Osmosensing by WNK Kinases

With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.     

Cloning of Clr, a new family of lectin-like genes localized between mouse Nkrp1a and Cd69

We report the identification of a novel family of genes, named Clr, encoding C-type lectin-like molecules, which maps in the natural killer (NK) gene complex (NKC) on mouse Chromosome 6. Genomic sequence analysis indicates the presence of at least seven members between Nkrpla and Cd69. By RT-PCR, at least three members of the family are expressed on interleukin-2-activated NK cells. Sequence analysis revealed complete open reading frames of 203-205 amino acids, with a carboxyl-terminal C-type lectin-like carbohydrate recognition domain (CRD). The CRDs of the Clr proteins exhibit a significant degree of homology with the known NKC-encoded NK-cell receptors. However, a key cysteine usually present in the CRD is missing in the Clr proteins, suggesting that their ligands and functions are distinct from other molecules encoded in the NKC.     

Differential effects of warming and nitrogen fertilization on soil respiration and microbial dynamics in switchgrass croplands

The mechanistic understanding of warming and nitrogen (N) fertilization, alone or in combination, on microbially mediated decomposition is limited. In this study, soil samples were collected from previously harvested switchgrass (Panicum virgatum L.) plots that had been treated with high N fertilizer (HN: 67 kg N ha^?1) and those that had received no N fertilizer (NN) over a 3‐year period. The samples were incubated for 180 days at 15 °C and 20 °C, during which heterotrophic respiration, δ¹³C of CO₂, microbial biomass (MB), specific soil respiration rate (R_s: respiration per unit of microbial biomass), and exoenzyme activities were quantified at 10 different collections time. Employing switchgrass tissues (referred to as litter) with naturally abundant ¹³C allowed us to partition CO₂ respiration derived from soil and amended litter. Cumulative soil respiration increased significantly by 16.4% and 4.2% under warming and N fertilization, respectively. Respiration derived from soil was elevated significantly with warming, while oxidase, the agent for recalcitrant soil substrate decomposition, was not significantly affected by warming. Warming, however, significantly enhanced MB and R_s indicating a decrease in microbial growth efficiency (MGE). On the contrary, respiration derived from amended litter was elevated with N fertilization, which was consistent with the significantly elevated hydrolase. N fertilization, however, had little effect on MB and R_s, suggesting little change in microbial physiology. Temperature and N fertilization showed minimal interactive effects likely due to little differences in soil N availability between NN and HN samples, which is partly attributable to switchgrass biomass N accumulation (equivalent to ~53% of fertilizer N). Overall, the differential individual effects of warming and N fertilization may be driven by physiological adaptation and stimulated exoenzyme kinetics, respectively. The study shed insights on distinct microbial acquisition of different substrates under global temperature increase and N enrichment.     

A scalable system for production of functional pancreatic progenitors from human embryonic stem cells

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50-100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.     

Comparison of extracellular Escherichia coli AppA phytases expressed in Streptomyces lividans and Pichia pastoris

A phytase gene (appA) from Escherichia coli was cloned into Streptomyces lividans and expressed as an extracellular protein which was then compared with the same enzyme expressed in Pichia pastoris. The phytase expressed in S. lividans was not glycosylated and had a molecular mass of 45 kDa. Compared with the glycosylated phytase expressed in P. pastoris, this non-glycosylated phytase was 25–50% less active (p<0.05) at pH 2 to 3.5 or at 45 and 55 °C, but 50% more active (p<0.05) at 75 °C. The thermo-tolerance of the non-glycosylated phytase was 26 and 48% higher (p<0.05) than that of the glycosylated phytase at 45 and 55 °C, but was 80 and 94% lower (p<0.05) at 65 ° and 75 °C, respectively.     

