Sodium + potassium-activated ATPase of mammalian brain. Regulation of phosphatase activity.

1. The K+-nitrophenylphosphatase activity associated with mammalian brain (Na+ + K+)-ATPase displays K+ activation curves that have intermediary plateaus and maxima in the presence of less than saturating concentrations of Na+. Zero Na+ and saturating Na+ produce sigmoid K+-activation curves with low and high K+ affinities respectively. 2. ATP inhibits K+-activated nitrophenylphosphatase through both competitive and non-competitive mechanisms. ATP is synergistic with Na+ in the mechanism which converts the enzyme from low to high K+ affinity. 3. The Na+ and K+ interactions can be accounted for by equations which describe a model with separate regulatory sites for Na+ and K+ and with K+- requiring catalytic site which is only accessible in one of the two principal conformational stages of the enzyme. 4. The effects of ATP can be accounted for by the same model through interactions at a single nucleotide binding site. Inhibition which is competitive with K+ and non-competitive with substrate arises from stabilization of the inactive enzyme conformation. Inhibition which is non-competitive with K+ and competitive with substrate results from interactions with the active enzyme conformation. The synergism between Na+ and ATP appears to arise as a consequence of the formation of phosphoryl enzyme. 5. A model for (Na+ + K+)-ATPase is discussed which involves in-phase coupling of subunit interactions as suggested by these studies.     

Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ.

Interaction of lectins with a detergent-solubilized ATPase from eel electric organ was studied. Concanavalin A, which binds to alpha-mannosides, altered the rate of enzyme migration in agar and inhibited the formation of an antigen-antibody precipitate: other lectins had no such effects. Concanavalin A similar amounts partially inhibited (Na+ + K+)-ATPase; this inhibition was reversible by alpha-methylglucoside. There was no corresponding effect of concanavalin A on the potassium p-nitrophenylphosphatase. Concanavalin A also did not interfere with ouabain binding. Thus, concanavalin A binds to an antigenic region also involved in Na+ and/or ATP binding, but does not interact with a K+ site.     

ER-lysosome lipid transfer protein VPS13C/PARK23 prevents aberrant mtDNA-dependent STING signaling

Mutations in VPS13C cause early-onset, autosomal recessive Parkinson’s disease (PD). We have established that VPS13C encodes a lipid transfer protein localized to contact sites between the ER and late endosomes/lysosomes. In the current study, we demonstrate that depleting VPS13C in HeLa cells causes an accumulation of lysosomes with an altered lipid profile, including an accumulation of di-22:6-BMP, a biomarker of the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. In addition, the DNA-sensing cGAS-STING pathway, which was recently implicated in PD pathogenesis, is activated in these cells. This activation results from a combination of elevated mitochondrial DNA in the cytosol and a defect in the degradation of activated STING, a lysosome-dependent process. These results suggest a link between ER-lysosome lipid transfer and innate immune activation in a model human cell line and place VPS13C in pathways relevant to PD pathogenesis.     

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.     

Beyond ecosystem modeling: A roadmap to community cyberinfrastructure for ecological data‐model integration

In an era of rapid global change, our ability to understand and predict Earth's natural systems is lagging behind our ability to monitor and measure changes in the biosphere. Bottlenecks to informing models with observations have reduced our capacity to fully exploit the growing volume and variety of available data. Here, we take a critical look at the information infrastructure that connects ecosystem modeling and measurement efforts, and propose a roadmap to community cyberinfrastructure development that can reduce the divisions between empirical research and modeling and accelerate the pace of discovery. A new era of data‐model integration requires investment in accessible, scalable, and transparent tools that integrate the expertise of the whole community, including both modelers and empiricists. This roadmap focuses on five key opportunities for community tools: the underlying foundations of community cyberinfrastructure; data ingest; calibration of models to data; model‐data benchmarking; and data assimilation and ecological forecasting. This community‐driven approach is a key to meeting the pressing needs of science and society in the 21st century.     

