Causative Genes in Amyotrophic Lateral Sclerosis and Protein Degradation Pathways: a Link to Neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a disease caused by the degeneration of motor neurons (MNs) leading to progressive muscle weakness and atrophy. Several molecular pathways have been implicated, such as glutamate-mediated excitotoxicity, defects in cytoskeletal dynamics and axonal transport, disruption of RNA metabolism, and impairments in proteostasis. ALS is associated with protein accumulation in the cytoplasm of cells undergoing neurodegeneration, which is a hallmark of the disease. In this review, we focus on mechanisms of proteostasis, particularly protein degradation, and discuss how they are related to the genetics of ALS. Indeed, the genetic bases of the disease with the implication of more than 30 genes associated with familial ALS to date, together with the important increase in understanding of endoplasmic reticulum (ER) stress, proteasomal degradation, and autophagy, allow researchers to better understand the mechanisms underlying the selective death of motor neurons in ALS. It is clear that defects in proteostasis are involved in this type of cellular degeneration, but whether or not these mechanisms are primary causes or merely consequential remains to be clearly demonstrated. Novel cellular and animal models allowing chronic expression of mutant proteins, for example, are required. Further studies linking genetic discoveries in ALS to mechanisms of protein clearance will certainly be crucial in order to accelerate translational and clinical research towards new therapeutic targets and strategies.

Production and characterization of monoclonal antibodies specific for canine CD138 (syndecan-1) for nuclear medicine preclinical trials on spontaneous tumours.

We isolated 11 antibodies specific for canine CD138 (cCD138) to validate the interest of CD138 antigen targeting in dogs with spontaneous mammary carcinoma. The affinity of the monoclonal antibodies in the nanomolar range is suitable for immunohistochemistry and nuclear medicine applications. Four distinct epitopes were recognized on cCD138 by this panel of antibodies. CD138 expression in canine healthy tissues is comparable to that reported in humans. CD138 is frequently expressed in canine mammary carcinomas corresponding to the human triple negative breast cancer subtype, with cytoplasmic and membranous expression. In canine diffuse large B-cell lymphoma, CD138 expression is associated with the 'non-germinal center' phenotype corresponding to the most aggressive subtype in humans. This homology of CD138 expression between dogs and humans confirms the relevance of tumour-bearing dogs as spontaneous models for nuclear medicine applications, especially for the evaluation of new tumour targeting strategies for diagnosis by phenotypic imaging and radio-immunotherapy.

Comparative analysis of multiple myeloma treatment by CD138 antigen targeting with bismuth-213 and Melphalan chemotherapy.

INTRODUCTION: Multiple myeloma (MM) is a B-cell malignancy of terminally differentiated plasma cells within the bone marrow. Despite intense research to develop new treatments, cure is almost never achieved. Alpha-radioimmunotherapy (RIT) has been shown to be effective in vivo in a MM model. In order to define where alpha-RIT stands in MM treatment, the aim of this study was to compare Melphalan, MM standard treatment, with alpha-RIT using a [213Bi]-anti-mCD138 antibody in a syngeneic MM mouse model. METHODS: C57BL/KaLwRij mice were grafted with 1 × 10(6) 5T33 murine MM cells. Luciferase transfected 5T33 cells were used for in vivo localization. The first step of the study was to assess the dose-response of Melphalan 21 days after engraftment. The second step consisted in therapeutic combination: Melphalan followed by RIT at day 22 or day 25 after engraftment. Toxicity (animal weight, blood cell counts) and treatment efficacy were studied in animals receiving no treatment, injected with Melphalan alone, RIT alone at day 22 or day 25 (3.7 MBq of [213Bi]-anti-CD138) and Melphalan combined with alpha-RIT. RESULTS: Fifty percent of untreated mice died by day 63 after MM engraftment. In mice treated with Melphalan alone, only the 200 µg dose improved median survival. No animal was cured after Melphalan treatment whereas 60% of the mice survived with RIT alone at day 22 after tumor engraftment with only slight and reversible hematological radiotoxicity. No therapeutic effect was observed with alpha-RIT 25 days after engraftment. Melphalan and alpha-RIT combination does not improve overall survival compared to RIT alone, and results in increased leukocyte and red blood cell toxicity. CONCLUSIONS: Alpha-RIT seems to be a good alternative to Melphalan. Association of these two treatments provides no benefit. The perspectives of this work would be to evaluate RIT impact on the regimens incorporating the novel agents bortezomide, thalidomide and lenalidomide.

Influence of pegylation and hapten location at the surface of radiolabelled liposomes on tumour immunotargeting using bispecific antibody.

