blood pressure after neonatal intensive care unit admission. Logistic regression analysis was done to assess the impact of IVF compared to other maternal and infant factors. RESULTS: 1423 patients (195 IVF and 1228 non-IVF patients) were included in this study. The incidence of preterm labour was significantly higher in multiples after IVF than in spontaneously conceived multiples. In the IVF group, there were significantly more multiples. Mortality, pulmonary morbidity and cerebral morbidity did not differ among patients after IVF and naturally conceived patients. Also, there were no significant differences for the secondary outcomes, except for a significantly higher initial pH value in multiples after IVF between 1000-1499 g. CONCLUSION: IVF treatment was not associated with adverse outcome in very low birth weight infants. IVF, preterm birth, VLBW, singletons, multiples, outcome.

Comparison of neonatal MRI examinations with and without an MR-compatible incubator: advantages in examination feasibility and clinical decision-making.

PURPOSE: To assess the utility of an MRI-compatible incubator (INC) by comparing. METHODS: In a retrospective study, the clinical and radiological aspects of 129 neonatal MRI examinations during a 3 year period were analyzed. Routine protocols including fast spin-echo T2-weighted (w) sequences, axial T1w, Gradient-echo, diffusion sequences, and 3D T1 gradient-echo sequences were performed routinely, angiography and spectroscopy were added in some cases. Diffusion-tensor imaging was done in 50% of the babies examined in the INC and 26% without INC. Sequences, adapted from fetal MR-protocols were done in infants younger than 32 gestational weeks. Benefit from MR-information with respect to further management was evaluated. RESULTS: The number of the examinations increased (30-99), while the mean age (43-38, 8 weeks of gestational age) and weight (3308-2766 g) decreased significantly with the use of the MR-compatible incubator. The mean imaging time (34, 43-30, 29 min) decreased, with a mean of one additionally performed sequence in the INC group. All infants received sedatives according to our anaesthetic protocol preceding imaging, but a repeated dose was never necessary (10% without INC) using the INC. Regarding all cases, MR-based changes in clinical management were initiated in 58%, while in 57% of cases the initial ultrasound diagnosis was changed or further specified. CONCLUSIONS: The use of the INC enables the MR access of unstable infants with suspect CNS problems to the management, of whom is improved by MR information to significantly higher percentage, than without INC.

Haemodynamic changes and stress responses of piglets to surgery during total intravenous anaesthesia with propofol and fentanyl.

The purpose of the study was to assess the haemodynamic (blood pressure and heart rate) changes and stress responses (serum cortisol and serum amyloid A [SAA] concentrations) to surgery in piglets during total intravenous anaesthesia (TIVA) with propofol and fentanyl. After preanaesthetic medication with intramuscular midazolam (0.5 mg/kg body mass), ketamine (10 mg/kg) and butorphanol (0.5 mg/kg) anaesthesia was induced in five piglets, with intravenous propofol (1 mg/kg) followed by tracheal intubation and mechanical lung ventilation. Soft tissue surgery was performed in the jugular and inguinal regions during TIVA with propofol (8 mg/kg/h) and fentanyl (35 microg/kg/h). Anaesthesia was maintained for 300 min after surgery as the piglets were the control group of a project involving extracorporeal membrane oxygenation. Mean plasma cortisol concentration decreased significantly (P<0.05) from 59+/-39.9 nmol/L (mean+/-1 SD) before surgery to 7.5+/-2.5 nmol/L 300 min after end of surgical procedure. The mean SAA concentrations increased over the same period from 1.6+/-2.3 microg/mL to 4.2+/-5.6 microg/mL without statistical significance. The baseline (presurgery) mean arterial pressure (MAP) was 72+/-9 mmHg compared with 72+/-11 mmHg 300 min after end of surgery. Neither heart rate nor lactate concentrations changed significantly over the same time points: heart rate was 104+/-11 and 103+/-15 beats/min whereas mean lactate concentrations were reduced from 1.14+/-0.45 mmol/L to 0.90+/-0.22 mmol/L. Haemodynamic stability, a decrease in serum cortisol and a non-statistically significant rise in mean SAA concentrations suggest that the anaesthetic described suppresses the stress response of piglets to surgery without adverse cardiovascular effects. Therefore, it may prove useful in cardiovascular research.

Linezolid for treatment of catheter-related cerebrospinal fluid infections in preterm infants.

Ventriculostomy-related cerebrospinal fluid (CSF) infection remains a major problem in neonatal intensive care. The spectrum of pathogens causing these infections is dominated by coagulase-negative staphylococci, and vancomycin is the antibiotic of choice for treatment. However, vancomycin is known to have only poor penetration into the CSF when applied intravenously and is therefore being applied intraventricularly. The oxazolidinone linezolid has antibacterial activity against most drug-resistant Gram-positive bacteria and has been shown to have excellent penetration into the CSF in adults. Here, its successful use in five neonates with infected ventriculostomies is described.

Hippocampal levels of gamma-enolase, C-1-tetrahydrofolate synthase and serotransferrin fluctuate over the estrous cycle in the rat.

Hippocampal functions vary across the estrous cycle but metabolic changes at the protein level have not been systematically studied so far. It was therefore the aim of the study to screen expression of metabolic proteins mainly represented by metabolic enzymes in the hippocampus over the estrous cycle and in males. Female and male OFA Sprague-Dawley rats were used and female estrous phases were determined by vaginal smears, according to which females were separated into groups of proestrous, estrous, early and late metestrous and diestrous. Proteins were extracted from hippocampal tissue and separated on two-dimensional gel electrophoresis followed by identification with mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Comparative analysis of protein levels was carried out by quantifying protein spot volumes by means of specific software. Levels of one expression form of gamma-enolase were different between diestrous and early metestrous; C-1-tetrahydrofolate synthase levels were elevated in proestrous as compared with estrous and serotransferrin levels were increased in diestrous as compared with proestrous, estrous, metestrous and in males. The outcome of estrous cycle- and gender-dependent protein fluctuations is relevant for the interpretation of previous and future work as well as for the design of further studies at the protein level in the hippocampus.

Identification and characterisation of arsenite (+3 Oxidation State) methyltransferase (AS3MT) in mouse neuroblastoma cell line N1E-115.

Handling and detoxification of metals by enzymes is a major issue that is not in the focus of current biomedical research concepts. The finding of the presence of arsenic (+3 Oxidation State) methyltransferase (AS3MT) in neuroblastoma cells NE-115 as a high abundance protein made us investigate primary structure of AS3MT reflecting an example of metal-handling in eucaryotes. Proteins extracted from NE-115 cells were run on 2-DE followed by two different mass spectrometrical methods. High sequence coverage was obtained by multiple protease digestion and a sequence conflict was solved at arginine 335. These findings are important when future studies on this enzyme are designed at the protein level and in particular, when antibodies against this protein will be generated.