The synthesis of reduced metabolites of aldosterone by subcellular fractions of rat kidney: effects of antimineralocorticoids

Subcellular fractionation of male rat kidney revealed that the nuclear and plasma membrane fractions isolated from the 1,000 g pellet retained a significant proportion of the aldosterone ring-A reducing activity. Improved HPLC solvent systems separated all six possible ring-A reduced metabolites of aldosterone and revealed that 80-90% of the reduced metabolites synthesized by purified nuclei and plasma membranes were 5 alpha-reduced compounds consisting of 5 alpha-DHA and 3 alpha,5 alpha-THA in ratios of 1:2 (nuclei) and 1:1 (membranes). The 105,000 g cytosol also synthesized significant quantities of reduced, hydroxylated, and conjugated metabolites of aldosterone. In contrast, the majority of the reduced metabolites of aldosterone synthesized by kidney cytosol were 5 beta-products, consisting principally of 5 beta-DHA and smaller quantities of 3 alpha,5 beta-THA and 3 beta,5 beta-THA. The synthesis of reduced aldosterone metabolites in the cytosol, nuclear, and plasma membrane fraction was inhibited by both 5 and 50 microM concentrations of the antimineralocorticoids, progesterone, K+-canrenoate, and corticosterone. Progesterone was the strongest inhibitor of the synthesis of 5 alpha-DHA and 3 alpha,5 alpha-THA in both nuclei and plasma membranes. The overall order of inhibition of the synthesis of ring-A reduced metabolites in the kidney subcellular fractions was progesterone greater than K+-canrenoate greater than corticosterone; both progesterone and K+-canrenoate inhibited 5 alpha-reduction more than 5 beta-reduction.     

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na⁺ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na⁺. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4''-Diisothiocyano-2,2''-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.     

Landscape Position Influences Microbial Composition and Function via Redistribution of Soil Water across a Watershed

Subalpine forest ecosystems influence global carbon cycling. However, little is known about the compositions of their soil microbial communities and how these may vary with soil environmental conditions. The goal of this study was to characterize the soil microbial communities in a subalpine forest watershed in central Montana (Stringer Creek Watershed within the Tenderfoot Creek Experimental Forest) and to investigate their relationships with environmental conditions and soil carbonaceous gases. As assessed by tagged Illumina sequencing of the 16S rRNA gene, community composition and structure differed significantly among three landscape positions: high upland zones (HUZ), low upland zones (LUZ), and riparian zones (RZ). Soil depth effects on phylogenetic diversity and β-diversity varied across landscape positions, being more evident in RZ than in HUZ. Mantel tests revealed significant correlations between microbial community assembly patterns and the soil environmental factors tested (water content, temperature, oxygen, and pH) and soil carbonaceous gases (carbon dioxide concentration and efflux and methane concentration). With one exception, methanogens were detected only in RZ soils. In contrast, methanotrophs were detected in all three landscape positions. Type I methanotrophs dominated RZ soils, while type II methanotrophs dominated LUZ and HUZ soils. The relative abundances of methanotroph populations correlated positively with soil water content (R = 0.72, P < 0.001) and negatively with soil oxygen (R = −0.53, P = 0.008). Our results suggest the coherence of soil microbial communities within and differences in communities between landscape positions in a subalpine forested watershed that reflect historical and contemporary environmental conditions.     

Pathway of inactivation of cholecystokinin octapeptide (CCK-8) by synaptosomal fractions

Cholecystokinin octapeptide (CCK-8) was degraded by peptidases present in intact synaptosomes isolated from rat cortex and hypothalamus. Most of the degrading activity was present in the cytoplasmic fraction although a small amount (7%) was membrane-bound. Products of the degradation were isolated by HPLC and characterized by amino acid analysis. The Met³-Gly⁴ bond was the main primary site of cleavage giving rise to Asp-Tyr(SO₃H)-Met and Gly-Trp-Met-Asp-Phe-NH₂. These products appeared to be further degraded by sequential removal of amino-terminal residues. The Asp¹-Tyr² and Asp⁷-Phe⁸ bonds were also sites of cleavage. p-Chloromercuribenzoate was the most effective inhibitor (90% inhibition) of CCK-8 degradation by synaptosomal peptidases at the concentrations tested.This peptidase activity in synaptosomes may be important in the regulation of levels of the neuropeptide CCK-8 at the synapse. Identification of the sites of cleavage of CCK-8 on incubation with synaptosomes will assist in the isolation and characterization of the enzymes involved.     

Improvement of mammalian cell culture performance through surfactant enabled concentrated feed media

The design of basal and feed media in mammalian cell culture is paramount towards ensuring acceptable upstream process performance in various operation modes, especially fed‐batch culture. Mammalian cell culture media designs have evolved from the classical formulations designed by Eagle and Ham, to today's formulations designed from continuous improvement and statistical frameworks. Feed media is especially important for ensuring robust cell growth, productivity, and ensuring the product quality of recombinant therapeutics are within acceptable ranges. Numerous studies have highlighted the benefit of various media designs, supplements, and feed addition strategies towards the resulting cell culture process. In this work we highlight the use of a top‐down level approach towards feed media design enabled by the use of select surfactants for the targeted enrichment of a chemically defined feed media. The use of the enriched media was able to improve product titers at g/L levels, without adversely impacting the growth of multiple Chinese Hamster Ovary cell lines or the product quality of multiple recombinant antibodies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1023–1033, 2013