Nitric oxide synthetic pathway and cGMP levels are altered in red blood cells from end-stage renal disease patients

Red blood cells (RBCs) enzymatically produce nitric oxide (NO) by a functional RBC-nitric oxide synthase (RBC-NOS). NO is a vascular key regulatory molecule. In RBCs its generation is complex and influenced by several factors, including insulin, acetylcholine, and calcium. NO availability is reduced in end-stage renal disease (ESRD) and associated with endothelial dysfunction. We previously demonstrated that, through increased phosphatidylserine membrane exposure, ESRD-RBCs augmented their adhesion to human cultured endothelium, in which NO bioavailability decreased. Since RBC-NOS-dependent NO production in ESRD is unknown, this study aimed to investigate RBC-NOS levels/activation, NO production/bioavailability in RBCs from healthy control subjects (C, N = 18) and ESRD patients (N = 27). Although RBC-NOS expression was lower in ESRD-RBCs, NO, cyclic guanosine monophosphate (cGMP), RBC-NOS Serine1177 phosphorylation level and eNOS/Calmodulin (CaM)/Heat Shock Protein-90 (HSP90) interaction levels were higher in ESRD-RBCs, indicating increased enzyme activation. Conversely, following RBCs stimulation with insulin or ionomycin, NO and cGMP levels were significantly lower in ESRD- than in C-RBCs, suggesting that uremia might reduce the RBC-NOS response to further stimuli. Additionally, the activity of multidrug-resistance-associated protein-4 (MRP4; cGMP-membrane transporter) was significantly lower in ESRD-RBCs, suggesting a possible compromised efflux of cGMP across the ESRD-RBCs membrane. This study for the first time showed highest basal RBC-NOS activation in ESRD-RBCs, possibly to reduce the negative impact of decreased NOS expression. It is further conceivable that high NO production only partially affects cell function of ESRD-RBCs maybe because in vivo they are unable to respond to physiologic stimuli, such as calcium and/or insulin

Standardized protocol to evaluate pollen polypeptides as bee food source

International audienceBees mainly rely on pollen for their protein resources. As these molecules are essential for numerous aspects of bee physiology like ovary development and larval growth, their quantification and determination are crucial to evaluate diet quality. However, the term “protein” has been used to mention crude protein, total amino acids, or protein sensu stricto (i.e., polypeptides of molecular weight >10,000 Da). In addition to this ambiguity, current methods for protein quantification suffer from bias due to nonprotein nitrogen and protein-to-protein variations. A reliable and nondestructive method to quantify the pollen polypeptides is then essential to estimate bee food source. The present paper aims (a) to detail such a protocol, (b) to evaluate its efficiency, and (c) to confront its results to those returned by traditional methods of protein estimation. Our protocol clearly overrides some bias of previous methods and is highly reliable. Results show the high variability in content of pollen polypeptides and suggest that the main part of the proteinaceous nitrogen is from oligopeptides. They also highlight that hand-collected pollen is a better matrice than pollen loads to estimate the polypeptides of pollen as bee food source

In silico analysis of ESTs from roots of Rangpur lime (Citrus limonia Osbeck) under water stress

CitEST project resulted in the construction of cDNA libraries from different Citrus sp. tissues under various physiological conditions. Among them, plantlets of Rangpur lime were exposed to hydroponic conditions with and without water stress using PEG6000. RNA from roots was obtained and generated a total of 4,130 valid cDNA reads, with 2,020 from the non-stressed condition and 2,110 from the stressed set. Bioinformatic analyses measured the frequency of each read in the libraries and yielded an in silico transcriptional profile for each condition. A total of 40 contigs were differentially expressed and allowed to detect up-regulated homologue sequences to well known genes involved in stress response, such as aquaporins, dehydrin, sucrose synthase, and proline-related synthase. Some sequences could not be classified by using FunCat and remained with an unknown function. A large number of sequences presented high similarities to annotated genes involved with cell energy, protein synthesis and cellular transport, suggesting that Rangpur lime may sustain active cell growth under stressed condition. The presence of membrane transporters and cell signaling components could be an indication of a coordinated morphological adaptation and biochemical response during drought, helping to explain the higher tolerance of this rootstock to water stress