The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins

BACKGROUND:Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. MATERIALS AND METHODS: Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay. RESULTS: Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II). CONCLUSIONS:On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition.     

Number,position, diameter and initial direction of growth of primary roots in Musa

To understand soil colonization by a root system, information is needed on the architecture of the root system. In monocotyledons, soil exploration is mainly due to the growth of adventitious primary roots. Primary root emergence in banana was quantified in relation to shoot and corm development. Root emergence kinetics were closely related to the development of aerial organs. Root position at emergence on the corm followed an asymptotic function of corm dry weight, so that the age of each root at a given time could be deduced from its position. Root diameter at emergence was related to the position of the roots on the corm, with younger roots being thicker than older ones. However, root diameters were not constant along a given root, but instead decreased with the distance to the base; roots appear to be conical in their basal and apical parts. Root growth directions at emergence were variable, but a high proportion of the primary roots emerged with a low angle to the horizontal. Further research is needed to evaluate whether these initial trajectories are conserved during root development. Results presented in this study are in good agreement with those reported for other monocotyledons such as maize and rice. They give quantitative information that will facilitate the development of models of root system architecture in banana.     

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.     

Prediction of Recurrence and Survival for Triple-Negative Breast Cancer (TNBC) by a Protein Signature in Tissue Samples

To date, there is no available targeted therapy for patients who are diagnosed with triple-negative breast cancers (TNBC). The aim of this study was to identify a new specific target for specific treatments. Frozen primary tumors were collected from 83 adjuvant therapy-naive TNBC patients. These samples were used for global proteome profiling by iTRAQ-OFFGEL-LC-MS/MS approach in two series: a training cohort (n = 42) and a test set (n = 41). Patients who remains free of local or distant metastasis for a minimum of 5 years after surgery were classified in the no-relapse group; the others were in the relapse group. OPLS and Kaplan–Meier analyses were performed to select candidate markers, which were validated by immunohistochemistry. Three proteins were identified in the training set and validated in the test set by Kaplan–Meier method and immunohistochemistry (IHC): TrpRS as a good prognostic markers and DP and TSP1 as bad prognostic markers. We propose the establishment of an IHC test to calculate the score of TrpRS, DP, and TSP1 in TNBC tumors to evaluate the degree of aggressiveness of the tumors. Finally, we propose that DP and TSP1 could provide therapeutic targets for specific treatments.Triple-negative breast cancers (TNBC)¹ are defined by a lack of expression of estrogen (ER), progesterone (PR), and HER2/neu receptors and account for about 15% of all breast cancers. This subtype is associated with poor prognosis (1) in terms of distant free survival (DFS) and overall survival (OS), and to date, there is no clinically available targeted therapy for patients diagnosed with TNBC. Because of the absence of specific treatment guidelines for this group of patients, TNBC are managed with standard adjuvant chemotherapy (2), which, however, seems to be less effective in those cancers. In order to improve survival, it is important to determine new specific-targeted treatment.A proteomic analysis has several inherent advantages over a genomic approach in that measured mRNA levels do not necessarily correlate to corresponding protein levels. In addition, protein detection is probably also more reflective of the tumor microenvironment. Several proteomic studies have been conducted on TNBC (3–5), but no proteomic study was conducted on large cohorts including the clinical outcome of the patients, except a recent comparative proteome analysis that identified a 11-protein signature for aggressive TNBC in a large cohort of 93 microdissected tumors (6). Although microdissection was necessary to elucidate the contribution of TNBC cells, it did not reflect the tumor with its microenvironment that is increasingly described as fundamental to explain the tumor outcome. Thus, it is now recognized that carcinomas derive from phenomena that occur in tissues, not in individual cancer cells. From this perspective, the microenvironment becomes an integral, essential part of the tumor (7, 8). In this context, taking into account the tumor microenvironment, we investigated a cohort of 83 TNBC samples without microdissection by a quantitative proteomic approach using iTRAQ labeling. Based on clinical data, we established a protein signature of the most aggressive tumors. From these differentially expressed proteins, some of them appeared to be potential therapeutic targets.     