INTRODUCTION: This paper proposes liposomes as a potential new tool for radioimmunotherapy in solid tumours with a two step targeting system. Tumour pretargeting is obtained by using a monoclonal bispecific antibody (BsmAb, anti CEA x anti-DTPA-In) and pegylated liposomes containing lipid-hapten (DSPE-DTPA-In or DSPE-PEG-DTPA-In). To optimise at the same time in vivo behaviour and specific targeting, the study focuses on the liposome formulation in order to determine more precisely the role of pegylation on both the blood half-life and the specific recognition with the BsmAb. METHODS: Different liposome formulations containing two PEG length (1000 and 2000) in varying amount (1.5-6 mol%) were prepared with DTPA directly coupled to DSPE or at the end of the PEG chain (DSPE-DTPA or DSPE-PEG-DTPA). Liposomes were immobilized on an L1 chip to measure by SPR (Surface Plasmon Resonance) the effect of pegylation on the BsmAb recognition of the DTPA-In hapten. Pharmacokinetic studies were performed in mice. Tumour targeting was studied in nude mice xenografted with human colorectal adenocarcinoma cells that express CEA, and doubly radiolabelled liposomes (with (111)In and (125)I) injected 24h after the BsmAb. RESULTS: The best in vitro apparent dissociation constant was obtained with liposomes bearing DTPA at the end of the PEG chain (KD=6.3 nM), which showed significant specific tumour uptake after BsmAb injection (8.6 ± 2.4% ID/g at 24h versus 4.5 ± 0.5%ID/g for passive targeting, α=0.01). All tumour/organ ratios were superior to 1 at 24h for this formulation, except for the spleen. CONCLUSION: The feasibility of specific tumour targeting in mice with a BsmAb and radiolabelled liposomes was demonstrated and the interest of SPR to predict their targeting performance in vivo was highlighted. This original and new approach provides promising prospects for the radioimmunotherapy of solid tumours.

Cholera with severe renal failure in an Italian tourist returning from Cuba, July 2013.

In July 2013, an Italian tourist returning from Cuba was hospitalised in Trieste, Italy, for cholera caused by Vibrio cholerae O1 serotype Ogawa with severe renal failure. An outbreak of cholera was reported in Cuba in January 2013. Physicians should consider the diagnosis of cholera in travellers returning from Cuba presenting with acute watery diarrhoea.

Insights into Populus XIP aquaporins: evolutionary expansion, protein functionality, and environmental regulation.

A novel category of major intrinsic proteins which share weak similarities with previously identified aquaporin subfamilies was recently identified in land plants, and named X (for unrecognized) intrinsic proteins (XIPs). Because XIPs are still ranked as uncharacterized proteins, their further molecular characterization is required. Herein, a systematic fine-scale analysis of XIP sequences found in flowering plant databases revealed that XIPs are found in at least five groups. The phylogenetic relationship of these five groups with the phylogenetic organization of angiosperms revealed an original pattern of evolution for the XIP subfamily through distinct angiosperm taxon-specific clades. Of all flowering plant having XIPs, the genus Populus encompasses the broadest panel and the highest polymorphism of XIP isoforms, with nine PtXIP sequences distributed within three XIP groups. Comprehensive PtXIP gene expression patterns showed that only two isoforms (PtXIP2;1 and PtXIP3;2) were transcribed in vegetative tissues. However, their patterns are contrasted, PtXIP2;1 was ubiquitously accumulated whereas PtXIP3;2 was predominantly detected in wood and to a lesser extent in roots. Furthermore, only PtXIP2;1 exhibited a differential expression in leaves and stems of drought-, salicylic acid-, or wounding-challenged plants. Unexpectedly, the PtXIPs displayed different abilities to alter water transport upon expression in Xenopus laevis oocytes. PtXIP2;1 and PtXIP3;3 transported water while other PtXIPs did not.

Scanographic features of gastrointestinal stromal tumors.

