Probing the structural basis for differential KCNQ1 modulation by KCNE1 and KCNE2

KCNE1 associates with KCNQ1 to increase its current amplitude and slow the activation gating process, creating the slow delayed rectifier channel that functions as a “repolarization reserve” in human heart. The transmembrane domain (TMD) of KCNE1 plays a key role in modulating KCNQ1 pore conductance and gating kinetics, and the extracellular juxtamembrane (EJM) region plays a modulatory role by interacting with the extracellular surface of KCNQ1. KCNE2 is also expressed in human heart and can associate with KCNQ1 to suppress its current amplitude and slow the deactivation gating process. KCNE1 and KCNE2 share the transmembrane topology and a high degree of sequence homology in TMD and surrounding regions. The structural basis for their distinctly different effects on KCNQ1 is not clear. To address this question, we apply cysteine (Cys) scanning mutagenesis to TMDs and EJMs of KCNE1 and KCNE2. We analyze the patterns of functional perturbation to identify high impact positions, and probe disulfide formation between engineered Cys side chains on KCNE subunits and native Cys on KCNQ1. We also use methanethiosulfonate reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Our data suggest that the TMDs of both KCNE subunits are at about the same location but interact differently with KCNQ1. In particular, the much closer contact of KCNE2 TMD with KCNQ1, relative to that of KCNE1, is expected to impact the allosteric modulation of KCNQ1 pore conductance and may explain their differential effects on the KCNQ1 current amplitude. KCNE1 and KCNE2 also differ in the relationship between their EJMs and KCNQ1. Although the EJM of KCNE1 makes intimate contacts with KCNQ1, there appears to be a crevice between KCNQ1 and KCNE2. This putative crevice may perturb the electrical field around the voltage-sensing domain of KCNQ1, contributing to the differential effects of KCNE2 versus KCNE1 on KCNQ1 gating kinetics.     

Continuous microalgae cultivation in a photobioreactor

New biomass sources for alternative fuels has become a subject of increasing importance as the nation strives to resolve the economic and strategic impacts of limited fossil fuel resources on our national security, environment, and global climate. Algae are among the most promising non‐food‐crop‐based biomass feedstocks. However, there are currently no commercially viable microalgae‐based production systems for biofuel production that have been developed, as limitations include less‐than optimal oil content, growth rates, and cultivation techniques. While batch studies are critical for determining basic growth phases and characteristics of the algal species, steady‐state studies are necessary to better understand and measure the specific growth parameters. This study evaluated the effects of dilution rate on microalgal biomass productivity, lipid content, and fatty acid profile under steady‐state conditions with continuous illumination and carbon dioxide supplemention for two types of algae. Continuous cultures were conducted for more that 3 months. Our results show that the productivity of Chlorella minutissima varied from 39 to 137 mg/L/day (dry mass) when the dilution rate varied from 0.08 to 0.64 day^?1. The biomass productivity of C. minutissima reached a maximum value (137 mg/L/day) at a dilution rate of 0.33 day^?1, while the productivity of Dunaliella tertiolecta varied from 46 to 91 mg/L/day at a dilution rate of 0.17 to 0.74 day^?1. The biomass productivity of D. tertiolecta reached a maximum value of 91 mg/L/day at a dilution rate of 0.42 day^?1. Moreover, the lipid content had no significant change with various dilution rates. Biotechnol. Bioeng. 2012; 109: 2468–2474. © 2012 Wiley Periodicals, Inc.     

Demographic History of Shorea curtisii (Dipterocarpaceae) Inferred from Chloroplast DNA Sequence Variations

We assessed the variability of chloroplast DNA sequences in populations of the dipterocarp forest tree, Shorea curtisii. This species is widely distributed in hill and coastal hill dipterocarp forests of the Malay Peninsula, whereas isolated populations are found in the coastal hills of north Borneo. Two chloroplast DNA regions (1555 bp of trnH‐psbA‐trnK and 925 bp of trnL‐trnF) were sequenced from 123 individuals collected from six Malay Peninsula and two Bornean populations. There were 15 chloroplast haplotypes derived from 16 polymorphic sites. A haplotype network revealed two distinct haplogroups that correlate with two geographic regions, the Malay Peninsula and Borneo. These two haplogroups differed by a number of mutations, and no haplotypes were shared between populations from the different geographic regions. This suggests an ancient diversification of these haplogroups, and that long‐distance seed dispersal was unlikely to have occurred during the Pleistocene when the Sunda Shelf was a contiguous landmass. Phylogenetic analysis of the haplotypes together with those found in other Shorea species showed that two haplogroups in S. curtisii appear in different positions of the phylogenetic tree. This could be explained by the persistence of ancestral polymorphisms or by ancient chloroplast capture. Low levels of genetic differentiation were found between populations within each geographic region. Signature of a bottleneck followed by demographic expansion was detected in the Malay Peninsula haplogroup. The presence of two distinct evolutionary lineages in the different regions suggests that they should be managed independently to conserve the major sources of genetic diversity in S. curtisii.     

