Neuroacanthocytosis associated with a defect of the 4.1R membrane protein

Background

Neuroacanthocytosis (NA) denotes a heterogeneous group of diseases that are characterized by nervous system abnormalities in association with acanthocytosis in the patients' blood. The 4.1R protein of the erythrocyte membrane is critical for the membrane-associated cytoskeleton structure and in central neurons it regulates the stabilization of AMPA receptors on the neuronal surface at the postsynaptic density. We report clinical, biochemical, and genetic features in four patients from four unrelated families with NA in order to explain the cause of morphological abnormalities and the relationship with neurodegenerative processes.

Case presentation

All patients were characterised by atypical NA with a novel alteration of the erythrocyte membrane: a 4.1R protein deficiency. The 4.1R protein content was significantly lower in patients (3.40 ± 0.42) than in controls (4.41 ± 0.40, P < 0.0001), reflecting weakened interactions of the cytoskeleton with the membrane. In patients IV:1 (RM23), IV:3 (RM15), and IV:6 (RM16) the 4.1 deficiency seemed to affect the horizontal interactions of spectrin and an impairment of the dimer self-association into tetramers was detected. In patient IV:1 (RM16) the 4.1 deficiency seemed to affect the skeletal attachment to membrane and the protein band 3 was partially reduced.

Conclusion

A decreased expression pattern of the 4.1R protein was observed in the erythrocytes from patients with atypical NA, which might reflect the expression pattern in the central nervous system, especially basal ganglia, and might lead to dysfunction of AMPA-mediated glutamate transmission.     

p66SHC promotes T cell apoptosis by inducing mitochondrial dysfunction and impaired Ca2+ homeostasis

p66Shc, a redox enzyme that enhances reactive oxygen species (ROS) production by mitochondria, promotes T cell apoptosis. We have addressed the mechanisms regulating p66Shc-dependent apoptosis in T cells exposed to supraphysiological increases in [Ca2+]c. p66Shc expression resulted in profound mitochondrial dysfunction in response to the Ca2+ ionophore A23187, as revealed by dissipation of mitochondrial transmembrane potential, cytochrome c release and decreased ATP levels. p66Shc expression also caused a dramatic alteration in the cells' Ca2+-handling ability, which resulted in Ca2+ overload after A23187 treatment. The impairment in Ca2+ homeostasis was ROS dependent and caused by defective Ca2+ extrusion due at least in part to decreased plasma membrane ATPase (PMCA) expression. Both effects of p66Shc required Ca2+-dependent serine-36 phosphorylation. The mitochondrial effects of p66Shc were potentiated by but not strictly dependent on the rise in [Ca2+]c. Thus, Ca2+-dependent p66Shc phosphorylation causes both mitochondrial dysfunction and impaired Ca2+ homeostasis, which synergize in promoting T cell apoptosis.     

Gastrointestinal stromal tumor of the anal canal: an unusual presentation

Background

Gastrointestinal stromal tumors (GIST) of the stomach are the most frequent followed by those of the intestinal tract, while colon and rectum represent rare sites. GIST of the anal canal are extremely rare. They have been studied along with GIST of the rectum, as a single entity, and along with them they represent 5% of GIST. GIST arising from the anal canal account for only 2%–8% of the anorectal GIST. Thus anal GIST must be considered an exceptional case.

Case presentation

A 78-year-old man was referred to our Institution for an anal mass, in absence of any symptom. The patient was treated by local excision. An histological diagnosis of a low grade GIST was made. No further treatment was necessary. No local recurrence of distant metastases were found at follow-up.

Conclusion

At the moment, only ten cases of c-kit positive anal GIST are reported in the literature. These few data are not sufficient to establish a widely accepted approach for this neoplasia.We recommend to perform an initial local excision, to define the risk of aggressive behavior and the resection margins and proceed to a more aggressive treatment, if the GIST belongs to high or very high risk group. The role of adjuvant therapy is still uncertain. Although inhibitors of tyrosine-kinase receptor needs further studies before their routine use, their role in case of distant or local recurrence has been accepted. Patients' close follow up is mandatory to disclose as soon as possible local recurrences or metastases.

     

Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies

The promyelocytic leukaemia (PML) tumour suppressor protein epitomizes the PML-nuclear body (PML-NB) and is crucially required for the proper assembly of this macromolecular nuclear structure. Unlike other, more specialized subnuclear structures such as Cajal and Polycomb group bodies, PML-NBs are functionally promiscuous and have been implicated in the regulation of diverse cellular functions. PML-NBs are dynamic structures that favour the sequestration and release of proteins, mediate their post-translational modifications and promote specific nuclear events in response to various cellular stresses. Recent data suggest that PML-NBs may be heterogeneous in composition, mobility and function.     