PURPOSE: To present the scanographic features of gastrointestinal stromal tumor (GIST) and to discuss their differential diagnosis. PATIENTS AND METHODS: A retrospective study of 45 patients who underwent surgery for GIST between January 1990 and March 2006 was performed. RESULTS: Patient age was 64 years on average. The most common symptoms were abdominal pain and gastrointestinal bleeding. Tumors were located in the stomach in 28 patients (body: 19, antrum: 5, fundus: 4), the small intestine in 13 (jejunum: 6, duodenum: 4, ileum: 3), the rectum in two and the small bowel mesentery in two. Computed tomography showed a large (average size: 9.2 cm, range 3.3-30 cm) exophytic extragastric lobulated mass with an associated wall thickening in 35 cases (78%). The pattern was an endoluminal polyp (average size: 3.2 cm, range 2.2-5.5 cm) in eight cases (18%). The two mesenteric stromal tumors (4%) were seen as well-delimited lobulated large masses (3 and 12 cm). The enhancement was peripheral with central hemorrhagic, necrotic and cystic areas in 37 cases (82%). Mucosal ulceration was seen in 18 cases (40%) and ascites in five (11%). Peritoneal spread and liver metastasis were demonstrated in three patients (7%). Calcification, metastatic lymphadenopthy, venous thrombosis or vascular invasion were not seen. CONCLUSION: Scanographic features of GIST can suggest the diagnosis of GIST before surgery.

Doubly radiolabeled liposomes for pretargeted radioimmunotherapy.

The aim of this study was to design liposomes as radioactivity carriers for pretargeted radioimmunotherapy with favorable pharmacokinetic parameters. To monitor the liposomes integrity in vivo, their surface was radiolabeled with indium-111 bound to DTPA-derivatized phosphatidylethanolamine (DSPE-DTPA) and the aqueous phase was labeled by using an original active loading technique of radioiodinated Bolton-Hunter reagent (BH) that reacts with pre-encapsulated arginine to form a positively charged conjugate ((125)I-BH-arginine). Different formulations of doubly radiolabeled liposomes were tested in vitro and in vivo to evaluate radiolabeling stability, integrity of the vesicles and their pharmacokinetics. Radiolabeling yields were high (surface >75%, encapsulation >60%) and stable (>85% after 24 h in serum 37 degrees C). In vivo, the pharmacokinetic behavior of doubly radiolabeled liposomes was strongly dependant on the formulation. Blood clearance of PEGylated liposomes (DSPC/Chol/DSPE-DTPA/DSPE-PEG5%) was 0.15 mL/h compared to a conventional formulation (DSPC/Chol/DSPE-DTPA: clearance 1.44 mL/h). Non-encapsulated BH-arginine conjugate was quickly eliminated in urine (clearance 6.04 mL/h). Blood kinetics of the two radionuclides were similar and radiochromatographic profiles of mice serum confirmed the integrity of circulating liposomes. The significant reduction of activity uptake in organs after liposome catabolism (liver and spleen), achieved by the rapid renal elimination of (125)I-BH-arginine, should bring significant improvements for targeted radionuclide therapy with sterically-stabilized liposomes.

A voxel-based mouse for internal dose calculations using Monte Carlo simulations (MCNP).

Murine models are useful for targeted radiotherapy pre-clinical experiments. These models can help to assess the potential interest of new radiopharmaceuticals. In this study, we developed a voxel-based mouse for dosimetric estimates. A female nude mouse (30 g) was frozen and cut into slices. High-resolution digital photographs were taken directly on the frozen block after each section. Images were segmented manually. Monoenergetic photon or electron sources were simulated using the MCNP4c2 Monte Carlo code for each source organ, in order to give tables of S-factors (in Gy Bq-1 s-1) for all target organs. Results obtained from monoenergetic particles were then used to generate S-factors for several radionuclides of potential interest in targeted radiotherapy. Thirteen source and 25 target regions were considered in this study. For each source region, 16 photon and 16 electron energies were simulated. Absorbed fractions, specific absorbed fractions and S-factors were calculated for 16 radionuclides of interest for targeted radiotherapy. The results obtained generally agree well with data published previously. For electron energies ranging from 0.1 to 2.5 MeV, the self-absorbed fraction varies from 0.98 to 0.376 for the liver, and from 0.89 to 0.04 for the thyroid. Electrons cannot be considered as 'non-penetrating' radiation for energies above 0.5 MeV for mouse organs. This observation can be generalized to radionuclides: for example, the beta self-absorbed fraction for the thyroid was 0.616 for I-131; absorbed fractions for Y-90 for left kidney-to-left kidney and for left kidney-to-spleen were 0.486 and 0.058, respectively. Our voxel-based mouse allowed us to generate a dosimetric database for use in preclinical targeted radiotherapy experiments.

Molecular physiology of aquaporins in plants.