Low WSS Induces Intimal Thickening,while Large WSS Variation and Inflammation Induce Medial Thinning,in an Animal Model of Atherosclerosis

Objective

Atherosclerotic plaque development in the arterial wall is the result of complex interaction between the wall’s endothelial layer and blood hemodynamics. However, the interaction between hemodynamic parameters and inflammation in plaque evolution is not yet fully understood. The aim of the present study was to investigate the relation between wall shear stress (WSS) and vessel wall inflammation during atherosclerotic plaque development in a minipig model of carotid stenosis.

Methods

A surgical procedure was performed to create left common carotid artery stenosis by placement of a perivascular cuff in minipigs under atherogenic diet. Animals were followed up on 3T MRI, 1 week after surgery and 3, 6, and 8 months after initiation of the diet. Computational fluid dynamics simulation estimated WSS distribution for the first imaging point. Vascular geometries were co-registered for direct comparison of plaque development and features (Gadolinium- and USPIO-Contrast Enhanced MRI, for permeability and inflammation respectively) with the initial WSS. Histological analysis was performed and sections were matched to MR images, based on spatial landmarks.

Results

Vessel wall thickening, permeability and inflammation were observed distally from the stenosis. They were eccentric and facing regions of normal wall thickness. Histological analysis confirmed eccentric plaque formation with lipid infiltration, intimal thickening and medial degradation. High phagocytic activity in the stenosis region was co-localized with high WSS, corresponding to intense medial degradation observed on histology samples.

Conclusion

Lower WSS promotes atherosclerotic plaque development distal to an induced stenosis. Vascular and perivascular inflammation locations were predominant in the high WSS stenosis segment, where medial thinning was the major consequence.     

Is caseinomacropeptide from milk proteins,an inhibitor of gastric secretion?

The aim of this work was to study, in vivo, the effect of the ingestion of not glycosylated caseinomacropeptide (CMP) on gastric secretion. In Experiments #1 and #2, 7 calves fitted with a gastric pouch received either a diet without CMP (C diet) or C diet in which CMP was introduced (equal to and 5 folds that of CMP quantity contained in cow milk, diets CMP1 and CMP5, respectively). In Experiment #3, 2 calves (with gastric pouch) were fed C diet followed by an “iv perfusion” of CMP. In Experiment #4, 25 calves fed either C, CMP1 or CMP5 diets were fitted with a blood catheter for sample collections. The quantities of daily gastric secretions seemed few modified by CMP ingestion but the profile of these secretions was changed along the day. The most important result is that CMP can inhibit gastric secretions (mainly hydrochloric acid) stimulated by the meal, but there was no dose-dependent response. No similar observations were obtained after perfusion of CMP in jugular vein. CMP was not detected in blood. Results obtained in our experiments are not in favor of its significant intestinal absorption. Gastrin, somatostatin and VIP could be implicated in the mechanisms of regulation.     

Rapid odorant release in mammalian odour binding proteins facilitates their temporal coupling to odorant signals

We have measured the effect of rat odorant-binding protein 1 on the rates of ligand uptake and liquid-to-air transfer rates with a set of defined odorous compounds. Comparison of observed rate constants (k_obs) with data simulated over a wide range of different kinetic and thermodynamic regimes shows that the data do not agree with the previously held view of a slow off-rate regime (k_off <  0.0004 s^− 1). We propose that a rapid k_off would be a necessary requirement for such a system, since slow odorant-release rates would result in significant decorrelation between the olfactory world and odour perception.     

Vasculitis: mechanisms involved and clinical manifestations

Systemic vasculitis, an inflammatory necrotizing disease of the blood vessel walls, can occur secondary to autoimmune diseases, including connective tissue diseases. Various pathogenic mechanisms have been implicated in the induction of vasculitis, including cell-mediated inflammation, immune complex-mediated inflammation and autoantibody-mediated inflammation. This inflammatory activity is believed to contribute to accelerated atherosclerosis, and also leads to increased risk for cardiovascular events in patients with rheumatoid arthritis and systemic lupus erythematosus. Endothelial cell activation is a common pathogenic pathway in the systemic vasculitis associated with rheumatoid arthritis and systemic lupus erythematosus, with elevated levels of endothelin-1 potentially inducing vascular dysregulation.     

TRP channel–associated factors are a novel protein family that regulates TRPM8 trafficking and activity

TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor–activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named “TRP channel–associated factors” (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity.