4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer

Calcium (Ca²⁺) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca²⁺ release was associated with the ER Ca²⁺ release channels, inositol 1,4,5?triphosphate receptor (IP₃R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca²⁺ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca²⁺ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca²⁺ concentration ([Ca²⁺]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca²⁺ release mechanism distinct from classical Ca²⁺ release channels.     

Purification and characterization of pea thioredoxin f expressed in Escherichia coli

The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide. This cDNA fragment was subcloned into a pET expression vector and used to transform E. coli cells. After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells. The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity. The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin. The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay. Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E. coli. At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.     

Intestinal humoral immunity and digestive opportunistic infections associated with AIDS]

The infectious diarrhea in AIDS is principaly due to Cryptosporidium. The study of the inflammatory and humoral immunity proteins reveal a high exudative enteropathy associated with an IgA, IgG and IgM intestinal immune response. However, this barrier of defence is not sufficient to eradicate the infectious agent.     

Growth Context and Fate of Axillary Meristems of Young Peach Trees. Influence of Parent Shoot Growth Characteristics and of Emergence Date

The relationship between several growth components of a shootand the fates of the axillary meristems (developing in the axilsof the leaves) borne by that shoot were studied, on first-ordershoots of young peach trees. A comprehensive picture of thoserelationships was obtained by a discriminant analysis. Shootgrowth at meristem emergence date was characterized by internodelength, leaf-production rate and leaf-unfolding duration. Allpossible fates of axillary meristems at the end of the growingseason (i.e. blind nodes, single vegetative or flower bud, budassociations, sylleptic or proleptic shoots) were considered.Shoot-elongation rate determined meristem fates quantitatively.The number of buds produced by a meristem increased when theshoot-elongation rate increased. Qualitatively, the fate of axillary meristems was related tothe balance between shoot-growth components. If the subtendingleaf unfolded slowly, sylleptic or proleptic shoots were morelikely to develop than bud associations, for high shoot-elongationrates; and flower buds were more frequent than vegetative buds,for low shoot-elongation rates. Compared to flower buds, blindnodes appeared for similar shoot-elongation rates but longerinternodes and lower leaf-production rates. The emergence dateslightly modified the relation between shoot growth and axillary-meristemfates, but the main features held true throughout the growingseason. The relationships between shoot growth and meristem fates mayresult from competitive interactions between the growing subtendingleaf and the developing axillary meristem. Growing conditionsmight also influence both shoot growth and meristem fates byfavouring either cell enlargement or cell division.Copyright1995, 1999 Academic Press Peach tree, Prunus persica (L.) Batsch, axillary meristem, meristem fate, branching, flowering, shoot growth, discriminant analysis, exploratory analysis     

Major Neutrophilia Observed in Acute Phase of Human Leptospirosis Is Not Associated with Increased Expression of Granulocyte Cell Activation Markers

It has long been known that pathogenic Leptospira can mobilize the immune system but the specific contribution of neutrophils to control the infectious challenge remains to be clarified. We herein analyzed the phenotype of circulating neutrophils of patients with leptospirosis and healthy controls for the expression of toll-like receptor (TLR) type 2 (TLR2, to sense the leptospiral LPS) and several activation markers: interleukin 8 chemokine receptor CD182 (CXCR2), CD11b of the integrin/opsonin complement receptor type 3 (CR3) and CD15 (ligand of the selectin). The plasmatic level of the main CD182 ligand, interleukin 8 (CXCL8), was measured by ELISA. Hospitalized leptospirosis cases showed marked neutrophilia, particularly in the most severe cases. Interestingly, TLR2 was significantly increased in leptospirosis but identical levels of CD182 and CD11b were detected when compared to controls. CD15 was significantly decreased on neutrophils in leptospirosis but returned to normal within 1 month. Basal levels of IL-8 were measured in control subjects and were not increased in leptospirosis cases at the initial stage of the disease. In conclusion, we observed that neutrophils failed to regulate the expression of several of the receptors involved in cell activation and recruitment. This study further emphasizes the paradigm that neutrophils may be impaired in their overall capacity to thwart bacterial infection in leptospirosis patients.     

