Jonathan Schoer

Intracellular Sterol Binding Proteins: Cholesterol Transport and Membrane Domains

Regulation intracellular cholesterol transport is not well understood. Part of the difficulty is that not only is the intracellular distribution of cholesterol not uniform, but even within membranes cholesterol has an asymmetric transbilayer and lateral distribution. Although both vesicular and protein mediated pathways for cholesterol movement are recognized, the role of intracellular cholesterol binding proteins in cytosolic cholesterol trafficking remains largely unresolved. Recent work from this and other laboratories demonstrates that these cholesterol binding proteins are involved in cholesterol uptake and intracellular trafficking. Immunocytochemical evidence supports the presence of significant amounts of proteins such as the sterol carrier protein-2 outside of peroxisomes.

A potential role for sterol carrier protein-2 in cholesterol transfer to mitochondria

Mitochondrial cholesterol oxidation rapidly depletes cholesterol from the relatively cholesterol-poor mitochondrial membranes. However, almost nothing is known regarding potential mechanism(s) whereby the mitochondrial cholesterol pool is restored. Since most exogenous cholesterol enters the cell via the lysosomal pathway, this could be a source of mitochondrial cholesterol. In the present study, an in vitro fluorescent sterol transfer assay was used to examine whether the lysosomal membrane could be a putative cholesterol donor to mitochondria. First, it was shown that spontaneous sterol transfer from lysosomal to mitochondrial membranes was very slow (initial rate, 0.316 +/- 0.032 pmol/min). This was due, in part, to the fact that 90% of the lysosomal membrane sterol was not exchangeable, while the remaining 10% also had a relatively long half-time of exchange t(1/2) = 202 +/- 19 min. Second, the intracellular sterol carrier protein-2 (SCP-2) and its precursor (pro-SCP-2) increased the initial rate of sterol transfer from the lysosomal to mitochondrial membrane by 5.2- and 2.0-fold, respectively, but not in the reverse direction. The enhanced sterol transfer was due to a 3.5-fold increase in exchangeable sterol pool size and to induction of a very rapidly (t(1/2) = 4.1 +/- 0.6 min) exchangeable sterol pool. Confocal fluorescence imaging and indirect immunocytochemistry colocalized significant amounts of SCP-2 with the mitochondrial marker enzyme cytochrome oxidase in transfected L-cells overexpressing SCP-2. In summary, SCP-2 and pro-SCP-2 both stimulated molecular sterol transfer from lysosomal to mitochondrial membranes, suggesting a potential mechanism for replenishing mitochondrial cholesterol pools depleted by cholesterol oxidation.

Exogenous Phosphorus Inputs Alter Complexity of Soil-Dissolved Organic Carbon in Agricultural Riparian Wetlands

High-strengthened farmland fertilization leads to mass inputs of nutrients and elements to agricultural riparian wetlands. The dissolved organic carbon (DOC) of such wetland sediments is an important intermediate in global carbon (C) cycling due to its role in connecting soil C pools with atmospheric CO2. But the impact of phosphorus (P) on sediment DOC is still largely unknown, despite increasing investigations to emphasize P interception by riparian wetlands. Here, we simulated the temporal influences of exogenous P on sediment DOC of riparian wetlands by integrating gradient P loading at rates of 0%, 5%, 10%, 20%, 30%, and 60% relative to the initial total phosphorus content of the sediment with the purpose of illustrating the role of external P on the complexity of soil DOC in terms of its amount and composition. After incubating for nine months, a dramatic linear correlation between Olsen-P and fluorescent and ultraviolet spectral indices considered DOC skeleton was observed. Together with a more microbial-derived origin of DOC and a reduction of DOC aromaticity or humicity, the excitation-emission matrix had shown a blue shift reflecting a trend towards a simpler molecular structure of sediment DOC after P addition. Meanwhile, the content of soil DOC and its ratio with total organic carbon (TOC) were also increased by P loading, coupled with enhanced values of highly labile organic carbon and two C-related enzymes. While TOC and recalcitrant organic carbon decreased significantly. Such implications of DOC amounts and composition stimulated by external P loading may enhance its bioavailability, thereby inducing an accelerated effect on soil C cycling and a potential C loss in response to global climate change.

Molecular and fluorescent sterol approaches to probing lysosomal membrane lipid dynamics.

