Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.     

Enhanced natriuretic effect of ADH in rats loaded with NaCl

ADH at doses 20 μU/100 g and 100 μU/100 g or 20 μU/100 g and 200 μU/100 g was injected intravenously into pentobarbital and alcohol anaesthetized rats loaded with either water or isotonic solution consisting of NaCl, glucose and ethanol. At the dose of 20 μU/100 g ADH retained water in both water and NaCl loaded animals. At high doses 100 μU/100 g in NaCl loaded rats and 200 μU/100 g in water loaded rats, ADH retained water and increased the renal excretion of Na⁺. That the natriuretic effect of ADH at the dose 20 μU/100 g was enhanced in NaCl loaded rats suggests that ADH is probably important in the regulation of Na⁺ content in the body when it is loaded with NaCl.     

Light induction of nonphotochemical quenching,CO2 fixation,and photoinhibition in woody and fern species adapted to different light regimes

We aimed to find out relations among nonphotochemical quenching (NPQ), gross photosynthetic rate (P _G), and photoinhibition during photosynthetic light induction in three woody species (one pioneer tree and two understory shrubs) and four ferns adapted to different light regimes. Pot-grown plants received 100% and/or 10% sunlight according to their light-adaptation capabilities. After at least four months of light acclimation, CO₂ exchange and chlorophyll fluorescence were measured simultaneously in the laboratory. We found that during light induction the formation and relaxation of the transient NPQ was closely related to light intensity, light-adaption capability of species, and P _G. NPQ with all treatments increased rapidly within the first 1–2 min of the light induction. Thereafter, only species with high P _G and electron transport rate (ETR), i.e., one pioneer tree and one mild shade-adapted fern, showed NPQ relaxing rapidly to a low steady-state level within 6–8 min under PPFD of 100 μmol(photon) m^?2 s^?1 and ambient CO₂ concentration. Leaves with low P _Gand ETR, regardless of species characteristics or inhibition by low CO₂ concentration, showed slow or none NPQ relaxation up to 20 min after the start of low light induction. In contrast, NPQ increased slowly to a steady state (one pioneer tree) or it did not reach the steady state (the others) from 2 to 30 min under PPFD of 2,000 μmol m^?2 s^?1. Under high excess of light energy, species adapted to or plants acclimated to high light exhibited high NPQ at the initial 1 or 2 min, and showed low photoinhibition after 30 min of light induction. The value of fastest-developing NPQ can be quickly and easily obtained and might be useful for physiological studies.     

Basal activation of p70S6K results in adipose-specific insulin resistance in protein-tyrosine phosphatase 1B -/- mice

Although protein-tyrosine phosphatase 1B (PTP-1B) is a negative regulator of insulin action, adipose tissue from PTP-1B-/- mice does not show enhanced insulin-stimulated insulin receptor phosphorylation. Investigation of glucose uptake in isolated adipocytes revealed that the adipocytes from PTP-1B-/- mice have a significantly attenuated insulin response as compared with PTP-1B+/+ adipocytes. This insulin resistance manifests in PTP-1B-/- animals older than 16 weeks of age and could be partially rescued by adenoviral expression of PTP-1B in null adipocytes. Examination of adipose signaling pathways found that the basal p70S6K activity was at least 50% higher in adipose from PTP-1B-/- mice compared with wild type animals. The increased basal activity of p70S6K in PTP-1B-/- adipose correlated with decreases in IR substrate-1 protein levels and insulin-stimulated Akt/protein kinase B activity, explaining the decrease in insulin sensitivity even as insulin receptor phosphorylation was unaffected. The insulin resistance of the of the PTP-1B-/- adipocytes could also be rescued by treatment with rapamycin, suggesting that in adipose the loss of PTP-1B results in basal activation of mTOR (mammalian target of rapamycin) complex 1 leading to a tissue-specific insulin resistance.     

Epidermal growth factor receptor and notch pathways participate in the tumor suppressor function of gamma-secretase

Gamma-secretase, a unique aspartyl protease, is required for the regulated intramembrane proteolysis of Notch and APP, pathways that are implicated, respectively, in the pathogenesis of cancer and Alzheimer disease. However, the mechanism whereby reduction of gamma-secretase causes tumors such as squamous cell carcinoma (SCC) remains poorly understood. Here, we demonstrate that gamma-secretase functions in epithelia as a tumor suppressor in an enzyme activity-dependent manner. Notch signaling is down-regulated and epidermal growth factor receptor (EGFR) is activated in SCC caused by genetic reduction of gamma-secretase. Moreover, the level of EGFR is inversely correlated with the level of gamma-secretase in fibroblasts, suggesting that the up-regulation of EGFR stimulates hyperproliferation in epithelia of mice with genetic reduction of gamma-secretase. Supporting this notion is our finding that the proliferative response of fibroblasts lacking gamma-secretase activity is more sensitive when challenged by either EGF or an inhibitor of EGFR as ompared with wild type cells. Interestingly, the up-regulation of EGFR is independent of Notch signaling, suggesting that the EGFR pathway functions in parallel with Notch in the tumorigenesis of SCC. Collectively, our results establish a novel mechanism linking the EGFR pathway to the tumor suppressor role of gamma-secretase and that mice with genetic reduction of gamma-secretase represent an excellent rodent model for clarifying pathogenesis of SCC and for testing therapeutic strategy to ameliorate this type of human cancer.     

Nitric oxide inhibits larval settlement in Amphibalanus amphitrite cyprids by repressing muscle locomotion and molting

Nitric oxide (NO) is a universal signaling molecule and plays a negative role in the metamorphosis of many biphasic organisms. Recently, the NO/cGMP (cyclic guanosine monophosphate) signaling pathway was reported to repress larval settlement in the barnacle Amphibalanus amphitrite. To understand the underlying molecular mechanism, we analyzed changes in the proteome of A. amphitrite cyprids in response to different concentrations of the NO donor sodium nitroprusside (SNP; 62.5, 250, and 1000 μM) using a label‐free proteomics method. Compared with the control, the expression of 106 proteins differed in all three treatments. These differentially expressed proteins were assigned to 13 pathways based on KEGG pathway enrichment analysis. SNP treatment stimulated the expression of heat shock proteins and arginine kinase, which are functionally related to NO synthases, increased the expression levels of glutathione transferases for detoxification, and activated the iron‐mediated fatty acid degradation pathway and the citrate cycle through ferritin. Moreover, NO repressed the level of myosins and cuticular proteins, which indicated that NO might inhibit larval settlement in A. amphitrite by modulating the process of muscle locomotion and molting.     

Nutritional evaluation of protein concentrates isolated from two red seaweeds: Hypnea charoides and Hypnea japonica in growing rats

Hydrobiologia - The nutritional values of protein concentrates (PCs) isolated from two subtropical red seaweeds, Hypnea charoides Lamouroux and H. japonica Tanaka, were evaluated in growing rats....