Phylogenetic analyses of Zostera species based on rbcL and matK nucleotide sequences: implications for the origin and diversification of seagrasses in Japanese waters

Seagrasses are composed of four families belonging to angiosperms and they are thought to become adaptive to aquatic life independently. Zosteraceae is one such family and because of the relatively high species diversity around Japan and Korea coast areas, the family might have arisen therefrom. To elucidate the origin and evolution of Zosteraceae which consists of three genera, Phyllospadix, Zostera, and Heterozostera, 2.8 kb nucleotide sequences of rbcL and matK genes in the chloroplast genome were examined for various species, including cosmopolitan Z. marina and endemic Z. caulescens. The phylogenetic analysis reveals the following three features. First, based on the synonymous nucleotide substitution rate of the rice chloroplast genome, we estimated the divergence times between Zosteraceae and its closest relative, Potamogetonaceae, and between different genera, Zostera and Phyllospadix, as approximately 100 million years (myr) and 36 myr, respectively, suggesting that Zosteraceae emerged somewhere in the period from 36 myr ago to 100 myr ago. Second, two subgenera of Zostera, Zostera and Zosterella, exhibit their reciprocal monophyly and appear to have differentiated from each other approximately 33 myr ago. However, the third genus Heterozostera branched off only 5 myr ago from the stem lineage leading to Zosterella and this seems too recent in comparison with the ancient divergence of the two subgenera. Third, we estimated the most recent common ancestor of subgenus Zostera as 6 myr. In Z. marina four haplotypes were found in the sample and have diversified in the past 1.5 myr. One haplotype is shared by both sides of the Japan Archipelago and its closely related haplotypes occur also in eastern Pacific Ocean. Based on these phylogeographic analyses, we propose a provisional age related classification of Zosteraceae to argue the origin and evolution.     

Statistical models of the overdispersed molecular clock

The most commonly used statistical model to describe the rate constancy of molecular evolution (molecular clock) is a simple Poisson process in which the variance of the number of amino acid or nucleotide substitutions in a particular gene should be equal to the mean and henceforth the dispersion index, the ratio of the variance to the mean, should be equal to one. Recent sequence data, however, have shown that the substitutional process in molecular evolution is often considerably overdispersed and have called into question the generality of using a simple Poisson process. Several efforts have been made to develop more realistic models of molecular evolution. In this paper, I will show that the spatial (site-specific) variation in the rate of molecular evolution is an improbable cause of the overdispersion and then review various statistical models which take the temporal variation into account. Although these models do not immediately specify what the mechanisms of molecular evolution might be, they do make qualitatively different predictions and give some insight into their inference. One way to distinguish them is suggested. In addition, effects of selected substitutions that presumably occur after a major change in a molecule are quasi-quantitatively examined. It is most likely that the overdispersion of molecular clock is due either to a major molecular reconfiguration (fluctuating neutral space) led by a series of subliminal neutral changes or to selected substitutions fine-tuning a molecule after a major molecular change. Although the latter possibility, of course, violates the simplest neutrality assumption, it would not impair the neutral theory as a whole.     

Gastric inhibitory polypeptide as an endogenous factor promoting new bone formation after food ingestion

Calcium plays a fundamental role as second messenger in intracellular signaling and bone serves as the body's calcium reserve to tightly maintain blood calcium levels. Calcium in ingested meal is the main supply and inadequate calcium intake causes osteoporosis and bone fracture. Here, we describe a novel mechanism of how ingested calcium is deposited on bone. Meal ingestion elicits secretion of the gut hormone gastric inhibitory polypeptide (GIP) from endocrine K cells in the duodenum. Bone histomorphometrical analyses revealed that bone formation parameters in the mice lacking GIP receptor (GIPR(-/-)) were significantly lower than those of wild-type (GIPR(+/+)) mice, and that the number of osteoclasts, especially multinuclear osteoclasts, was significantly increased in GIPR(-/-) mice, indicating that GIPR(-/-) mice have high-turnover osteoporosis. In vitro examination showed the percentage of osteoblastic cells undergoing apoptosis to be significantly decreased in the presence of GIP. Because GIPR(-/-) mice exhibited an increased plasma calcium concentration after meal ingestion, GIP directly links calcium contained in meal to calcium deposition on bone.     

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.     

Dual-gradient high-performance liquid chromatography for identification of cytosolic high-mannose-type free glycans

It has been shown that free oligosaccharides derived from N-linked glycans accumulate in the cytosol of animal cells. Most of the glycans have only a single GlcNAc at their reducing termini (Gn1 glycans), whereas the original N-glycans retain N,N′-diacetylchitobiose at their reducing termini (Gn2 glycans). Under the conditions of high-performance liquid chromatography (HPLC) mapping established for pyridylamine (PA)-labeled Gn2 N-glycans, Gn1 glycans are not well retained on reversed-phase HPLC, making simultaneous analysis of Gn1 and Gn2 glycans problematic. We introduced a dual gradient (i.e., pH and butanol gradient) for the separation of Gn1 and Gn2 glycans in a single reversed-phase HPLC. Determination of elution time for various standard Gn2 high-mannose-type glycans, as well as Gn1 glycans found in the cytosol of animal cells, showed that elution of Gn1 and Gn2 glycans could be separated. Sufficient separation for most of the structural isomers could be achieved for Gn1 and Gn2 glycans. This HPLC, therefore, is a powerful method for identification of the structures of PA-labeled glycans, especially Gn1-type glycans, isolated from the cytosol of animal cells.     

Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically

 We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. They were then stained with 4′,6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P=0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients. Accepted: 29 April 1997