Morphological and physiological differences among cultivation lines of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> in a common garden experiment using a tank culture system

Undaria pinnatifida is grown for food and industrial materials worldwide; therefore, advanced breeding is needed to meet quality and productivity requirements. In this study, we examined regional lines of U. pinnatifida from five cultivation sites in Japan with different environmental conditions: Oga (OGA, the northern Sea of Japan coast), Hirota Bay (HRT, the northeastern Pacific coast), Matsushima Bay (MAT, the northeastern Pacific coast), Naruto (the Seto Inland Sea coast) and Shimonoseki (SIM, the southern Sea of Japan coast). The sporophytes of these lines were cultured in a tank culture system under controlled environmental conditions, and their morphological characteristics, nutrient uptake kinetics (V _max, K _s and V _max/K _s ), and carbon, nitrogen and phosphorus contents were determined. Sporophytes from MAT grew faster, whereas those from SIM were smaller than those from the other sites. Although the blade thickness of sporophytes cultivated in the sea significantly differs among cultivation sites in the previous study, there was no significant difference in blade thickness among the regional lines cultivated in the tank. Sporophytes from OGA had the greatest V _max/K _s values and significantly greater nitrogen contents than the other lines. Therefore, the morphological characteristics of MAT and SIM sporophytes, and the nutrient uptake kinetics of OGA sporophytes may have a genetic origin. This indicates that these lines may represent useful resources for selective breeding, with MAT sporophytes providing faster growth and OGA sporophytes being well-adapted to low-nutrient conditions.     

Paradoxical gain‐of‐function mutant of the G‐protein‐coupled receptor PROKR2 promotes early puberty

The human genome encodes ~750 G‐protein‐coupled receptors (GPCRs), including prokineticin receptor 2 (PROKR2) involved in the regulation of sexual maturation. Previously reported pathogenic gain‐of‐function mutations of GPCR genes invariably encoded aberrant receptors with excessive signal transduction activity. Although in vitro assays demonstrated that an artificially created inactive mutant of PROKR2 exerted paradoxical gain‐of‐function effects when co‐transfected with wild‐type proteins, such a phenomenon has not been observed in vivo. Here, we report a heterozygous frameshift mutation of PROKR2 identified in a 3.5‐year‐old girl with central precocious puberty. The mutant mRNA escaped nonsense‐mediated decay and generated a GPCR lacking two transmembrane domains and the carboxyl‐terminal tail. The mutant protein had no in vitro signal transduction activity; however, cells co‐expressing the mutant and wild‐type PROKR2 exhibited markedly exaggerated ligand‐induced Ca²⁺ responses. The results indicate that certain inactive PROKR2 mutants can cause early puberty by enhancing the functional property of coexisting wild‐type proteins. Considering the structural similarity among GPCRs, this paradoxical gain‐of‐function mechanism may underlie various human disorders.     

Origin of the novel chemoreceptor Aesthetasc "Y" in Ostracoda: morphogenetical thresholds and evolutionary innovation

SUMMARY The morphology and developmental processes of the two types of ostracod chemoreceptors, the Aesthetasc "Y" and the "Grouped setae," were compared. Cypridoidea and Pontocypridoidea, belonging to Cypridocopina, have a large baseball bat-like seta as an autapomorphic character on the second antenna, whereas most ostracod taxa with plesiomorphic characters bear "Grouped setae" consisting of multiple setae on the second antenna. Their budding positions, morphology, and ontogenetic changes were compared, and our deduction is that the Aesthetasc "Y" originated from "Grouped setae-like" organ in the Paleozoic. The morphogenetic processes in the molting period of these chemoreceptors were compared at the cellular level. The observations suggest that the "Grouped setae" are formed by hypodermal cells and share sheath cells corresponding to those of the Aesthetasc "Y" as a common constraint in the molting process of setae. We conclude that modification of the morphogenetic processes in the molting period of the "Grouped setae" gave rise to the Aesthetasc "Y" as a novel organ in the evolutionary pathway of the Ostracoda.     