Prospects for prostate cancer imaging and therapy using high-affinity TRPM8 activators

One of the best-studied temperature-gated channels is transient receptor potential melastatin 8 (TRPM8), which is activated by cold and cooling agents, such as menthol. Besides inducing a cooling sensation in sensory neurons, TRPM8 channel activation also plays a major role in physiopathology. Indeed, TRPMP8 expression increases in early stages of prostate cancer and its involvement in prostate cell apoptosis has recently been demonstrated. Thus, as TRPM8 is a tumor marker with significant potential use in diagnosis, as well as a target for cancer therapy, there is a need for new TRPM8-specific ligands. In this study, we investigated the action of "WS" compounds on TRPM8 channels. We compared the affinity of these molecules to that of menthol and icilin. This enabled us to identify new TRPM8 agonists. The menthol analog with the highest affinity, WS-12, had an EC(50) value about 2000 times lower than that of menthol and is, therefore, the highest-affinity TRPM8 ligand known to date. Finally, incorporating a fluorine atom in the WS-12 retained 75% of the activity of the parent compound. The high affinity of this new TRPM8 ligand and the possibility of incorporating a radiohalogen could thus be useful for diagnosis, monitoring and, perhaps, even therapy of prostate cancer.     

Quantitative Flow Cytometric Evaluation of Maximal Cryptosporidium parvum Oocyst Infectivity in a Neonate Mouse Model

The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.     

Wharton’s jelly‐derived stromal cells and their cell therapy applications in allogeneic haematopoietic stem cell transplantation

For decades, mesenchymal stromal cells (MSCs) have been of great interest in the fields of regenerative medicine, tissue engineering and immunomodulation. Their tremendous potential makes it desirable to cryopreserve and bank MSCs to increase their accessibility and availability. Postnatally derived MSCs seem to be of particular interest because they are harvested after delivery without ethical controversy, they have the capacity to expand at a higher rate than adult‐derived MSCs, in which expansion decreases with ageing, and they have demonstrated immunological and haematological supportive properties similar to those of adult‐derived MSCs. In this review, we focus on MSCs obtained from Wharton''s jelly (the mucous connective tissue of the umbilical cord between the amniotic epithelium and the umbilical vessels). Wharton''s jelly MSCs (WJ‐MSCs) are a good candidate for cellular therapy in haematology, with accumulating data supporting their potential to sustain haematopoietic stem cell engraftment and to modulate alloreactivity such as Graft Versus Host Disease (GVHD). We first present an overview of their in‐vitro properties and the results of preclinical murine models confirming the suitability of WJ‐MSCs for cellular therapy in haematology. Next, we focus on clinical trials and discuss tolerance, efficacy and infusion protocols reported in haematology for GVHD and engraftment.     

Plant-water regime management in a wetland: consequences for a floating vegetation-nesting bird, whiskered tern Chlidonias hybridus

In this study we investigated the interplay between water level management, floating macrophytic vegetation and nesting whiskered tern (Chlidonias hybridus) during 8 years (1995–2002) at a shallow macrophyte-dominated lake in western France. The specific question was to see if slight increases in the water regime of the lake (three scenarios), as part of a restoration programme, affect the timing of nesting and occupation of colonies by whiskered terns by way of changes in biomass of waterlily (Nymphaea alba) beds where colonies always establish. During the study period, egg-laying dates were progressively delayed up to 40–50 days (mid-May to early July) in relation to late appearance of suitable waterlily biomass (R ² = 0.67, p = 0.01) associated with high spring water levels (1.10–1.25 m vs. 0.95 m). Simultaneously some nesting attempts were observed in sub-optimal habitats (adjacent wet grasslands) in high flooding conditions leading either to poor breeding success or colony desertion. We concluded that there is a need for compromise between the experimental water levels used to control waterlily biomass and the nesting requirements of the whiskered tern population in this lake of international importance (260–510 pairs in 1995–2002, ≥ 1% Ramsar level).