Endosymbionts in paramecium

Paramecium species are extremely valuable organisms to enable experiments for the reestablishment of endosymbiosis. This is investigated in two different systems, the first with Paramecium caudatum and the endonuclear symbiotic bacterium Holospora species. Although most endosymbiotic bacteria cannot grow outside the host cell as a result of their reduced genome size, Holospora species can maintain their infectivity for a limited time. We found that an 89-kDa periplasmic protein has an important function for Holospora's invasion into the target nucleus, and that Holospora alters the host gene expression; the host thereby acquires resistance against various stresses. The second system is the symbiosis between P. bursaria and symbiotic Chlorella. Alga-free P. bursaria and the algae retain the ability to grow without a partner. Consequently, endosymbiosis between the aposymbiotic host cells and the symbiotic algae can be reestablished easily by mixing them. We now found four checkpoints for the reestablishment of the endosymbiosis between P. bursaria and the algae. The findings in the two systems provide excellent opportunities for us to elucidate not only infection processes but also to assess the associations leading to eukaryotic cell evolution. This paper summarizes recent progresses on reestablishment of the primary and the secondary endosymbiosis in Paramecium.     

Impaired growth of pancreatic exocrine cells in transgenic mice expressing human activin betaE subunit

Activins, TGF-beta superfamily members, have multiple functions in a variety of cells and tissues. Recently, additional activin beta subunit genes, betaC and betaE, have been identified. To explore the role of activin E, we created transgenic mice overexpressing human activin betaE subunit. There were pronounced differences in the pancreata of the transgenic animals as compared with their wild-type counterparts. Pancreatic weight, expressed relative to total body weight, was significantly reduced. Histologically, adipose replacement of acini in the exocrine pancreas was observed. There was a significant decrease in the number of PCNA-positive cells in the acinar cells, indicating reduced proliferation in the exocrine pancreas of the transgenic mice. However, quantitative pancreatic morphometry showed that the total number and mass of the islets of the transgenic mice were comparable with those of the nontransgenic control mice. Our findings suggest a role for activin E in regulating the proliferation of pancreatic exocrine cells.     

Can an arbitrary sequence evolve towards acquiring a biological function?

Abstract To explore the possibility that an arbitrary sequence can evolve towards acquiring functional role when fused with other pre-existing protein modules, we replaced the D2 domain of the fd-tet phage genome with the soluble random polypeptide RP3-42. The replacement yielded an fd-RP defective phage that is six-order magnitude lower infectivity than the wild-type fd-tet phage. The evolvability of RP3-42 was investigated through iterative mutation and selection. Each generation consists of a maximum of ten arbitrarily chosen clones, whereby the clone with highest infectivity was selected to be the parent clone of the generation that followed. The experimental evolution attested that, from an initial single random sequence, there will be selectable variation in a property of interest and that the property in question was able to improve over several generations. fd-7, the clone with highest infectivity at the end of the experimental evolution, showed a 240-fold increase in infectivity as compared to its origin, fd-RP. Analysis by phage ELISA using anti-M13 antibody and anti-T7 antibody revealed that about 37-fold increase in the infectivity of fd-7 was attributed to the changes in the molecular property of the single polypeptide that replaced the D2 domain of the g3p protein. This study therefore exemplifies the process of a random polypeptide generating a functional role in rejuvenating the infectivity of a defective bacteriophage when fused to some preexisting protein modules, indicating that an arbitrary sequence can evolve toward acquiring a functional role. Overall, this study could herald the conception of new perspective regarding primordial polypeptides in the field of molecular evolution.     

Structure-activity relationship of benzo[b]thiophene-2-sulfonamide derivatives as novel human chymase inhibitors

We have identified a new class of chymase inhibitor through a substituent analysis of MWP00965, which we previously discovered by in silico screening. TY-51076 (7) showed high potency (IC(50)=56 nM) and excellent selectivity for chymase compared to chymotrypsin and cathepsin G (>400-fold). The synthesis and structure-activity relationship of this class are described.     

Synthesis and preliminary biological evaluation of truncated zoanthenol analogues

Zoanthamines are a family of marine alkaloids that have complex heptacyclic structures and are reported to be interleukin-6 modulators. While the structure of zoanthamines, especially the ABC-ring portion, is similar to that of steroids, the CDEFG-ring portion, composed of aminoacetal and lactone core, is a unique structural element. In this report, we designed and synthesized ABC-ring 6 and CDEFG-ring 7, which are truncated analogues of the northern and southern hemispheres of zoanthenol 5, respectively, and which incorporate all of the functionality of each hemisphere. A preliminary SAR study suggested that the hydrochloride of the CEFG-ring portion is an active pharmacophore for suppressing the growth of interleukin-6-dependent MH60 cells.     

