Phosphoinositides in membrane traffic.

Phosphoinositides serve as direct local modulators or recruiters of the protein machineries that control membrane trafficking. In the past year, examples of phosphoinositide effectors include regulators of small GTPases in coat assembly, dynamin in clathrin coated vesicle formation and FYVE finger proteins in endocytic membrane traffic. A novel phosphoinositide appears to regulate effectors involved in the formation of multivesicular endosomes.

An atypical presentation of diffuse midline pontine glioma in a middle age patient: Case report.

INTRODUCTION: Diffuse midline glioma is a newly WHO defined entity (grade IV) (Louis et al., 2016) which includes diffuse intrinsic pontine glioma (DIPG) reported in pediatric population and, occasionally, in young adults. Here, we present a detailed description of an atypical case of diffuse midline glioma in a 53 years old woman. CASE REPORT: A caucasian woman aged 53 from Ukraine, was referred to another neurological department complaining of 3 months history of progressive postural instability and gait impairment with frequent falling. Magnetic resonance demonstrated two brainstem lesions, hyperintense in FLAIR with "patchy" peripheral enhancement, leptomeningeal and cranial nerves enhancement. CSF was normal. Due to positive antinuclear antibodies test (ANA 1:360), intravenous steroid treatment was administered and reported to initially improve the patient condition. However, the following weeks the lady worsened. Imaging features were unchanged. Because quantiferon test resulted positive, MRI-Spectroscopy showed an inflammatory pattern and MRI perfusion study and brain FDG-PET, were normal, tubercolar granulomatous hypothesis was initially favored. Antitubercular therapy with isoniazid, pyrazinamide, ethambutol and rifampicin was started without any clinical improvement. Hence, the biopsy was proposed. The procedure revealed a diffuse midline pontine glioma. Considering the advanced stage of the disease, radiotherapy was not indicated. Patient died after eight months from the onset of neurological disturbances. CONCLUSION: Our case shows that diffuse midline glioma is a CNS tumor not limited to young population but occurring also in middle aged patients with an insidious pattern. We therefore recommend to perform biopsy at very early stages in patients with atypical brainstem lesions.

GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells.

DNA mismatch recognition and binding in human cells has been thought to be mediated by the hMSH2 protein. Here it is shown that the mismatch-binding factor consists of two distinct proteins, the 100-kilodalton hMSH2 and a 160-kilodalton polypeptide, GTBP (for G/T binding protein). Sequence analysis identified GTBP as a new member of the MutS homolog family. Both proteins are required for mismatch-specific binding, a result consistent with the finding that tumor-derived cell lines devoid of either protein are also devoid of mismatch-binding activity.

Mutations of GTBP in genetically unstable cells.

The molecular defects responsible for tumor cell hypermutability in humans have not yet been fully identified. Here the gene encoding a G/T mismatch-binding protein (GTBP) was localized to within 1 megabase of the related hMSH2 gene on chromosome 2 and was found to be inactivated in three hypermutable cell lines. Unlike cells defective in other mismatch repair genes, which display widespread alterations in mononucleotide, dinucleotide, and other simple repeated sequences, the GTBP-deficient cells showed alterations primarily in mononucleotide tracts. These results suggest that GTBP is important for maintaining the integrity of the human genome and document molecular defects accounting for variation in mutator phenotype.

Microtubules are involved in bafilomycin A1-induced tubulation and Rab5-dependent vacuolation of early endosomes.

The small GTPase Rab5 is an important regulator of membrane fusion in the early endocytic pathway. Here we have studied at the light microscopy level the morphology of early endosomes in MDCK cells stably expressing a GTPase-deficient Rab5 mutant, Rab5 Q79L, N-terminally tagged with a myc-epitope. These cells contain large vacuoles, readily visible by phase-contrast microscopy. Confocal immunofluorescence microscopy showed the presence of the epitopetagged protein on large perinuclear vacuoles, as well as on smaller peripheral structures. A subset of the perinuclear vacuoles appeared to colocalize with the late endosomal GTPase, Rab7. In addition, a population of very large Rab7-positive, Rab5 Q79L-negative structures were observed, suggesting that an increase in the size of early endosomes may be accompanied by an increased size of later or more mature endocytic structures. Using antibodies against the myc epitope and the early endosomal autoantigen EEA1 as markers, we found that endosomes in wild-type and mutant MDCK cells rapidly tubulate in the presence of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPase. Elongated or tubular endosomes partially colocalized with microtubules and were redistributed upon preincubation with the microtubule depolymerizing agent nocodazole before bafilomycin A1 treatment. Treatment of the Rab5 Q79L expressing cells with nocodazole alone led to a spatial redistribution and a significant decrease in the size of EEA1-positive structures, whereas their number increased. These results implicate microtubules in the bafilomycin A1-induced tubulation of endosomes as well as in the vacuolation of endosomes caused by Rab5 Q79L.

The targeting information of the mitochondrial outer membrane isoform of cytochrome b5 is contained within the carboxyl-terminal region.

Two isoforms of mammalian cytochrome b5, which have homologous cytosolic amino-terminal catalytic domains, are located one on endoplasmic reticulum (ER b5) the other on mitochondrial outer membranes (OM b5). A cDNA coding for the previously unknown carboxyl-terminal domain of OM b5 was cloned and a chimera between the catalytic domain of ER b5 and the carboxyl-terminal region of OM b5 was expressed in cultured mammalian cells. The chimera localized to mitochondria, indicating that the carboxyl-terminal 43 amino acids of OM b5 contain sufficient information to target the catalytic domain of ER b5 to the mitochondrial outer membrane.

