Proteomic analysis of rat atrial secretory granules: a platform for testable hypotheses

The recent development of powerful proteomic tools has enabled investigators to directly examine the population of proteins present in defined biological systems. We report here the first proteomic analysis of atrial secretory granules. Approximately 100 distinct protein components of the atrial secretory granule proteome were detected using subcellular fractionation and one-dimensional SDS-PAGE in conjunction with peptide mass fingerprinting by MALDI-TOF mass spectrometry. Of this number, 61 proteins were clearly identified by high probability data matches and repeated observation. The majority of the proteome was found to be membrane-associated with the most prominent proteins being peptidylglycine alpha-amidating monooxygenase (PAM) and pro-atrial natriuretic peptide (pro-ANP). This proteomic analysis of the rat atrium secretory granule produced an assembly of proteins with a diverse array of reported functions. The identified proteins fall into seven functional categories: (1) granular transport, docking and fusion; (2) signal transduction; (3) calcium-binding/calcium-dependent; (4) cellular architecture/chaperoning; (5) peptide/protein processing; (6) hormone; (7) proton transport. The novel finding of several protein processing enzymes and signal transduction proteins offer new perspectives on how pro-ANP is stored and processed to ANP during release. Accordingly, defining the proteome of the atrial secretory granule provides a framework for the development of new hypotheses that address key mechanisms governing granule function and ANP secretion.     

Association of Right Ventricular Pressure and Volume Overload with Non-Ischemic Septal Fibrosis on Cardiac Magnetic Resonance

Background

Non-ischemic fibrosis (NIF) on cardiac magnetic resonance (CMR) has been linked to poor prognosis, but its association with adverse right ventricular (RV) remodeling is unknown. This study examined a broad cohort of patients with RV dysfunction, so as to identify relationships between NIF and RV remodeling indices, including RV pressure load, volume and wall stress.

Methods and Results

The population comprised patients with RV dysfunction (EF<50%) undergoing CMR and transthoracic echo within a 14 day (5±3) interval. Cardiac structure, function, and NIF were assessed on CMR. Pulmonary artery systolic pressure (PASP) was measured on echo. 118 patients with RV dysfunction were studied, among whom 47% had NIF. Patients with NIF had lower RVEF (34±10 vs. 39±9%; p = 0.01) but similar LVEF (40±21 vs. 39±18%; p = 0.7) and LV volumes (p = NS). RV wall stress was higher with NIF (17±7 vs. 12±6 kPa; p<0.001) corresponding to increased RV end-systolic volume (143±79 vs. 110±36 ml; p = 0.006), myocardial mass (60±21 vs. 53±17 gm; p = 0.04), and PASP (52±18 vs. 41±18 mmHg; p = 0.001). NIF was associated with increased wall stress among subgroups with isolated RV (p = 0.005) and both RV and LV dysfunction (p = 0.003). In multivariable analysis, NIF was independently associated with RV volume (OR = 1.17 per 10 ml, [CI 1.04–1.32]; p = 0.01) and PASP (OR = 1.43 per 10 mmHg, [1.14–1.81]; p = 0.002) but not RV mass (OR = 0.91 per 10 gm, [0.69–1.20]; p = 0.5) [model χ² = 21; p<0.001]. NIF prevalence was higher in relation to PA pressure and RV dilation and was > 6-fold more common in the highest, vs. the lowest, common tertile of PASP and RV size (p<0.001).

Conclusion

Among wall stress components, NIF was independently associated with RV chamber dilation and afterload, supporting the concept that NIF is linked to adverse RV chamber remodeling.     

A life cycle assessment framework combining nutritional and environmental health impacts of diet: a case study on milk

Purpose

While there has been considerable effort to understand the environmental impact of a food or diet, nutritional effects are not usually included in food-related life cycle assessment (LCA).

Methods

We developed a novel Combined Nutritional and Environmental Life Cycle Assessment (CONE-LCA) framework that evaluates and compares in parallel the environmental and nutritional effects of foods or diets. We applied this framework to assess human health impacts, expressed in Disability Adjusted Life Years (DALYs), in a proof-of-concept case study that investigated the environmental and nutritional human health effects associated with the addition of one serving of fluid milk to the present average adult US diet. Epidemiology-based nutritional impacts and benefits linked to milk intake, such as colorectal cancer, stroke, and prostate cancer, were compared to selected environmental impacts traditionally considered in LCA (global warming and particulate matter) carried to a human health endpoint.

Results and discussion

Considering potential human health effects related to global warming, particulate matter, and nutrition, within the context of this study, findings suggest that adding one serving of milk to the current average diet could result in a health benefit for American adults, assuming that existing foods associated with substantial health benefits are not substituted, such as fruits and vegetables. The net health benefit is further increased when considering an iso-caloric substitution of less healthy foods (sugar-sweetened beverages). Further studies are needed to test whether this conclusion holds within a more comprehensive assessment of environmental and nutritional health impacts.

Conclusions

This case study provides the first quantitative epidemiology-based estimate of the complements and trade-offs between nutrition and environment human health burden expressed in DALYs, pioneering the infancy of a new approach in LCA. We recommend further testing of this CONE-LCA approach for other food items and diets, especially when making recommendations about sustainable diets and food choices.

