Distinct subcellular localization of tRNA-derived fragments in the infective metacyclic forms of Trypanosoma cruzi

Small non-coding RNAs derived from transfer RNAs have been identified as a broadly conserved prokaryotic and eukaryotic response to stress. Their presence coincides with changes in developmental state associated with gene expression regulation. In the epimastigote form of Trypanosoma cruzi, tRNA fragments localize to posterior cytoplasmic granules. In the infective metacyclic form of the parasite, we found tRNA-derived fragments to be abundant and evenly distributed within the cytoplasm. The fragments were not associated with polysomes, suggesting that the tRNA-derived fragments may not be directly involved in translation control in metacyclics.

Congenital ataxia and hemiplegic migraine with cerebral edema associated with a novel gain of function mutation in the calcium channel CACNA1A.

Mutations in the CACNA1A gene, encoding the α1 subunit of the voltage-gated calcium channel Ca(V)2.1 (P/Q-type), have been associated with three neurological phenotypes: familial and sporadic hemiplegic migraine type 1 (FHM1, SHM1), episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 (SCA6). We report a child with congenital ataxia, abnormal eye movements and developmental delay who presented severe attacks of hemiplegic migraine triggered by minor head traumas and associated with hemispheric swelling and seizures. Progressive cerebellar atrophy was also observed. Remission of the attacks was obtained with acetazolamide. A de novo 3 bp deletion was found in heterozygosity causing loss of a phenylalanine residue at position 1502, in one of the critical transmembrane domains of the protein contributing to the inner part of the pore. We characterized the electrophysiology of this mutant in a Xenopus oocyte in vitro system and showed that it causes gain of function of the channel. The mutant Ca(V)2.1 activates at lower voltage threshold than the wild type. These findings provide further evidence of this molecular mechanism as causative of FHM1 and expand the phenotypic spectrum of CACNA1A mutations with a child exhibiting severe SHM1 and non-episodic ataxia of congenital onset.

Current cytogenetic map of the common shrew, Sorex araneus L.: localization of 7 genes and 4 microsatellites

Here we present information on the assignment of 7 genes, ACADVL, ADORA3, ATP7A, MTMR4, MYH2, HBB, TSPAN-3, and 4 common shrew microsatellites to chromosomes of the common shrew (Sorex araneus) and on the current status of its cytogenetic map. Comparative mapping data were used for the analysis of evolutionary chromosomal rearrangements in the common shrew genome.

Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca(2+) fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca(2+)-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα(+)/calbindin(+) cells were closely surrounded by NAPE-PLD(+) fiber varicosities. No pyramidal PPARα(+)/calbindin(+) cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD(+)/calretinin(+) cells were specifically detected in CA3. NAPE-PLD(+) puncta surrounded the calretinin(+) cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions.

Gain-of-function mutations in PDR1, a regulator of antifungal drug resistance in Candida glabrata, control adherence to host cells.

Candida glabrata is an emerging opportunistic pathogen that is known to develop resistance to azole drugs due to increased drug efflux. The mechanism consists of CgPDR1-mediated upregulation of ATP-binding cassette transporters. A range of gain-of-function (GOF) mutations in CgPDR1 have been found to lead not only to azole resistance but also to enhanced virulence. This implicates CgPDR1 in the regulation of the interaction of C. glabrata with the host. To identify specific CgPDR1-regulated steps of the host-pathogen interaction, we investigated in this work the interaction of selected CgPDR1 GOF mutants with murine bone marrow-derived macrophages and human acute monocytic leukemia cell line (THP-1)-derived macrophages, as well as different epithelial cell lines. GOF mutations in CgPDR1 did not influence survival and replication within macrophages following phagocytosis but led to decreased adherence to and uptake by macrophages. This may allow evasion from the host's innate cellular immune response. The interaction with epithelial cells revealed an opposite trend, suggesting that GOF mutations in CgPDR1 may favor epithelial colonization of the host by C. glabrata through increased adherence to epithelial cell layers. These data reveal that GOF mutations in CgPDR1 modulate the interaction with host cells in ways that may contribute to increased virulence.

Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca(2+) and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca(2+)-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D28k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB(+) 1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin(+) cells (granular and pyramidal neurons), and calretinin(+) and parvalbumin(+) interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin(+) principal cells in the dentate gyrus and CA1, and in the calretinin(+) and parvalbumin(+) interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL(+) terminals were only observed around CA1 calbindin(+) pyramidal cells, CA1/3 calretinin(+) interneurons and CA3 parvalbumin(+) interneurons localized in the pyramidal cell layers. Interestingly, calbindin(+) pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.