NADH-cytochrome b5 reductase and cytochrome b5 isoforms as models for the study of post-translational targeting to the endoplasmic reticulum.

Cytochrome b5 and NADH-cytochrome b5 reductase are integral membrane proteins with cytosolic active domains and short membrane anchors, which are inserted post-translationally into their target membranes. Both are produced as different isoforms, with different localizations, in mammalian cells. In the rat, the reductase gene generates two transcripts by an alternative promoter mechanism: a ubiquitous mRNA coding for the myristylated membrane-bound form, and an erythroid mRNA which generates both the soluble form and a nonmyristylated membrane-binding form. The available evidence indicates that the ubiquitous myristylated form binds to the cytosolic face of both outer mitochondrial membranes and ER. In contrast, two genes code for two homologous forms of cytochrome b5, one of which is found on outer mitochondrial membranes, the other on the ER. The gene specifying the ER form probably also generates an erythroid-specific mRNA by alternative splicing, which codes for soluble cytochrome b5. Possible molecular mechanisms responsible for the observed localizations of these different enzyme isoforms are discussed.

FYVE finger proteins as effectors of phosphatidylinositol 3-phosphate.

Phosphatidylinositol 3-phosphate (PtdIns(3)P), generated via the phosphorylation of phosphatidylinositol by phosphatidylinositol 3-kinase (PI 3-kinase), plays an essential role in intracellular membrane traffic. The underlying mechanism is still not understood in detail, but the recent identification of the FYVE finger as a protein domain that binds specifically to PtdIns(3)P provides a number of potential effectors for PtdIns(3)P. The FYVE finger (named after the first letter of the four proteins containing it; Fab1p, YOTB, Vac1p and EEA1) is a double-zinc binding domain that is conserved in more than 30 proteins from yeast to mammals. It is found in several proteins involved in intracellular traffic, and FYVE finger mutations that affect zinc binding are associated with the loss of function of several of these proteins. The interaction of FYVE fingers with PtdIns(3)P may serve three alternative functions: First, to recruit cytosolic FYVE finger proteins to PtdIns(3)P-containing membranes (in concert with accessory molecules); second, to enrich for membrane bound FYVE finger proteins into PtdIns(3)P containing microdomains within the membrane; and third, to modulate the activity of membrane bound FYVE finger proteins.

Hodgkin's disease developing in a patient with angioimmunoblastic lymphadenopathy with dysproteinemia--a case report.

The case of a woman presenting the clinical and pathologic phenomena of angioimmunoblastic lymphadenopathy (AILD) with dysproteinemia is reported. The patient developed lesions in the lymph nodes, skin, lungs, liver and spleen, and her response to steroid and cyclophosphamide therapy was poor. At autopsy, microscopic findings in the mediastinal and abdominal lymph nodes were consistent with the diagnosis of Hodgkin's disease. Whereas the development of immunoblastic lymphoma is frequent in AILD, Hodgkin's disease is far less common. It is argued that malignant lymphoma in AILD may be the consequence of chronically depressed lymphocyte functions.

A saturated N-acylethanolamine other than N-palmitoyl ethanolamine with anti-inflammatory properties: a neglected story...

N-acylethanolamines, which include the endocannabinoid anandamide and the cannabinoid receptor-inactive saturated compounds N-palmitoyl ethanolamine and N-stearoyl ethanolamine, are ethanolamines of long-chain fatty acids degraded by fatty acid amide hydrolase (FAAH) known to accumulate in degenerating tissues and cells. Whilst much evidence supports a protective anti-inflammatory role of both anandamide and N-palmitoyl ethanolamine, very little information is available with regard to the bioactivity of N-stearoyl ethanolamine. Employing a murine model of passive IgE-induced cutaneous anaphylaxis, we have found that N-stearoyl ethanolamine is endowed with marked anti-inflammatory properties in vivo, supporting the hypothesis that endogenous N-stearoyl ethanolamine is, in analogy to N-palmitoyl ethanolamine, a bioactive signalling lipid capable of downregulating allergic inflammation in the skin. This effect, although mimicked by synthetic, non-selective, CB(1)/CB(2) receptor agonists, such as WIN55, 212-2, was not sensitive to CB(1) or CB(2) receptor antagonists, but rather was fully reversed by capsazepine, a competitive antagonist of the TRPV1 receptor. Moreover, CB(1) receptor antagonists, although effective in antagonising the WIN55,212-2-induced hypothermia, did not reduce the anti-inflammatory effect of WIN55,212-2, whilst CB(2) receptor antagonists, per se inactive, potentiated the WIN55,212-2 effect, suggesting an involvement of non-CB(1)/CB(2) receptors in the anti-inflammatory action of WIN55,212-2. All this, together with demonstration of FAAH as a major regulator of the in vivo concentrations of saturated N-stearoyl ethanolamine, in addition to N-palmitoyl ethanolamine, raise the speculation that pharmacological treatments with saturated N-acylethanolamines such as N-stearoyl ethanolamine, or alternatively FAAH inhibitors able to increase their local concentration, rather than selective CB receptor agonists, might be of promising therapeutic benefit in reducing allergic inflammation in the skin.

Initial decoherence in solid state qubits.

We study decoherence due to low frequency noise in Josephson qubits. Non-Markovian classical noise due to switching impurities determines inhomogeneous broadening of the signal. The theory is extended to include effects of high-frequency quantum noise, due to impurities or to the electromagnetic environment. The interplay of slow noise with intrinsically non-Gaussian noise sources may explain the rich physics observed in the spectroscopy and in the dynamics of charge based devices.