     

Early cell death induced by Clostridium difficile TcdB: Uptake and Rac1‐glucosylation kinetics are decisive for cell fate

Toxin A and Toxin B (TcdA/TcdB) are large glucosyltransferases produced by Clostridium difficile. TcdB but not TcdA induces reactive oxygen species‐mediated early cell death (ECD) when applied at high concentrations. We found that nonglucosylated Rac1 is essential for induction of ECD since inhibition of Rac1 impedes this effect. ECD only occurs when TcdB is rapidly endocytosed. This was shown by generation of chimeras using the trunk of TcdB from a hypervirulent strain. TcdB from hypervirulent strain has been described to translocate from endosomes at higher pH values and thus, meaning faster than reference type TcdB. Accordingly, intracellular delivery of the glucosyltransferase domain of reference TcdB by the trunk of TcdB from hypervirulent strain increased ECD. Furthermore, proton transporters such as sodium/proton exchanger (NHE) or the ClC‐5 anion/proton exchanger, both of which contribute to endosomal acidification, also affected cytotoxic potency of TcdB: Specific inhibition of NHE reduced cytotoxicity, whereas transfection of cells with the endosomal anion/proton exchanger ClC‐5 increased cytotoxicity of TcdB. Our data suggest that both the uptake rate of TcdB into the cytosol and the status of nonglucosylated Rac1 are key determinants that are decisive for whether ECD or delayed apoptosis is triggered.     

SCF E3-mediated autoubiquitination negatively regulates activity of Cdc34 E2 but plays a nonessential role in the catalytic cycle in vitro and in vivo

One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless (K0)Cdc34(DeltaC) is indistinguishable from Cdc34(DeltaC) in ubiquitination of the prototype SCF(Cdc4) substrate Sic1 in vitro, and replacement of the CDC34 gene with either the (K0)cdc34(DeltaC) or the cdc34(DeltaC) allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.     

Plasma heat shock protein 60 and cardiovascular disease risk: the role of psychosocial, genetic, and biological factors

The Whitehall Study is a prospective epidemiological study of cardiovascular risk factors in healthy members of the British Civil Service, which has identified psychological distress as a major risk factor for coronary heart disease. The levels of circulating Hsp60 in 860 participants from the Whitehall cohort and 761 individuals diagnosed with diabetes have been measured and related to psychological, biological, and genetic factors. In the Whitehall participants, concentrations of Hsp60 ranged from undetectable to mg/mL levels. Circulating Hsp60 correlated with total and low-density lipoprotein (LDL) cholesterol and was positively associated with a flattened slope of cortisol decline over the day. Levels of this stress protein also correlated with measures of psychological stress including psychological distress, job demand, and low emotional support. Mass spectrometric analysis of circulating immunoreactive Hsp60 reveal that it is predominantly the intact protein with no mitochondrial import peptide, suggesting that this circulating protein emanates from mitochondria. The Hsp60 is stable when added to plasma and the levels in the circulation of individuals are remarkably constant over a 4-year period, suggesting plasma levels are partly genetically controlled. Sequence analysis of the HSP60-HSP10 intergenic promoter region identified a common variant 3175 C>G where the G allele had a frequency of 0.30 and was associated with higher Hsp60 levels in 761 type 2 diabetic patients. The extended range of plasma Hsp60 concentrations in the general population is genuine and is likely to be related to genetic, biological, and psychosocial risk factors for coronary artery disease.     

Mu- and kappa-opioids induce the differentiation of embryonic stem cells to neural progenitors

Growth factors, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected mu-opioid receptor (MOR-1) and kappa-opioid receptor (KOR-1) expression and immunoreactivity in embryonic stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell differentiation and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages.     

Ventilatory acclimatization in response to very small changes in PO2 in humans.

Ventilatory acclimatization to hypoxia (VAH) consists of a progressive increase in ventilation and decrease in end-tidal Pco(2) (Pet(CO(2))). Underlying VAH, there are also increases in the acute ventilatory sensitivities to hypoxia and hypercapnia. To investigate whether these changes could be induced with very mild alterations in end-tidal Po(2) (Pet(O(2))), two 5-day exposures were compared: 1) mild hypoxia, with Pet(O(2)) held at 10 Torr below the subject's normal value; and 2) mild hyperoxia, with Pet(O(2)) held at 10 Torr above the subject's normal value. During both exposures, Pet(CO(2)) was uncontrolled. For each exposure, the entire protocol required measurements on 13 consecutive mornings: 3 mornings before the hypoxic or hyperoxic exposure, 5 mornings during the exposure, and 5 mornings postexposure. After the subjects breathed room air for at least 30 min, measurements were made of Pet(CO(2)), Pet(O(2)), and the acute ventilatory sensitivities to hypoxia and hypercapnia. Ten subjects completed both protocols. There was a significant increase in the acute ventilatory sensitivity to hypoxia (Gp) after exposure to mild hypoxia, and a significant decrease in Gp after exposure to mild hyperoxia (P < 0.05, repeated-measures ANOVA). No other variables were affected by mild hypoxia or hyperoxia. The results, when combined with those from other studies, suggest that Gp varies linearly with Pet(O(2)), with a sensitivity of 3.5%/Torr (SE 1.0). This sensitivity is sufficient to suggest that Gp is continuously varying in response to normal physiological fluctuations in Pet(O(2)). We conclude that at least some of the mechanisms underlying VAH may have a physiological role at sea level.