Répartition du tissu adipeux: implications cliniques [Localization of adipose tissue: clinical implications]

The excessive accumulation of the adipose tissue is at the origin of the obesity. However its severity has no direct correlation with the comorbidities. These last ones are rather linked to the type of distribution of the fat than to its total quantity. The morphological and functional analysis of the adipose tissue reveals specific differences in its localization. The adipose tissue is thus a complex organ constituted by several cell types having various capacities of hypertrophy, hyperplasia and differentiation. While the first one is more predominant in the subcutaneous compartment, where the cell size is big, the others are more specific of the visceral adipocytes. Finally the severity of the obesity is linked to hypertrophy, while the comorbidities are associated with the capacity of proliferation and differentiation.

Gain-of-function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation.

The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel TRPV6. The TRPV6 gene was completely sequenced in 170 renal calcium stone patients. The frequency of an ancestral TRPV6 haplotype consisting of three non-synonymous polymorphisms (C157R, M378V, M681T) was significantly higher (P = 0.039) in calcium stone formers (8.4%; derived = 502, ancestral = 46) compared to non-stone-forming individuals (5.4%; derived = 645, ancestral = 37). Mineral metabolism was investigated on four different calcium regimens: (i) free-choice diet, (ii) low calcium diet, (iii) fasting and (iv) after a 1 g oral calcium load. When patients homozygous for the derived haplotype were compared with heterozygous patients, no differences were found with respect to the plasma concentrations of 1,25-vitamin D, PTH and calcium, and the urinary excretion of calcium. In one stone-forming patient, the ancestral haplotype was found to be homozygous. This patient had absorptive hypercalciuria. We therefore expressed the ancestral protein (157R+378V+681T) in Xenopus oocytes and found a significantly enhanced calcium permeability when tested by a (45)Ca(2+) uptake assay (7.11 +/- 1.93 versus 3.61 +/- 1.01 pmol/min/oocyte for ancestral versus derived haplotype, P < 0.01). These results suggest that the ancestral gain-of-function haplotype in TRPV6 plays a role in calcium stone formation in certain forms of absorptive hypercalciuria.

The apical localization of transcribing RNA polymerases on supercoiled DNA prevents their rotation around the template.

The interaction of Escherichia coli RNA polymerase with supercoiled DNA was visualized by cryo-electron microscopy of vitrified samples and by classical electron microscopy methods. We observed that when E. coli RNA polymerase binds to a promoter on supercoiled DNA, this promoter becomes located at an apical loop of the interwound DNA molecule. During transcription RNA polymerase shifts the apical loop along the DNA, always remaining at the top of the moving loop. This relationship between RNA polymerase and the supercoiled template precludes circling of the RNA polymerase around the DNA and prevents the growing RNA transcript from becoming entangled with the template DNA.

Generation and characterization of function-blocking anti-ectodysplasin A (EDA) monoclonal antibodies that induce ectodermal dysplasia.

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.

Immunoscintigraphic localization of inflammatory lesions: concept, radiolabelling and in vitro testing of a granulocyte specific antibody.

Current nuclear medicine techniques for the localization of inflammatory processes are based on injection of 111In labelled autologous granulocytes which need to be isolated and radiolabelled in vitro before reinjection. A new technique is presented here that obviates the need for cell isolation by the direct intravenous injection of a granulocyte specific 123I labelled monoclonal antibody. In this publication the basic parameters of the antibody granulocyte interaction are described. Antibody binding does not inhibit vital functions of the granulocytes, such as chemotaxis and superoxide generation. Scatchard analysis of binding data reveals an apparent affinity of the antibody for granulocytes of 6.8 X 10(9) l/mol and approximately 7.1 X 10(4) binding sites per cell. Due to the high specificity of the antibody, the only expected interference is from CEA producing tumors.

Loss-of-Function Mutations in PTPN11 Cause Metachondromatosis, but Not Ollier Disease or Maffucci Syndrome.

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a "second hit," that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome.

In vivo localization of a bispecific antibody which targets human T lymphocytes to lyse human colon cancer cells.

