Purification of capping protein using the capping protein binding site of CARMIL as an affinity matrix

Capping protein (CP) is a ubiquitously expressed, heterodimeric actin binding protein that is essential for normal actin dynamics in cells. The existing methods for purifying native CP from tissues and recombinant CP from bacteria are time-consuming processes that involve numerous conventional chromatographic steps and functional assays to achieve a homogeneous preparation of the protein. Here, we report the rapid purification of Acanthamoeba CP from amoeba extracts and recombinant mouse CP from E. coli extracts using as an affinity matrix GST-fusion proteins containing the CP binding site from Acanthamoeba CARMIL and mouse CARMIL-1, respectively. This improved method for CP purification should facilitate the in vitro analysis of CP structure, function, and regulation.     

Arabidopsis Cor15am is a chloroplast stromal protein that has cryoprotective activity and forms oligomers

Many plants acquire increased freezing tolerance when they are exposed to nonfreezing temperatures of a certain duration. This process is known as cold acclimation and allows plants to protect themselves from freezing injury. A wide variety of polypeptides are induced during cold acclimation, among which is one encoded by COR15A in Arabidopsis (Arabidopsis thaliana). Previous studies showed that the COR15A gene encodes a small, plastid-targeted polypeptide that is processed to a mature form called Cor15am. In this study, we examined the biochemical properties and activities of Cor15am in more detail. We provide evidence that Cor15am localizes almost exclusively to the chloroplast stroma. In addition, the cold-regulated accumulation of Cor15am is affected by chloroplast functionality. Both gel-filtration chromatography and protein cross-linking reveal that Cor15am forms oligomers in the stroma of chloroplasts. Although Cor15am accumulates in response to low temperature, cold acclimation is not a prerequisite for oligomerization of Cor15am. Structural analysis suggests that Cor15am is composed of both ordered and random structures, and can stay soluble with small structural change after boiling and freeze-thaw treatments. Recombinant Cor15am exhibits in vitro cryoprotection of a freeze-labile enzyme, l-lactate dehydrogenase. Furthermore, Cor15am is capable of associating with l-lactate dehydrogenase in vitro and with potential stromal substrates in vivo. On the basis of these results, we propose that Arabidopsis Cor15am is a cryoprotective protein that forms oligomers in the chloroplast stroma, and that direct association of Cor15am with its substrates is part of its cryoprotective mechanism.     

Stay cool: habitat selection of a cyclopoid copepod in a north temperate oligotrophic lake

1. We studied the vertical distributions of Cyclops cf. sibiricus in Lake Toya, a north temperate oligotrophic lake. During the winter circulation period, their distribution was vertically homogeneous both day and night. During the summer stratification period, C. cf. sibiricus stayed below the thermocline. Diel vertical migration (DVM) was pronounced in advanced developmental stages, although the upper limit of the migration became deeper as the thermocline gradually descended. This seasonal change was observed throughout the 4‐year study period, implying that thermal structure is the primary determinant of C. cf. sibiricus distribution. 2. In a field experiment, C. cf. sibiricus incubated in the summer epilimnion, which most of the population never experience, developed faster and grew better than in their original habitat. We consider that trans‐thermocline DVM would not have evolved because of possible disadvantages such as the cost of migration offset the benefit observed in the field experiment. The rapid temperature change at the thermocline may act as a swimming‐cost estimator for the copepod. 3. Low food availability in deep water during the summer stratification period seemed to determine the lower limit of C. cf. sibiricus distribution, and the copepod minimised the risks of predation by fish via DVM. These results suggest that C. cf. sibiricus modified their distribution seasonally to obtain maximum benefit in terms of individual fitness.     