Porting the synthetic D‐glucaric acid pathway from Escherichia coli to Saccharomyces cerevisiae

D‐Glucaric acid can be produced as a value‐added chemical from biomass through a de novo pathway in Escherichia coli. However, previous studies have identified pH‐mediated toxicity at product concentrations of 5 g/L and have also found the eukaryotic myo‐inositol oxygenase (MIOX) enzyme to be rate‐limiting. We ported this pathway to Saccaromyces cerevisiae, which is naturally acid‐tolerant and evaluate a codon‐optimized MIOX homologue. We constructed two engineered yeast strains that were distinguished solely by their MIOX gene – either the previous version from Mus musculus or a homologue from Arabidopsis thaliana codon‐optimized for expression in S. cerevisiae – in order to identify the rate‐limiting steps for D‐glucaric acid production both from a fermentative and non‐fermentative carbon source. myo‐Inositol availability was found to be rate‐limiting from glucose in both strains and demonstrated to be dependent on growth rate, whereas the previously used M. musculus MIOX activity was found to be rate‐limiting from glycerol. Maximum titers were 0.56 g/L from glucose in batch mode, 0.98 g/L from glucose in fed‐batch mode, and 1.6 g/L from glucose supplemented with myo‐inositol. Future work focusing on the MIOX enzyme, the interplay between growth and production modes, and promoting aerobic respiration should further improve this pathway.     

The choline transporter resurfaces: new roles for synaptic vesicles?

Presynaptic choline uptake is vital to sustained neuronal acetylcholine (ACh) release; however, only with the recent cloning of choline transporters (CHTs) (i.e., SLC5A7), has a picture emerged of the regulatory pathways supporting CHT modulation. Studies arising from the development of CHT-specific antibodies reveal a large, intracellular reserve of CHT proteins, localized to ACh-containing, synaptic vesicles. The intersection of mechanisms supporting vesicular ACh release and choline uptake demonstrates an elegant mechanism for linking regulation of CHT membrane density to rates of ACh release. Furthermore, these studies point to control of the CHT endocytic process as an important target for novel therapeutics that could offset functional deficits in disorders bearing diminished cholinergic tone, including myasthenias and dementias.     

Stimulation of lecithin:cholesterol acyltransferase activity by apolipoprotein A-II in the presence of apolipoprotein A-I

Various combinations of incorporation and addition of apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) individually or together to a defined lecithin-cholesterol (250/12.5 molar ratio) liposome prepared by the cholate dialysis procedure were used to study the effect of apo A-II on lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity of both purified enzyme preparations and plasma. When apo A-I (0.1-3.0 nmol/assay) alone was incorporated or added to the liposome, apo A-I effectively activated the enzyme. By contrast, when apo A-II (0.1-3.0 nmol/assay) alone was incorporated into or added to the liposome, apo A-II exhibited minimal activation of LCAT activity, approximately 1% of the activity obtained by an equal amount of apo A-I. Addition of apo A-II (0.1-3.0 nmol/assay) together with apo A-I (0.8 nmol/assay) to the liposome reduced the LCAT activity to approximately 30% of the level obtained with addition of apo A-I alone. On the other hand, addition of apo A-II (0.1-3.0 nmol/assay) or addition of lecithin-cholesterol liposome containing apo A-II (0.1-3.0 nmol/assay) to lecithin-cholesterol liposome containing apo A-I (0.8 nmol/assay) did not significantly alter apo A-I activation of LCAT activity. However, when the same amounts (0.1-3.0 nmol/assay) of apo A-II were incorporated together with apo A-I (0.8 nmol/assay) into the liposome, apo A-II significantly stimulated LCAT activity as compared to activity obtained with incorporation of apo A-I alone. The maximal stimulation was obtained with 0.4 nmol apo A-II/assay for both purified and plasma enzyme. At this apo A-II concentration, approximately 4-fold and 1.8-fold stimulation was observed for purified enzyme and plasma enzyme, respectively. These results indicated that apo A-II must be incorporated together with apo A-I into lecithin-cholesterol liposomes to exert its stimulatory effect on LCAT activity and that apo A-II in high-density lipoprotein may play an important role in the regulation of LCAT activity.     

Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.