Large‐scale patterns in summer diffusive CH4 fluxes across boreal lakes,and contribution to diffusive C emissions

Lakes are a major component of boreal landscapes, and whereas lake CO₂ emissions are recognized as a major component of regional C budgets, there is still much uncertainty associated to lake CH₄ fluxes. Here, we present a large‐scale study of the magnitude and regulation of boreal lake summer diffusive CH₄ fluxes, and their contribution to total lake carbon (C) emissions, based on in situ measurements of concentration and fluxes of CH₄ and CO₂ in 224 lakes across a wide range of lake type and environmental gradients in Québec. The diffusive CH₄ flux was highly variable (mean 11.6 ± 26.4 SD mg m^?2 d^?1), and it was positively correlated with temperature and lake nutrient status, and negatively correlated with lake area and colored dissolved organic matter (CDOM). The relationship between CH₄ and CO₂ concentrations fluxes was weak, suggesting major differences in their respective sources and/or regulation. For example, increasing water temperature leads to higher CH₄ flux but does not significantly affect CO₂ flux, whereas increasing CDOM concentration leads to higher CO₂ flux but lower CH₄ flux. CH₄ contributed to 8 ± 23% to the total lake C emissions (CH₄ + CO₂), but 18 ± 25% to the total flux in terms of atmospheric warming potential, expressed as CO₂‐equivalents. The incorporation of ebullition and plant‐mediated CH₄ fluxes would further increase the importance of lake CH₄. The average Q₁₀ of CH₄ flux was 3.7, once other covarying factors were accounted for, but this apparent Q₁₀ varied with lake morphometry and was higher for shallow lakes. We conclude that global climate change and the resulting shifts in temperature will strongly influence lake CH₄ fluxes across the boreal biome, but these climate effects may be altered by regional patterns in lake morphometry, nutrient status, and browning.     

High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli

Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lack of knowledge about its structure, specific biological function, and role in cardiovascular disease. Here we present a novel Escherichia coli-based recombinant expression system that produces highly pure mature human apoA-II at substantial yields. A Mxe GyrA intein containing a chitin binding domain was fused at the C terminus of apoA-II. A 6× histidine-tag was also added at the fusion protein's C terminus. After rapid purification on a chitin column, intein auto-cleavage was induced under reducing conditions, releasing a peptide with only one extra N-terminal Met compared with the sequence of human mature apoA-II. A pass through a nickel chelating column removed any histidine-tagged residual fusion protein, leaving highly pure apoA-II. A variety of electrophoretic, mass spectrometric, and spectrophotometric analyses demonstrated that the recombinant form is comparable in structure to human plasma apoA-II. Similarly, recombinant apoA-II is comparable to the plasma form in its ability to bind and reorganize lipid and promote cholesterol efflux from macrophages via the ATP binding cassette transporter A1. This system is ideal for producing large quantities of recombinant wild-type or mutant apoA-II for structural or functional studies.     

Spinal muscular atrophy pathogenic mutations impair the axonogenic properties of axonal-survival of motor neuron

The axonal survival of motor neuron (a-SMN) protein is a truncated isoform of SMN1, the spinal muscular atrophy (SMA) disease gene. a-SMN is selectively localized in axons and endowed with remarkable axonogenic properties. At present, the role of a-SMN in SMA is unknown. As a first step to verify a link between a-SMN and SMA, we investigated by means of over-expression experiments in neuroblastoma-spinal cord hybrid cell line (NSC34) whether SMA pathogenic mutations located in the N-terminal part of the protein affected a-SMN function. We demonstrated here that either SMN1 missense mutations or small intragenic re-arrangements located in the Tudor domain consistently altered the a-SMN capability of inducing axonal elongation in vitro. Mutated human a-SMN proteins determined in almost all NSC34 motor neurons the growth of short axons with prominent morphologic abnormalities. Our data indicate that the Tudor domain is critical in dictating a-SMN function possibly because it is an association domain for proteins involved in axon growth. They also indicate that Tudor domain mutations are functionally relevant not only for FL-SMN but also for a-SMN, raising the possibility that also a-SMN loss of function may contribute to the pathogenic steps leading to SMA.     

1H NMR metabolomics analysis of glioblastoma subtypes: correlation between metabolomics and gene expression characteristics

Glioblastoma multiforme (GBM) is the most common form of malignant glioma, characterized by unpredictable clinical behaviors that suggest distinct molecular subtypes. With the tumor metabolic phenotype being one of the hallmarks of cancer, we have set upon to investigate whether GBMs show differences in their metabolic profiles. (1)H NMR analysis was performed on metabolite extracts from a selection of nine glioblastoma cell lines. Analysis was performed directly on spectral data and on relative concentrations of metabolites obtained from spectra using a multivariate regression method developed in this work. Both qualitative and quantitative sample clustering have shown that cell lines can be divided into four groups for which the most significantly different metabolites have been determined. Analysis shows that some of the major cancer metabolic markers (such as choline, lactate, and glutamine) have significantly dissimilar concentrations in different GBM groups. The obtained lists of metabolic markers for subgroups were correlated with gene expression data for the same cell lines. Metabolic analysis generally agrees with gene expression measurements, and in several cases, we have shown in detail how the metabolic results can be correlated with the analysis of gene expression. Combined gene expression and metabolomics analysis have shown differential expression of transporters of metabolic markers in these cells as well as some of the major metabolic pathways leading to accumulation of metabolites. Obtained lists of marker metabolites can be leveraged for subtype determination in glioblastomas.