Interisland Evolution of <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom Phospholipase A<Subscript>2</Subscript> Isozymes

Abstract Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A₂ (PLA₂), named PL-Y, was isolated from Okinawa T. flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA₂ from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA₂, named PLA-B′, which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp⁴⁹]PLA₂ isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima), and PLA-B′ (Amami-Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the β1-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different environments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA₂, named PLA2, ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA2 gene is a prototype among T. flavoviridis venom PLA₂ isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA₂ isozymes are clearly separated into three groups, PLA2 type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Basic [Asp⁴⁹]PLA₂-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys⁴⁹]PLA₂ type.     

Analysis of DNA-ploidy using laser scanning cytometer in brain tumors and its clinical application

There have been few reports on investigation of the cell kinetics of brain tumors using a laser scanning cytometer (LSC). We compared DNA analysis using a flow cytometer (FCM) with that by LSC using established cell lines of brain tumors, and we demonstrated the similarity of both analytical results. The DNA-index (DI) and proliferating index (PI) of various brain tumors were determined, and the results showed that these indices reflected the malignancy of the tumors. The DI values were higher for astrocytoma grade IV than for astrocytoma grade II, frequently showing an aneuploid pattern. The determination of DNA-ploidy using LSC, together with cryopathological diagnosis, was considered to be useful for deciding the area to be resected for glioma perioperatively and to be worth applying clinically.     

Age-dependent in situ hepatic and gill CYP1A activity in the see-through medaka (Oryzias latipes)

We used a recently introduced strain of medaka, the see-through medaka, whose internal organs can be seen through the skin, to develop an in situ toxicity assay of ethoxyresorufin-O-deethylase (EROD) activity that detected fluorescence from resorufin, a metabolite of ethoxyresorufin and thus an indicator of CYP1A activity. EROD activity in the liver and gills of 2-week post-hatch see-through medaka exposed simultaneously to various concentrations of 3-methylcholanthrene and 200 microg/L ethoxyresorufin for 24 h was proportional to the 3-methylcholanthrene dose. Activities in the liver and gills peaked at 40 microg/L of 3-methylcholanthrene and then decreased at higher doses, possibly because of 3-methylcholanthrene toxicity. At 1-week post-hatch stage, however, constant high EROD activity was observed in controls and at all 3-methylcholanthrene doses. Four-week post-hatch see-through medaka exhibited less EROD activity than 2-week post-hatch see-through medaka, and activity in the liver peaked at 100 microg/L of 3-methylcholanthrene. Adult see-through medaka were not suitable for fluorescence detection owing to their thick skin, muscle and/or tissue. In tests of oxidative activity response to ethoxyresorufin, 1-day and 1-week post-hatch see-through medaka exhibited high intrinsic EROD activity in the liver, gills, and other organs in the absence of 3-methylcholanthrene. This intrinsic activity declined with growth and explained the high constant EROD activity at 1-week post-hatch stage.     

Identification of the mRNA coding for the ACTH-beta-lipotropin precursor in a human ectopic ACTH-producing tumor

The mRNA coding for the ACTH-β-lipotropin precursor from a human ectopic ACTH-producing thymic carcinoid was identified by blot hybridization analysis with the bovine cDNA as a probe. The mRNA from the tumor had the same length (approximately 1,100 nucleotides) as that from the human pituitary. An additional hybridization-positive RNA species of a larger size was found in the tumor. Cell-free translation of the mRNA from the tumor as well as from the pituitary yielded a product with an apparent molecular weight of 38,000 that was reactive both with antibody to ACTH and with antibody to β-endorphin.     

The Comparative Effectiveness of Rodents and Dung Beetles as Local Seed Dispersers in Mediterranean Oak Forests