SOSUI-GramN: high performance prediction for sub-cellular localization of proteins in gram-negative bacteria

A predictive software system, SOSUI-GramN, was developed for assessing the subcellular localization of proteins in Gram-negative bacteria. The system does not require the sequence homology data of any known sequences; instead, it uses only physicochemical parameters of the N- and C-terminal signal sequences, and the total sequence. The precision of the prediction system for subcellular localization to extracellular, outer membrane, periplasm, inner membrane and cytoplasmic medium was 92.3%, 89.4%, 86.4%, 97.5% and 93.5%, respectively, with corresponding recall rates of 70.3%, 87.5%, 76.0%, 97.5% and 88.4%, respectively. The overall performance for precision and recall obtained using this method was 92.9% and 86.7%, respectively. The comparison of performance of SOSUI-GramN with that of other methods showed the performance of prediction for extracellular proteins, as well as inner and outer membrane proteins, was either superior or equivalent to that obtained with other systems. SOSUI-GramN particularly improved the accuracy for predictions of extracellular proteins which is an area of weakness common to the other methods.     

Describing size-related mortality and size distribution by nonparametric estimation and model selection using the Akaike Bayesian Information Criterion

When we calculate mortality along a gradient such as size, dividing into size classes and calculating rates for every class often involves a trade-off: fine class intervals produce fluctuating rates along the gradient, whereas broad ones may miss some trends within an interval. The same trade-off occurs when we want to illustrate size distribution by a histogram. This paper introduces nonparametric methods, published in a statistical journal, into forest ecology, in which the fine-class strategy is used in an extreme way: (1) a smoothly changing pattern is approximated by a fine step function, (2) the goodness-of-fit to the data and the smoothness along the gradient are formulated as a weighting sum within a Bayesian framework, (3) the Akaike Bayesian Information Criterion (ABIC) selects the weighting system that most appropriately balances the two demands, and (4) the values of the step function are optimized by the maximum likelihood method. The nonparametric estimates enable us to represent various patterns visually and, unlike parametric modeling, calculations do not demand the determination of a functional form. Mortality and size distribution analyses were conducted on 12-year forest tree monitoring data from a 4 ha permanent plot in an old-growth warm–temperate evergreen broad-leaved forest in Japan. From trees of 11 evergreen species with a diameter at breast height (DBH) greater than 5 cm, we found three types of trend with increasing DBH: decreasing, ladle-shaped and constant mortality. These patterns reflect variations in life history particular to each species.     

The effects of mutations in the carboxyl-terminal region on the catalytic activity of Escherichia coli signal peptidase I

Escherichia coli signal peptidase I (SPase I) is a membrane-bound serine endopeptidase that catalyses the cleavage of signal peptides from the pre-forms of membrane or secretory proteins. Our previous studies using chemical modification and site-directed mutagenesis suggested that Trp(300) and Arg(77), Arg(222), Arg(315) and Arg(318) are important for the proper and stable conformation of the active site of SPase I. Interestingly, many of these residues reside in the C-terminal region of the enzyme. As a continuation of these studies, we investigated in the present study the effects of mutations in the C-terminal region including amino acid residues at positions from 319 to 323 by deletions and site-directed mutagenesis. As a result, the deletion of the C-terminal His(323) was shown to scarcely affect the enzyme activity of SPase I, whereas the deletion of Gly(321)-His(323) or Ile(319)-His(323) as well as the point mutation of Ile(322) to alanine was shown to decrease significantly both the activity in vitro and in vivo without a big gross conformational change in the enzyme. These results suggest a significant contribution of Ile(322) to the construction and maintenance of the proper and critical local conformation backing up the active site of SPase I.     

Total HLA class I loss in a sarcomatoid renal carcinoma cell line caused by the coexistence of distinct mutations in the two encoding β<Subscript>2</Subscript>-microglobulin genes

In renal cell carcinoma (RCC), HLA class I downregulation has been found in about 40% of the lesions examined. Since only scanty information is available about the molecular basis of these defects, we have investigated the mechanism(s) underlying HLA class I antigen downregulation or loss in six RCC cell lines. Five of them express HLA class I antigens although at various levels; on the other hand, HLA class I antigens are not detectable on the remaining cell line, the RCC52 cell line, belonging to a sarcomatoid subtype, even following incubation with IFN-γ. β₂-microglobulin (β₂ m) was not detected in RCC52 cells. Surprisingly, RCC52 cells harbor two mutations in the β ₂ m genes in exon 1: a single G deletion (delG) in codon 6, which introduces a premature stop at codon 7, and a CT dinucleotide deletion (delCT), which leads to a premature stop at codon 55. Analysis of eight clonal sublines isolated from the RCC52 cell line showed that the two β ₂ m gene mutations are carried separately by RCC52 cell subpopulations. The delG/delCT double mutations were detected in two sublines with a fibroblast-like morphology, while the delCT mutation was detected in the remaining six sublines with an epithelial cell morphology. Furthermore, loss of heterozygosity (LOH) of the β ₂ m gene at STR D15S-209 was found only in the epithelioid subpopulation, indicating loss of one copy of chromosome 15. Immunostaining results of the tumor lesion from which the cell line RCC52 was originated were consistent with the phenotyping/molecular findings of the cultured cells. This is the first example of the coexistence of distinct β ₂ m defects in two different tumor subpopulations of a RCC, where loss of one copy of chromosome 15 occurs in one of the subpopulations with total HLA class I antigen loss. Chin-Hsuan Hsieh, Ya-Jan Hsu and Cheng-Keng Chuang contributed equally to the work.     

