The existence of all three ParaHox genes in the clitellate annelid, Perionyx excavatus

A ParaHox gene cluster is composed of three genes (Gsx, Xlox, and Cdx). It has been proposed that all three ParaHox genes were present in the last common ancestor to the lophotrochozoan protostomes and the deuterostomes and that gene loss event has occurred in the ecdysozoan lineage. In this paper, we report the existence of all three ParaHox genes in Perionyx excavatus, a clitellate annelid. Although orthologs of each of the three ParaHox genes were previously discovered from other lopotrochozoan taxa, this study constitutes the first reported isolation of all three ParaHox genes in the same clitellate species.Bum Joon Park and Sung-Jin Cho contributed equally to this work.     

Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein (~33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP (~2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.     

In vitro characterization of protein kinase CKII beta mutants defective in beta-beta dimerization

Protein kinase CKII is composed of two catalytic (alpha or alpha') subunits and two regulatory (beta) subunits. The beta subunit mediates tetramer formation through beta-beta homodimerization and alpha-beta heterodimerization. In a previous study R26 and R75, point mutants of CKIIb defective in beta-beta dimerization, were isolated. In the present work we characterized these CKIIbeta mutants in vitro. Purified R26 and R75 bound to CKIIalpha but were defective in binding to CKIIbeta. R75 stimulated the catalytic activity of CKII whereas R26 gave little stimulation, and poly-L-lysine increased the stimulation of catalytic activity by R26 or R75. Circular dichroism and intrinsic fluorescence data pointed to different conformational changes in R26 and R75. Molecular modeling of these mutants provides an explanation of the difference in their ability to interact with CKIIbeta and to activate CKIIalpha.     

Protection of palmitic acid-mediated lipotoxicity by arachidonic acid via channeling of palmitic acid into triglycerides in C2C12

Background

Excessive saturated fatty acids have been considered to be one of major contributing factors for the dysfunction of skeletal muscle cells as well as pancreatic beta cells, leading to the pathogenesis of type 2 diabetes.

Results

PA induced cell death in a dose dependent manner up to 1.5 mM, but AA protected substantially lipotoxicity caused by PA at even low concentration of 62 μM, at which monounsaturated fatty acids including palmitoleic acid (POA) and oleic acid (OA) did not protect as much as AA did. Induction of cell death by PA was resulted from mitochondrial membrane potential loss, and AA effectively blocked the progression of apoptosis. Furthermore, AA rescued significantly PA-impaired glucose uptake and -signal transduction of Akt in response to insulin.Based on the observations that polyunsaturated AA generated competently cellular droplets at low concentration within the cytosol of myotubes compared with other monounsaturated fatty acids, and AA-driven lipid droplets were also enhanced in the presence of PA, we hypothesized that incorporation of harmful PA into inert triglyceride (TG) may be responsible for the protective effects of AA against PA-induced lipotoxicity. To address this assumption, C2C12 myotubes were incubated with fluorescent probed-PA analogue 4, 4-difluoro-5, 7-dimethyl-4-boro-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY FL C16) in the presence of AA and their subsequent lipid profiles were analyzed. The analyses of lipids on thin layer chromatograpy (TLC) showed that fluorescent PA analogue was rapidly channeled into AA-driven TG droplets.

Conclusion

Taken together, it is proposed that AA diverts PA into inert TG, therefore reducing the availability of harmful PA into intracellular target molecules.     

Association between Blood Lead Levels and Age-Related Macular Degeneration

Purpose

To investigate the association between blood lead levels and prevalence of age-related macular degeneration (AMD).

Methods

A nationwide population-based cross-sectional study included 4,933 subjects aged over 40 years who participated in the 2008–2012 Korean National Health and Nutrition Examination Survey, and for whom fundus photographs were available. All participants underwent a standardized interview, evaluation of blood lead concentration, and a comprehensive ophthalmic examination. Digital fundus photographs (45°) were taken of both eyes under physiological mydriasis. All fundus photographs were graded using an international classification and grading system.

Results

Mean blood lead levels were 3.15 μg/dL in men and 2.27 μg/dL in women (P < 0.001). After adjusting for potential confounders including age, gender, smoking status, total cholesterol levels, triglyceride levels, heart problems and strokes, the adjusted odds ratio (OR) in women for any AMD was 1.86 (95% Confidence Interval [CI], 1.03–3.36) and for early AMD was 1.92 (95% CI, 1.06–3.48), for those in the highest quintile of lead level compared with the lowest quintile. In men, however, blood lead level was not significantly associated with AMD.

Conclusions

Blood lead levels were higher in men, but were only associated with AMD in women. Increased levels of blood lead may be involved in the pathogenesis of AMD development in women.     