The process of seed dispersal of many animal-dispersed plants is frequently mediated by a small set of biotic agents. However, the contribution that each of these dispersers makes to the overall recruitment may differ largely, with important ecological and management implications for the population viability and dynamics of the species implied in these interactions. In this paper, we compared the relative contribution of two local guilds of scatter-hoarding animals with contrasting metabolic requirements and foraging behaviours (rodents and dung beetles) to the overall recruitment of two Quercus species co-occurring in the forests of southern Spain. For this purpose, we considered not only the quantity of dispersed seeds but also the quality of the seed dispersal process. The suitability for recruitment of the microhabitats where the seeds were deposited was evaluated in a multi-stage demographic approach. The highest rates of seed handling and predation occurred in those microhabitats located under shrubs, mostly due to the foraging activity of rodents. However, the probability of a seed being successfully cached was higher in microhabitats located beneath a tree canopy as a result of the feeding behaviour of beetles. Rodents and beetles showed remarkable differences in their effectiveness as local acorn dispersers. Quantitatively, rodents were much more important than beetles because they dispersed the vast majority of acorns. However, they were qualitatively less effective because they consumed a high proportion of them (over 95%), and seeds were mostly dispersed under shrubs, a less suitable microhabitat for short-term recruitment of the two oak species. Our findings demonstrate that certain species of dung beetles (such as Thorectes lusitanicus), despite being quantitatively less important than rodents, can act as effective local seed dispersers of Mediterranean oak species. Changes in the abundance of beetle populations could thus have profound implications for oak recruitment and community dynamics.     

Segmental distribution of genes harboring a CpG island-like region on rice chromosomes

In plant genomes, there exist discrete regions rich in CpG dinucleotides, namely CpG clusters. In rice, most of these CpG clusters are associated with genes. Rice genes are grouped into one of the five classes according to the position of an associated CpG cluster. Among them, class 1 genes, which harbor a CpG cluster at the 5′-terminus, share similarities with human genes having CpG islands. In the present study, by analyzing plant genome sequence data, primarily from rice, we investigated the chromosomal distribution of genes of each class, mainly class 1 genes. Class 1 genes were not uniformly distributed across the rice genome, but were clustered into discrete chromosomal segments. EST-based analysis of the distribution of expressed genes indicates that this segmental distribution of class 1 genes caused a preferential distribution of expressed genes within class 1 gene-rich segments. We then compared the methylation status of genes of each class to examine the possibility that differential DNA methylation, if any, is relevant to the observed differential expression level of genes inside and outside the class 1 segments. The difference in the methylation level between these genes was revealed to be fairly small, which does not support the above-mentioned possibility. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

RiceGAAS: an automated annotation system and database for rice genome sequence

An extensive effort of the International Rice Genome Sequencing Project (IRGSP) has resulted in rapid accumulation of genome sequence, and >137 Mb has already been made available to the public domain as of August 2001. This requires a high-throughput annotation scheme to extract biologically useful and timely information from the sequence data on a regular basis. A new automated annotation system and database called Rice Genome Automated Annotation System (RiceGAAS) has been developed to execute a reliable and up-to-date analysis of the genome sequence as well as to store and retrieve the results of annotation. The system has the following functional features: (i) collection of rice genome sequences from GenBank; (ii) execution of gene prediction and homology search programs; (iii) integration of results from various analyses and automatic interpretation of coding regions; (iv) re-execution of analysis, integration and automatic interpretation with the latest entries in reference databases; (v) integrated visualization of the stored data using web-based graphical view. RiceGAAS also has a data submission mechanism that allows public users to perform fully automated annotation of their own sequences. The system can be accessed at http://RiceGAAS.dna.affrc.go.jp/.     

New molecular tumor markers for endometrial cancer

Although the International Federation of Gynecology and Obstetrics officially changed the classification system of endometrial cancer from a clinically staged to a surgically staged disease in 1988, optimal management of patients with endometrial cancer is still controversial. Gynecologists happen to experience that patients with tumors that are identical in grade and stage often have significantly different clinical outcomes or responses to therapy. In order to identify an objective biological factor correlating with tumor aggressiveness, many tumor markers have been investigated. So far, CA125 is one of the most reliable tumor marker for adenocarcinoma of the uterus and frequently used in a clinical setting. Recently, with the advent of molecular biological techniques, many genes and regions of the genome related to endometrial cancer have been identified. We undertook a genome-wide screening to detect genetic changes by comparative genomic hybridization (CGH) in primary endometrioid cancers, since CGH analysis provides comprehensive information concerning relative chromosomal losses and gains in tumors by a single hybridization. In this paper, the usefulness of serum tumor markers and the new promising molecular tumor markers for endometrial cancer are discussed.