Acetylation-dependent regulation of mitochondrial ALDH2 activation by SIRT3 mediates acute ethanol-induced eNOS activation

Moderate alcohol consumption has beneficial effects on endothelial nitric-oxide synthase (eNOS) activation, which can engender an array of anti-atherogenic actions. Here we show that in human aortic endothelial cells (HAECs), rapid activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) mediates ethanol-induced eNOS activation by preventing reactive oxygen species (ROS) accumulation. Furthermore, activation of ALDH2 by ethanol is due to its hyperacetylation by SIRT3 inactivation. These data suggest that ethanol-induced eNOS activation in HAECs may be dependent on ALDH2 hyperacetylation by SIRT3 inactivation.     

E3 ubiquitin ligase tripartite motif 38 negatively regulates TLR-mediated immune responses by proteasomal degradation of TNF receptor-associated factor 6 in macrophages

Activation of TLR signaling in the innate immune cells is critical for the elimination of invading microorganisms. However, uncontrolled activation may lead to autoimmune and inflammatory diseases. In this article, we report the identification of tripartite motif (TRIM) 38 as a negative feedback regulator in TLR signaling by targeting TNFR-associated factor 6 (TRAF6). TRIM38 was induced by TLR stimulation in an NF-κB-dependent manner in macrophages. Knockdown of TRIM38 expression by small interfering RNA resulted in augmented activation of NF-κB and MAPKs, and enhanced expression of proinflammatory cytokines, whereas overexpression of TRIM38 has an opposite effect. As an E3 ligase, TRIM38 bound to TRAF6 and promoted K48-linked polyubiquitination, which led to the proteasomal degradation of TRAF6. Consistently, knockdown of TRIM38 expression resulted in higher protein level of TRAF6 in primary macrophages. Our findings defined a novel function for TRIM38 to prevent excessive TLR-induced inflammatory responses through proteasomal degradation of TRAF6.     

Recruitment of a distinct but related set of VH sequences into the murine CD21hi/CD23- marginal zone B cell repertoire to that seen in the class-switched antibody response

Development and maintenance of cells in the murine follicular and marginal zone compartments is thought to involve differing levels of stimulation of the BCR, although it is still not clear which BCR ligands mediate these events. How the delineation between naive and Ag experienced B cell populations relates to cell phenotype and how precise or blurred this delineation is, is also not well understood. In this study, using PCR to analyze the Ab response to phenyl-oxazolone in the mouse, we show that the Ab repertoire of CD21(hi)/CD23(-) marginal zone B cells shows persistent increase in levels of particular IgM after immunization with foreign Ag. Further, we show that these IgMs have different but related VH/CDR3 sequences from those seen in the class-switched response to oxazolone that we have also analyzed. We also detect an effect of Ag on the follicular B cell repertoire that is less persisting. These results provide evidence consistent with the signal-strength model of mature B cell development being extended to include stimulation by foreign Ag, and also further the known zone of influence of foreign Ag on the B cell compartment.     

Sphingosine-1-phosphate lyase expression in embryonic and adult murine tissues

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in immunity, inflammation, angiogenesis, and cancer. S1P lyase (SPL) is the essential enzyme responsible for S1P degradation. SPL augments apoptosis and is down-regulated in cancer. SPL generates a S1P chemical gradient that promotes lymphocyte trafficking and as such is being targeted to treat autoimmune diseases. Despite growing interest in SPL as a disease marker, antioncogene, and pharmacological target, no comprehensive characterization of SPL expression in mammalian tissues has been reported. We investigated SPL expression in developing and adult mouse tissues by generating and characterizing a β-galactosidase-SPL reporter mouse combined with immunohistochemistry, immunoblotting, and enzyme assays. SPL was expressed in thymic and splenic stromal cells, splenocytes, Peyer's Patches, colonic lymphoid aggregates, circulating T and B lymphocytes, granulocytes, and monocytes, with lowest expression in thymocytes. SPL was highly expressed within the CNS, including arachnoid lining cells, spinal cord, choroid plexus, trigeminal nerve ganglion, and specific neurons of the olfactory bulb, cerebral cortex, midbrain, hindbrain, and cerebellum. Expression was detected in brown adipose tissue, female gonads, adrenal cortex, bladder epithelium, Harderian and preputial glands, and hair follicles. This unique expression pattern suggests SPL has many undiscovered physiological functions apart from its role in immunity.     

Interleukin-22 protects rat PC12 pheochromocytoma cells from serum deprivation-induced cell death

Interleukin-22 (IL-22), an IL-10 family cytokine, mediates the crosstalk between leukocytes and epithelial cells. Previous studies reported that IL-22 expresses in mouse brain, and the rat PC12 cells are responsive to IL-22 stimulation. However, the biological roles of IL-22 in neuronal cells remain largely unknown. We show here that IL-22 activates Stat3, p38 mitogen-activated protein kinases (MAPK), and Akt pathways and inhibits Erk/MAPK pathway in na?ve PC12 cells. We further demonstrate that IL-22 protects na?ve PC12 cells from serum starvation-induced cell death via the Jak1/Stat3 and Akt pathways. We also show that IL-22 has no effects on na?ve PC12 cell proliferation and cannot protect na?ve PC12 cells from 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity. However, IL-22 exerts a dose-dependent protective effect on MPP⁺-induced neurodegeneration in nerve growth factor-differentiated PC12 cells. Overall, our data suggest that IL-22 might play a role in neurological processes. To our knowledge, this is the first report showing that IL-22 confers a neuroprotective function, which may provide a new therapeutic option for treatment of neurodegenerative diseases.