In plants, membrane channels of the major intrinsic protein (MIP) super-family exhibit a high diversity with, for instance, 35 homologues in the model species Arabidopsis thaliana. As has been found in other organisms, plant MIPs function as membrane channels permeable to water (aquaporins) and in some cases to small nonelectrolytes. The aim of the present article is to integrate into plant physiology what has been recently learned about the molecular and functional properties of aquaporins in plants. Exhaustive compilation of data in the literature shows that the numerous aquaporin isoforms of plants have specific expression patterns throughout plant development and in response to environmental stimuli. The diversity of aquaporin homologues in plants can also be explained in part by their presence in multiple subcellular compartments. In recent years, there have been numerous reports that describe the activity of water channels in purified membrane vesicles, in isolated organelles or protoplasts, and in intact plant cells or even tissues. Altogether, these data suggest that the transport of water and solutes across plant membranes concerns many facets of plant physiology. Because of the high degree of compartmentation of plant cells, aquaporins may play a critical role in cell osmoregulation. Water uptake in roots represents a typical process in which to investigate the role of aquaporins in transcellular water transport, and the mechanisms and regulations involved are discussed.

Two-step targeting of xenografted colon carcinoma using a bispecific antibody and 188Re-labeled bivalent hapten: biodistribution and dosimetry studies.

UNLABELLED: Radioimmunotherapy (RIT) is currently being considered for the treatment of solid tumors. Although results have been encouraging for pretargeted 131I RIT with the affinity enhancement system (AES), the radionuclide used is not optimal because of its long half-life, strong gamma emission, poor specific activity, and low beta particle energy. 188Re, though unsuitable for direct antibody labeling, could be used with the AES two-step targeting technique. The purpose of this study was to compare the distribution and dosimetry of a bivalent hapten labeled with 188Re or 125I. For dosimetry calculations and biodistribution data, 125I was substituted for 131I. METHODS: After preliminary injection of a bispecific anticarcinoembryonic antigen (CEA) or antihapten antibody (Bs-mAb F6-679), AG 8.1 or AG 8.0 hapten radiolabeled with 188Re or 125I was injected into a nude mouse model grafted subcutaneously with a human colon carcinoma cell line (LS-174-T) expressing CEA. A dosimetry study was performed for each animal from the concentration of radioactivity in tumor and different tissues. RESULTS: Radiolabeling of AG 8.1 with 125I afforded a 40% yield with a specific activity of 11.1 MBq/nmol after purification. Radiolabeling of AG 8.0 with 188Re afforded a 72% yield with a specific activity of 31.82 MBq/nmol. In all experiments, the percentage of tumor uptake of 125I-AG 8.1 was always significantly greater than that of 188Re-AG 8.0. The corresponding tumor-to-tissue ratios reflected uptake values. The least favorable tumor-to-normal tissue ratios in the dosimetry study were 8.1 and 8.5 for 131I (tumor-to-blood ratio and tumor-to-kidney ratio, respectively) and 2.3 for 188Re (tumor-to-intestine ratio). CONCLUSION: This study indicates that 188Re can be used for radiolabeling of hapten in two-step radioimmunotherapy protocols with the AES technique. 188Re has a greater range than 131I, which should allow the treatment of solid tumors around 1 cm in diameter. Although the method used for hapten radiolabeling did not provide optimal tumor uptake, the use of a bifunctional chelating agent associated with AG 8.1 should solve this problem.

The high diversity of aquaporins reveals novel facets of plant membrane functions.

The past year has brought significant advances in the characterisation in plants of a large class of water-channel proteins called aquaporins. The capacity of some of these proteins to transport small non-electrolytes in addition to water, together with their broad range of sub-cellular localisations, provides new clues to explain the great diversity of aquaporins in plants. Recent studies on water transport in roots illustrate how the variety of aquaporin functions at the tissue level is being uncovered.

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

Disruption of putative anion channel gene AtCLC-a in Arabidopsis suggests a role in the regulation of nitrate content.

In animals and yeast, voltage-dependent chloride channels of the CLC family play a role in basic cellular functions such as epithelial transport, plasma membrane excitability, and control of pH and membrane potential in intracellular compartments. To assess the function of CLCs in plants, we searched for CLC insertion mutants in a library of Arabidopsis lines transformed by Agrobacterium tumefaciens transferred DNA (T-DNA). Using a polymerase chain reaction-based screening procedure, an Arabidopsis line that carries a T-DNA insertion within the C-terminus of the AtCLC-a coding sequence was identified. Progeny from this plant line, clca-1, showed dramatically altered transcription of the AtCLC-a gene. Plants homozygous for the clca-1 mutation exhibited normal development and a morphology indistinguishable from the wild-type. However, their capacity to accumulate nitrate under conditions of nitrate excess was reduced in roots and shoots, by approximately 50%, while chloride, sulphate and phosphate levels were similar to the wild-type. In addition, the herbicide chlorate, an analogue of nitrate, induced a faster and more pronounced chlorosis in mutant plants. Hypersensitivity to chlorate as well as decreased nitrate levels co-segregated with the T-DNA insertion. They were found at various time points of the clca-1 life cycle, supporting the idea that AtCLC-a has a general role in the control of the nitrate status in Arabidopsis. Concordant with such a function, AtCLC-a mRNA was found in roots and shoots, and its levels rapidly increased in both tissues upon addition of nitrate but not ammonium to the culture medium. The specificity of AtCLC-a function with respect to nitrate is further supported by a similar free amino acid content in wild-type and clca-1 plants. Although the cellular localization of AtCLC-a remains unclear, our results suggest that AtCLC-a plays a role in controlling the intracellular nitrate status.