Public perception of the cultural value of Satoyama landscape types in Japan

Using the public survey “The Top 100 Japanese Rural Landscapes” conducted by Asahi Shimbun Newspaper Company in 2008, this study attempted to analyze public perception of rural landscapes and their cultural value. A total of 3,024 nominated sites were given coordinates and combined with land use and topographic datasets using a GIS, and classified into several landscape types using cluster analysis. Keywords that appeared frequently in the nominations were extracted using a text-mining tool and used to interpret the cultural value of each type. As a result, the nominated sites were divided into six types. The majority of nominated sites were classified as Forest Type (forest 88%) and associated with the keywords traditional rural lifestyle, and sites to visit. Mixed Type (forest 50%, paddy field 20%, and other agricultural fields 10%) were associated with biodiversity and conservation activities, and Paddy Field Type (paddy field 60%) was significantly associated with Furusato (Home). Urban and Suburban Type sites (built-up land 50%) were concentrated in the Kanto Region and nominated mainly by local citizens for their nature activities. There was also the Other Agricultural Type, which made up 10% each of the nominations, and the Coastal Type, which was mostly nominated by ocean-related organizations and rarely nominated by the general public. Additionally, the concept of biodiversity seemed to be difficult for the public to understand. The results indicated that future studies should consider public perception of the variety of Satoyama landscapes and how it should be incorporated into future Satoyama management strategy.     

Effects of living at two ambient temperatures on 24-h blood pressure and neuroendocrine function among obese and non-obese humans: a pilot study

The effects of environmental temperature on blood pressure and hormones in obese subjects in Japan were compared in two seasons: summer vs winter. Five obese (BMI, 32?±?5 kg/m²) and five non-obese (BMI, 23 ±3 kg/m²) men participated in this experiment at latitude 35°10′ N and longitude 136°57.9′ E. The average environmental temperature was 29?±?1 °C in summer and 3?±?1 °C in winter. Blood samples were analyzed for leptin, ghrelin, catecholamines, thyroid stimulating hormone (TSH), free thyroxine (fT₄), free triiodothyronine (fT₃), total cholesterol, triglycerides, insulin and glucose. Blood pressure was measured over the course of 24 h in summer and winter. A Japanese version of the Profile of Mood States (POMS) questionnaire was also administered each season. Systolic and diastolic blood pressures in obese men were significantly higher in winter (lower environmental temperatures) than in summer (higher environmental temperatures). Noradrenaline and dopamine concentrations were also significantly higher at lower environmental temperatures in obese subjects, but ghrelin, TSH, fT₃, fT₄, insulin and glucose were not significantly different in summer and winter between obese and non-obese subjects. Leptin, total cholesterol and triglyceride concentrations were significantly higher in winter in obese than non-obese men. Results from the POMS questionnaire showed a significant rise in Confusion at lower environmental temperatures (winter) in obese subjects. In this pilot study, increased blood pressure may have been due to increased secretion of noradrenaline in obese men in winter, and the results suggest that blood pressure control in obese men is particularly important in winter.     

Measurement of resistant starch content in cooked rice and analysis of gelatinization and retrogradation characteristics

Digestion-resistant starch (RS) has many physiologic functions. The RS content is measured by enzymatically degrading flour samples according to the method of the Association of Official Analytical Chemists. Experiments have been performed with wheat, corn, and other grains, but there are no data for cooked rice grains in the form ingested by humans. Thus, we investigated a method to measure RS that is suitable for cooked rice grains using rice cultivars that are reported to differentially increase postprandial blood glucose in humans. Using a method for cooking individual rice grains and optimized enzyme reaction conditions, we established an RS measurement method. We also found that the amylopectin crystal condition affects the RS content measured using our method.     

Parkinson's disease-associated mutations in α-synuclein and UCH-L1 inhibit the unconventional secretion of UCH-L1

Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is an intracellular protein abundantly expressed in neurons, and a mutation in UCH-L1 has been identified in familial Parkinson’s disease. UCH-L1 has been detected in human cerebrospinal fluid, raising the possibility that UCH-L1 is secreted from neurons. In the present study, we showed that a portion of UCH-L1 is secreted from cultured cells. The secretion of D30K UCH-L1, which lacks ubiquitin binding activity, was decreased compared with that of wild-type UCH-L1, while the secretion of C90S UCH-L1, which lacks hydrolase activity, was not. Treatment with Brefeldin A, an inhibitor of vesicle transport from the endoplasmic reticulum to the Golgi, did not block the secretion of UCH-L1, indicating that UCH-L1 is secreted by an unconventional pathway. The UCH-L1 sequence from Leu-32 to Leu-39 is similar to the unconventional secretory signal sequence of engrailed 2, and substitution of the leucines within this region (L32S/L32A/L34S/L34A/L39S/L39A) reduced the secretion of UCH-L1. We found that the Parkinson’s disease-associated mutation I93M in UCH-L1 decreased the secretion of I93M UCH-L1. In addition, Parkinson’s disease-linked α-synuclein mutants reduced the secretion of endogenous UCH-L1. Our results indicate that the hydrolase activity is not necessary for the unconventional secretion of UCH-L1, and suggest that the ubiquitin binding activity and the sequence between Leu-32 and Leu-39 are involved in the secretion. Moreover, the secretion of UCH-L1 could be involved in the pathology of Parkinson’s disease.