A bispecific MAb was derived from the fusion of a hybridoma producing MAb CD3 with a hybridoma producing MAb L-DI (which is directed against a 41-kDa glycoprotein expressed in most gastro-intestinal and pancreatic carcinomas). Bispecific antibody molecules were isolated from parental antibody molecules by the use of hydroxylapatite-HPLC and shown to target human cytolytic T lymphocytes, irrespective of their original specificity, to specifically lyse human colon carcinoma cells. Localization studies in vivo using nude mice bearing human colon carcinoma xenografts showed significant accumulation of the HPLC-purified 125I-labelled bispecific antibodies into the tumor compared to 131I-labelled control CD3 antibody.

Metabolic compartmentalization in the human cortex and hippocampus: evidence for a cell- and region-specific localization of lactate dehydrogenase 5 and pyruvate dehydrogenase.

BACKGROUND: For a long time now, glucose has been thought to be the main, if not the sole substrate for brain energy metabolism. Recent data nevertheless suggest that other molecules, such as monocarboxylates (lactate and pyruvate mainly) could be suitable substrates. Although monocarboxylates poorly cross the blood brain barrier (BBB), such substrates could replace glucose if produced locally.The two key enzymatiques systems required for the production of these monocarboxylates are lactate dehydrogenase (LDH; EC1.1.1.27) that catalyses the interconversion of lactate and pyruvate and the pyruvate dehydrogenase complex that irreversibly funnels pyruvate towards the mitochondrial TCA and oxydative phosphorylation. RESULTS: In this article, we show, with monoclonal antibodies applied to post-mortem human brain tissues, that the typically glycolytic isoenzyme of lactate dehydrogenase (LDH-5; also called LDHA or LDHM) is selectively present in astrocytes, and not in neurons, whereas pyruvate dehydrogenase (PDH) is mainly detected in neurons and barely in astrocytes. At the regional level, the distribution of the LDH-5 immunoreactive astrocytes is laminar and corresponds to regions of maximal 2-deoxyglucose uptake in the occipital cortex and hippocampus. In hippocampus, we observed that the distribution of the oxidative enzyme PDH was enriched in the neurons of the stratum pyramidale and stratum granulosum of CA1 through CA4, whereas the glycolytic enzyme LDH-5 was enriched in astrocytes of the stratum moleculare, the alveus and the white matter, revealing not only cellular, but also regional, selective distributions. The fact that LDH-5 immunoreactivity was high in astrocytes and occurred in regions where the highest uptake of 2-deoxyglucose was observed suggests that glucose uptake followed by lactate production may principally occur in these regions. CONCLUSION: These observations reveal a metabolic segregation, not only at the cellular but also at the regional level, that support the notion of metabolic compartmentalization between astrocytes and neurons, whereby lactate produced by astrocytes could be oxidized by neurons.

MRI visualized neo-intimal dissection and co-localization of novel apoptotic markers apolipoprotein C-1, ceramide and caspase-3 in a Watanabe hyperlipidemic rabbit model

PURPOSE: Apoptotic arterial wall vascular smooth muscle cell death is known to contribute to plaque vulnerability and rupture. Novel apoptotic markers like apolipoprotein C-I have been implicated in apoptotic human vascular smooth muscle cell death via recruiting a neutral sphingomyelinase (N-SMase)-ceramide pathway. In vivo relevance of these observations in an animal model of plaque rupture has not been shown. METHODS AND RESULTS: Using Watanabe rabbits, we investigated three different groups (group 1, three normal Watanabe rabbits; group 2, six Watanabe rabbits fed with high cholesterol diet for 3 months; group 3, five Watanabe rabbits with similar diet but additional endothelial denudation). We followed progression of atherosclerosis to pharmacologically induced plaque rupture non-invasively using novel 3D magnetic resonance Fast-Field-Echo angiography (TR=7.2, TE=3.6 ms, matrix=512 x 512) and Fast-Spin-Echo vessel wall imaging methods (TR=3 heart beats, TE=10.5 ms, matrix=304 x 304) on 1.5 T MRI. MRI provided excellent image quality with good MRI versus histology vessel wall thickness correlation (r=0.8). In six animals of group 2/3 MRI detected neo-intimal dissection in the abdominal aorta which was accompanied by immuno-histochemical demonstration of concomitant aforementioned novel apoptotic markers, previously implicated in the apoptotic smooth muscle cell death in vitro. CONCLUSIONS: Our studies suggest a potential role for the signal transduction pathway involving apolipoprotein C-I for in vivo apoptosis and atherosclerotic plaque rupture visualized by MRI.