Anion channels in higher plants: functional characterization, molecular structure and physiological role.

Anion channels are well documented in various tissues, cell types and membranes of algae and higher plants, and current evidence supports their central role in cell signaling, osmoregulation, plant nutrition and metabolism. It is the aim of this review to illustrate through a few selected examples the variety of anion channels operating in plant cells and some of their regulation properties and unique physiological functions. In contrast, information on the molecular structure of plant anion channels has only recently started to emerge. Only a few genes coding for putative plant anion channels from the large chloride channel (CLC) family have been isolated, and current molecular data on these plant CLCs are presented and discussed. A major challenge remains to identify the genes encoding the various anion channels described so far in plant cells. Future prospects along this line are briefly outlined, as well as recent advances based on the use of knockout mutants in the model plant Arabidopsis thaliana to explore the physiological functions of anion channels in planta.

An abundant TIP expressed in mature highly vacuolated cells.

Aquaporins are water channel proteins found in vacuolar membranes and plasma membranes, and belong to the major intrinsic protein (MIP) family of proteins. In the present study, we purified a 75 kDa MIP protein from a crude fraction of spinach leaf intracellular membranes. Upon urea/SDS-PAGE, the 75 kDa protein appeared as a 21 kDa polypeptide, and the 75 kDa species therefore probably represents a tetramer. The corresponding cDNA was obtained by PCR cloning and had an open reading frame encoding a 25.1 kDa protein. The protein, So-deltaTIP, was most homologous to the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Using affinity-purified So-deltaTIP-specific peptide antibodies, we investigated the subcellular and tissue distribution of So-deltaTIP. So-deltaTIP was specifically located in the vacuolar membrane. It was abundant in most vacuolated cells in all vegetative organs, but was excluded from the leaf epidermis as well as from the root phloem parenchyma and meristem. In spite of the high sequence homology between delta-TIPs of spinach, Arabidopsis, sunflower and radish, their expression patterns were totally different. However, a comparison of the expression pattern of So-deltaTIP with that of more distantly related TIPs showed similarities with Arabidopsis gamma-TIP, which is expressed in zones of cell elongation/differentiation but excluded from meristematic tissues. Meristematic cells are characterized by many small vacuoles as opposed to elongating and mature cells, which generally harbour a single, large vacuole. Our results indicate that the expression of So-deltaTIP may be induced when the large vacuole is formed.

Validation of 213Bi-alpha radioimmunotherapy for multiple myeloma.

The efficacy of radioimmunotherapy (RIT) with beta emitters has been clinically demonstrated in the treatment of refractory forms of lymphoma. Alpha-emitting radionuclides with a short half-life are also good potential candidates for RIT directed at tumor targets easily accessible to radioimmunoconjugate molecules and small enough to benefit from the short range of alpha particles (<100 microm). The purpose of this study was to demonstrate the feasibility of ex vivo purging of multiple myeloma-invaded bone marrow. Tumor cells were targeted by a specific monoclonal antibody (B-B4) coupled to 213Bi by a chelating agent (pentaacetic triamine diethylene p-aminobenzyl acid). The efficacy of alpha-RIT was assessed in vitro by analysis of thymidine incorporation, cell mortality, apoptosis of myeloma cells, and the study of nonspecific irradiation of hematopoietic cell lines not recognized by B-B4-pentaacetic triamine diethylene p-aminobenzyl acid immunoconjugate. High dose-dependent cell mortality of myeloma cells was found with radiolabeled B-B4, and this mortality was total at 30 kBq/10(5) cells. Cells were found in apoptotic state at rates of up to 40% for a dose of 7.4 kBq/10(5) cells. Nonspecific mortality was low compared with specific mortality (up to 1%).