Pressure and Temperature Effects on Growth and Methane Production of the Extreme Thermophile Methanococcus jannaschii

The marine archaebacterium Methanococcus jannaschii was studied at high temperatures and hyperbaric pressures of helium to investigate the effect of pressure on the behavior of a deep-sea thermophile. Methanogenesis and growth (as measured by protein production) at both 86 and 90°C were accelerated by pressure up to 750 atm (1 atm = 101.29kPa), but growth was not observed above 90°C at either 7.8 or 250 atm. However, growth and methanogenesis were uncoupled above 90°C, and the high-temperature limit for methanogenesis was increased by pressure. Substantial methane formation was evident at 98°C and 250 atm, whereas no methane formation was observed at 94°C and 7.8 atm. In contrast, when argon was substituted for helium as the pressurizing gas at 250 atm, no methane was produced at 86°C. Methanogenesis was also suppressed at 86°C and 250 atm when the culture was pressurized with a 4:1 mix of H₂ and CO₂, although limited methanogenesis did occur when the culture was pressurized with H₂.     

Robust passive and active efflux of cellular cholesterol to a designer functional mimic of high density lipoprotein

The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1.     

Habitat Restoration as a Means of Controlling Non-Native Fish in a Mojave Desert Oasis

Non‐native fish generally cause native fish decline, and once non‐natives are established, control or elimination is usually problematic. Because non‐native fish colonization has been greatest in anthropogenically altered habitats, restoring habitat similar to predisturbance conditions may offer a viable means of non‐native fish control. In this investigation we identified habitats favoring native over non‐native fish in a Mojave Desert oasis (Ash Meadows) and used this information to restore one of its major warm water spring systems (Kings Pool Spring). Prior to restoration, native fishes predominated in warm water (25–32°C) stream and spring‐pool habitat, whereas non‐natives predominated in cool water (≤23°C) spring‐pool and marsh/slack water habitat. Native Amargosa pupfish (Cyprinodon nevadensis) and Ash Meadows speckled dace (Rhinichthys osculus nevadensis) inhabited significantly faster mean water column velocities (MWCV) and greater total depth (TD) than non‐native Sailfin molly (Poecilia latipinna) and Mosquitofish (Gambusia affinis) in warm water stream habitat, and Ash Meadows speckled dace inhabited significantly faster water than non‐natives in cool water stream habitat. Modification of the outflow of Kings Pool Spring from marsh to warm water stream, with MWCV, TD, and temperature favoring native fish, changed the fish composition from predominantly non‐native Sailfin molly and Mosquitofish to predominantly Ash Meadows pupfish. This result supports the hypothesis that restoring spring systems to a semblance of predisturbance conditions would promote recolonization of native fishes and deter non‐native fish invasion and proliferation.     

Improved strategies for rapid identification of chemically cross-linked peptides using protein interaction reporter technology

Protein interaction reporter (PIR) technology can enable identification of in vivo protein interactions with the use of specialized chemical cross-linkers, liquid chromatography, and high-resolution mass spectrometry. PIR-cross-linkers contain labile bonds that are specifically fragmented under low energy collision or photodissociation conditions in the mass spectrometer source, thus releasing cross-linked peptides. Successful analysis of PIR-cross-linked proteins requires the use of expected mathematical relationships between cross-linked complexes and released peptides after fragmentation of the labile PIR bonds. Presented here is a next-generation software tool, BLinks, for use in the analysis and identification of PIR-cross-linked proteins. BLinks is an advancement beyond our previous efforts by incorporation of chromatographic profiles that must match between cross-linked complexes and released peptides to enable estimation of p-values to help filter true relationships from complex data sets. Additionally, BLinks was used to incorporate Mascot database searching results from subsequent MS/MS analysis of the released peptides to facilitate identification of cross-linked proteins. BLinks was used in the analysis of human serum albumin, and 46 interpeptide relationships were found spanning 30 proximal residues with a 2.2% false discovery rate. BLinks was also used to track peptides involved in multiple, coeluting relationships that make accurate identification of protein interactions difficult. An additional 10 interpeptide relationships were identified despite poor correlation using the profiling tools provided with BLinks. Additionally, BLinks can be used to globally map all interpeptide relationships from the data analysis and customize subsequent analysis to target specific peptides of interest, thus making it a useful tool for both discovery of protein interactions and mapping protein topology.