Dongia rigui sp. nov., isolated from freshwater of a large wetland in Korea

A motile, curved to twisted rod-shaped aerobic bacterium, designated strain 04SU4-P^T, was isolated from freshwater collected from the Woopo wetland (Republic of Korea). Cells were observed to be Gram-stain negative, catalase negative and oxidase positive. The major fatty acids (>10 % of the total) were identified as C_19:0 ω8c cyclo (24.6 %), C_16:0 (24.3 %) and C_18:1 ω7c (13.1 %). The DNA G+C content was determined to be 71.5 mol%. The major polar lipids were identified as phosphatidylglycerol, phosphatidylethanolamine, one unknown phospholipid and one unknown aminolipid. The major ubiquinone was determined to be Q-10. A phylogenetic tree based on 16S rRNA gene sequences showed that strain 04SU4-P^T forms an evolutionary lineage within the genus Dongia and its nearest neighbour is Dongia mobilis LM22^T (98.0 % sequence similarity). Genomic DNA–DNA hybridization of stain 04SU4-P^T with D. mobilis LM22^T showed relatedness of only 34.2 %. The phenotypic characteristics indicate the strain 04SU4-P^T can be distinguished from the sole member of the genus Dongia. On the basis of the data presented in this study, strain 04SU4-P^T represents a novel species, for which the name Dongia rigui is proposed. The type strain is 04SU4-P^T (KCTC 23341^T = JCM 17521^T).     

Introduction of tyramide signal amplification (TSA) to pre-embedding nanogold-silver staining at the electron microscopic level.

The tyramide signal amplification (TSA) technique has been shown to detect scarce tissue antigens in light and electron microscopy. In this study we applied the TSA technique at the electron microscopic level to pre-embedding immunocytochemistry. This protocol was compared to the non-amplified protocol. With the TSA protocol, the labeling of GM130, a cis-Golgi matrix protein, was tested in a cell line and found to be highly sensitive and more enhanced than that with the simple protocol. Moreover, the gold particles were well localized to the cis-side of the Golgi apparatus in both the TSA and the simple protocol.     

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of β-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of β-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of β-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of β-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S₁ layer. At this stage, CW was strongly labeled in the S₁ layer, whereas no label was observed in the S₁ layer of NW and OW. Immunogold labeling showed that β-1-4-galactan in the S₁ layer of CW tracheids significantly decreased during the formation of the S₂ layer. Most β-1-4-galactan labeling was present in the outer S₂ region in mature CW tracheids, and was absent in the inner S₂ layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of β-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask β-1-4-galactan epitopes. The study clearly showed that β-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.     

AtCML8, a calmodulin-like protein, differentially activating CaM-dependent enzymes in Arabidopsis thaliana

Plants express many calmodulins (CaMs) and calmodulin-like (CML) proteins that sense and transduce different Ca²⁺ signals. Previously, we reported divergent soybean (Glycine max) CaM isoforms (GmCaM4/5) with differential abilities to activate CaM-dependent enzymes. To elucidate biological functions of divergent CaM proteins, we isolated a cDNA encoding a CML protein, AtCML8, from Arabidopsis. AtCML8 shows highest identity with GmCaM4 at the protein sequence level. Expression of AtCML8 was high in roots, leaves, and flowers but low in stems. In addition, the expression of AtCML8 was induced by exposure to salicylic acid or NaCl. AtCML8 showed typical characteristics of CaM such as Ca²⁺-dependent electrophoretic mobility shift and Ca²⁺ binding ability. In immunoblot analyses, AtCML8 was recognized only by antiserum against GmCaM4 but not by GmCaM1 antibodies. Interestingly, AtCML8 was able to activate phosphodiesterase (PDE) but did not activate NAD kinase. These results suggest that AtCML8 acts as a CML protein in Arabidopsis with characteristics similar to soybean divergent GmCaM4 at the biochemical levels.     

Urokinase-Type Plasminogen Activator Induces BV-2 Microglial Cell Migration Through Activation of Matrix Metalloproteinase-9

In response to brain injury, microglia migrate and accumulate in the affected sites, which is an important step in the regulation of inflammation and neuronal degeneration/regeneration. In this study, we investigated the effect of urokinase-type plasminogen activator (uPA) on the BV-2 microglial cell migration. At resting state, BV-2 microglial cells secreted uPA and the release of uPA was increased by ATP, a chemoattractant released from injured neuron. The migration of BV-2 cell was significantly induced by uPA and inhibited by uPA inhibitors. In this condition, uPA increased the activity of matrix metalloproteinase (MMP-9) and the inhibition of MMP activity with pharmacological inhibitors against either uPA (amiloride) or MMP (phenanthrolene and SB-3CT) effectively prevented BV2 cell migration. Interestingly, the level of MMP-9 protein and mRNA in the cell were not changed by uPA. These results suggest that the increase of MMP-9 activity by uPA is regulated at the post-translational level, possibly via increased activation of the enzyme. Unlike the uPA inhibitor, plasmin inhibitor PAI-1 only partially inhibited uPA-induced cell migration and MMP-9 activation. The incubation of recombinant MMP-9 with uPA resulted in the activation of MMP-9. These results suggest that uPA plays a critical role in BV-2 microglial cell migration by activating pro-MMP-9, in part by its direct action on MMP-9 and also in part by the activation of plasminogen/plasmin cascade.