Identification of the mouse paternally expressed imprinted gene Zdbf2 on chromosome 1 and its imprinted human homolog ZDBF2 on chromosome 2

In mammals, both the maternal and paternal genomes are necessary for normal embryogenesis due to parent-specific epigenetic modification of the genome during gametogenesis, which leads to non-equivalent expression of imprinted genes from the maternal and paternal alleles. In this study, we identified a paternally expressed imprinted gene, Zdbf2, by microarray-based screening using parthenogenetic and normal embryos. Expression analyses showed that Zdbf2 was paternally expressed in various embryonic and adult tissues, except for the placenta and adult testis, which showed biallelic expression of the gene. We also identified a differentially methylated region (DMR) at 10 kb upstream of exon 1 of the Zdbf2 gene and this differential methylation was derived from the germline. Furthermore, we also identified that the human homolog (ZDBF2) of the mouse Zdbf2 gene showed paternal allele-specific expression in human lymphocytes but not in the human placenta. Thus, our findings defined mouse chromosome 1 and human chromosome 2 as the loci for imprinted genes.     

The patch mosaic of an old-growth warm-temperate forest: patch-level descriptions of 40-year gap-forming processes and community structures

Old-growth forests consist of various types of small patches that reflect their own gap-forming process, which includes changes in environmental conditions occurring over several decades. We reconstructed the gap-forming processes that had occurred during a 40-year period for eight representative patches of an old-growth evergreen broad-leaved forest in Japan, and examined the current community structure. The selected patches were based on (1) changes in canopy heights estimated from aerial photographs taken in four different years, (2) long-term ecological research (LTER) monitoring records, and (3) a recent field survey, so that they sufficiently covered characteristic gap-forming processes such as a new gap, an old gap and consistently closed canopy. Height and diameter at breast height (DBH) were measured on all living trees taller than 1.3 m. In their height distributions, currently almost closed patches that were open in 1966 show a rotated sigmoid, whereas their DBH distributions are an inverse J-shape. In contrast, patches that have been consistently under a closed canopy exhibit gentle inverse J-shapes for both distributions. For species composition, there are no clear contrasts associated with the past gap-forming processes except for the existence of fast-growing deciduous species in large currently open patches. Our results suggested that the variation in several decades of gap-forming processes played a central role in the high patch diversity and the complex patch mosaic of the forest. Diverse gap-forming processes created micro-environmental heterogeneity both vertically and horizontally, and contributed to the maintenance of the species-rich, warm-temperate old-growth forest.     

Liquid chromatographic determination of acetylcholine based on pre‐column alkaline cleavage reaction and post‐column tris(2,2′‐bipyridyl)ruthenium(III) chemiluminescence detection

A method for the determination of acetylcholine (ACh) has been developed using liquid chromatography with chemiluminescence detection. This method is based on the pre‐column alkaline cleavage of ACh to form trimethylamine (TMA) and the post‐column tris(2,2′‐bipyridyl)ruthenium(III) chemiluminescence detection of TMA. ACh was converted to TMA with high yield at 180°C in the presence of lithium hydroxide, and the produced TMA was separated on a cation‐exchange/reversed‐phase dual‐functional column using a mixture of 0.2 m potassium phosphate buffer (pH 5.9) and acetonitrile (20:1, v/v) as the mobile phase. The eluate was online mixed with acidic tris(2,2′‐bipyridyl)ruthenium(III) solution, and the generated chemiluminescence was detected. The detection limit (signal‐to‐noise ratio = 3) for ACh was 0.80 nmol/mL, which corresponded to 1.1 pmol TMA per injection volume of 5 µL. This is simple and robust method that does not need an expensive device and unstable enzymes, and was applied to the determination of ACh in pharmaceutical formulations. Copyright © 2009 John Wiley & Sons, Ltd.