Although the most exogenous lipids enter the cell via the LDL-receptor pathway, the mechanism(s) whereby lipids leave the lysosome for transport to intracellular sites are not clearly resolved. As shown herein, expression of sterol carrier protein-2 (SCP-2) in transfected L-cells altered lysosomal membrane lipid distribution, dynamics, and response to lipid transfer proteins. SCP-2 expression decreased the mass of cholesterol and lyso-bis-phosphatidic acid [LBPA], as well as the ratios of cholesterol/phospholipid and polyunsaturated/monounsaturated fatty acids esterified to lysosomal membrane phospholipids. Concomitantly, a fluorescent sterol transfer assay showed that SCP-2 expression decreased the initial rates of spontaneous and SCP-2-mediated sterol transfer 5.5- and 3.8-fold, respectively, from lysosomal membranes isolated from SCP-2 expressing cells as compared to controls. SCP-2, sphingomyelinase, low density lipoprotein, and high density lipoprotein directly enhanced the initial rates of sterol transfer from isolated lysosomal membranes by 50-, 12-, 4-, and 5-fold, respectively. In contrast, albumin and cholesterol esterase had no effect on lysosomal sterol transfer. Spontaneous sterol was very slow, t(1/2)>4 days, regardless of the source of the lysosomal membrane, while SCP-2 added in vitro induced formation of rapid and slowly transferable sterol pools in lysosomal membranes of control cells. In contrast, SCP-2 did not induce formation of a rapidly transferable sterol domain in lysosomal membranes isolated from SCP-2 expressing cells. These data suggest that SCP-2 expression selectively shifted the distribution of lipids (cholesterol, LBPA, esterified polyunsaturated fatty acids) away from lysosomal membranes. Furthermore, the cholesterol depleted lysosomal membrane isolated from SCP-2 expressing cells was resistant to additional direct action of SCP-2 to further enhance sterol transfer and induce rapidly transferable sterol pools in the lysosomal membrane.

Deduction of arbitrary excitation temperatures for various analyte species in inductively coupled plasmas from vertically-resolved emission profiles

Abstract Vertically resolved profiles of excitation temperature (Texc) are derived using emission profiles from Fe I, Cr I, Mn I, Ba II and Ca II lines from an argon inductively coupled plasma (ICP). A calculation method is described with which the spatial behavior of Texc and the adherence of excited state populations to a Boltzmann distribution can be studied without transition probabilities for the lines monitored. In general, the various species all yield a hump-shaped Texc profile with maximum Texc at or near the peak of the intensity profile for “hard” lines. However, the Texc values obtained vary between species and also between energetically-different excited states of the same species, which indicates that the excited state populations deviate from a Boltzmann distribution.

Ion association by time-of-flight mass spectrometry: A study of V-P-O catalysts

The oxidation catalysts (VO2)P207 and β-VOPO4 are studied by laser desorption time-of flight mass spectrometry. Mass spectra of both positive and negative ions are reported for these compounds. Individual scans are tested for ion association, that is, the consistent appearance of a given pair of ions in many scans. The structurally simpler ⨿-VOPO4 shows fewer associations than either (VO)2P207 or an 18O-substituted analogue of β-VOPO4 even though the overall spectra from the three compounds do not differ greatly. These results indicate that the associations observed between ions may be sensitive to small differences in structure in the original specimen. Thus, the identification of ion association may provide information to supplement the mass spectrum of the sample.

Thermal electrolytic production of Mg from MgO: Reflections on commercial viability

We are exploring the commercial viability for producing Mg from MgO for which thermal energy is supplied to the cell as a substitute for some electric energy. The thermal input source may be concentrated sunlight or natural gas. Laboratory-scale electrochemical studies near 1250 K for two cell concepts show that we reached current densities above 0.5 A-cm−2at an overvoltage of 1.0 V. Current efficiency values exceeded 80%. The discussion of the relationship between these bench-top experimental results and the industrial potential of the process has been initiated.

Can Incongruent Studies Effectively Characterize Long-Term Water Quality?

Abstract Science-based measurements and studies are vital for the protection of freshwater resources, systems, and habitats. Scientists are trained to plan and implement projects to address environmental and public health challenges such as water quality problems. The most successful programs for public benefit require coordination between experienced scientists, resource managers, and interested citizens and include long-term goals. For the assessment and protection of resources, scientists should be assigned prominent roles, and administrators and others should utilize their strengths to avoid fragmented data collections, irregularities in measurements and follow-up, or abandonment of final objectives. The requirements for useful water quality monitoring projects are addressed in the context of the watershed management plans in the state of Indiana, with a focus on the Salt Creek watershed in Northwest Indiana, which drains into Lake Michigan.