Experimental evidence that lignin-modifying enzymes are essential for degrading plant cell wall lignin by Pleurotus ostreatus using CRISPR/Cas9

Lignin-modifying enzymes (LMEs), which include laccases (Lacs), manganese peroxidases (MnPs), versatile peroxidases (VPs), and lignin peroxidases (LiPs), have been considered key factors in lignin degradation by white-rot fungi because they oxidize lignin model compounds and depolymerize synthetic lignin in vitro. However, it remains unclear whether these enzymes are essential/important in the actual degradation of natural lignin in plant cell walls. To address this long-standing issue, we examined the lignin-degrading abilities of multiple mnp/vp/lac mutants of Pleurotus ostreatus. One vp2/vp3/mnp3/mnp6 quadruple-gene mutant was generated from a monokaryotic wild-type strain PC9 using plasmid-based CRISPR/Cas9. Also, two vp2/vp3/mnp2/mnp3/mnp6, two vp2/vp3/mnp3/mnp6/lac2 quintuple-gene mutants, and two vp2/vp3/mnp2/mnp3/mnp6/lac2 sextuple-gene mutants were generated. The lignin-degrading abilities of the sextuple and vp2/vp3/mnp2/mnp3/mnp6 quintuple-gene mutants on the Beech wood sawdust medium reduced drastically, but not so much for those of the vp2/vp3/mnp3/mnp6/lac2 mutants and the quadruple mutant strain. The sextuple-gene mutants also barely degraded lignin in Japanese Cedar wood sawdust and milled rice straw. Thus, this study presented evidence that the LMEs, especially MnPs and VPs, play a crucial role in the degradation of natural lignin by P. ostreatus for the first time.     

A potent endocytosis inhibitor Ikarugamycin up-regulates TNF production

Ikarugamycin (IK) is an antibiotic which has been reported to have a variety of functions, such as inhibition of clathrin-mediated endocytosis (CME), anti-tumor effects and regulation of the immune system. Whether IK influences cytokine production is poorly understood. We have investigated the relationship between IK and production of tumor necrosis factor-α (TNF). TNF plays a pivotal role in pathogenesis of many diseases. Although the dynamics of soluble TNF (sTNF) has been widely explored so far, the functions of the membrane form of TNF (mTNF) have not been fully elucidated. We demonstrated that IK increases the amount of mTNF and prolongs the duration of TNF expression. This effect is unrelated to the shedding activity of disintegrin and metalloproteinase domain-containing protein 17 (ADAM 17). Our results revealed that there is a mechanism to terminate inflammation at the cellular level which IK dysregulates. Furthermore, IK can be a tool to study TNF signaling due to its effect of increasing mTNF expression.     

Heterogeneous distribution of a floating-leaved plant,Trapa japonica,in Lake Mikata,Japan, is determined by limitations on seed dispersal and harmful salinity levels

Factors affecting the within-lake distribution of Trapa japonica were analysed in Lake Mikata, Japan, by integrating remote sensing analyses, field surveys, and laboratory experiments. The T. japonica bed has been expanding since 2006 and covered more than 60 % of the lake’s area from 2008 to 2010. However, two parts of the lake, the upper and lower areas, retained open water, even during recent years. A survey of lake-bottom sediments revealed a heterogeneous seed distribution. Although seed density exceeded 13 seeds/m² in the lake’s central area, no seeds were observed in the upper and lower areas. A seed-bag retrieval experiment showed that 75.6 % of seeds at the upper site germinated when seeds were artificially introduced, whereas 6.7 % of seeds germinated at the lower site. These results suggest that seed dispersal opportunities are the primary limitation on the distribution of T. japonica in the upper area. Brackish water was found in springs in the lower area, reflecting an adverse inflow of water from a downstream brackish lake. Laboratory experiments revealed significant adverse effects of water salinity on germination and early growth of T. japonica. Based on these results, we concluded that the heterogeneous distribution of T. japonica within the lake was determined by a combination of two factors: limitations on seed dispersal determined by the inflowing river and harmful salinity levels caused by inflows of seawater from the Sea of Japan via a downstream brackish lake.     

Heme oxygenase activity in liver microsomes of the bullfrog, Rana catesbeiana, and its change during the tadpole development

Heme oxygenase in the liver microsomes of tadpole and adult bullfrog, Rana catesbeiana, was studied in relation to hemoglobin metabolism during tadpole development. The results obtained are as follows. 1. The specific activity of the enzyme of premetamorphic tadpole liver was comparable to that of adult frog liver. The apparent Km for methemalbumin was about 25 microM for both tadpole and frog enzymes. 2. The enzyme activity was stimulated in vivo by the injection of methemalbumin, phenylhydrazine and triiodothyronine to the animals. 3. A marked increase in the enzyme activity was found in the liver of tadpole during prometamorphic stage and metamorphic climax.