Correlation of the Period Length of Circadian Rhythms with the Length of Immaturity in Paramecium bursaria

The circadian photoaccumulation rhythm of thirty strains of Paramecium bursaria collected at different places in Japan and China were measured with a microcomputer assisted data collection apparatus. Although most strains showed a period of 23-26 hours in LL, we found two strains of conspicuously different periods; a short period strain (UK1, 21.8 hr) and a long period strain (T316, 28.7 hr). F1 progeny from a cross between the short and the long period strains showed an intermediate period of about 24.7 hours (range 22.5-25.8 hr). The character was not distributed in a Mendelian ratio among the F1 progeny. We isolated a mutant (E2) with short period (21.8 hr) from the stock strain Kz1 by treatment with nitrosoguanidine (MNNG). The progeny of crosses between E2 and UK1, and between E2 and T316 exhibited the short period and the normal period phenotype respectively. Moreover, the progeny from a cross between E2 and a wild type strain (Sj2w) became sexually mature about 25 fissions after conjugation. This length of immaturity is much shorter than that of the progeny from wild type strains (about 50 fissions). This early maturation character was inherited to progeny in a Mendelian ratio. Homozygotes for the early maturation allele (EM2) exhibited mating ability about 15 fissions after conjugation. These data suggest that there is a correlation between the period length of the circadian rhythm and the length of immaturity after conjugation in Paramecium bursaria.     

Mutations Affecting Acetylcholine Levels in the Nematode Caenorhabditis elegans

Gene cha-1.unc-17 of the nematode Caenorhabditis elegans is a complex gene, consisting of at least two complementation groups. One part (cha-1 region) of the gene encodes the enzyme choline acetyltransferase (ChAT), but the function of the other part (unc-17 region) is still unclear. We measured the ChAT activity and ACh levels of the cha-1 and unc-17 complex gene mutants. We show here that alterations in ACh levels, rather than the ChAT activity, reflect abnormal phenotypes accompanying cha-1.unc-17 mutations, that is, the decreased ACh levels in cha-1 mutations and abnormal accumulation in unc-17 mutations. Our results suggest that the unc-17 region may encode functions necessary for storage and/or release of ACh at the presynaptic level.     

The secondary structure of the colicin E3 immunity protein as studied by 1H-1H and 1H-15N two-dimensional NMR spectroscopy.

By performing 1H-1H and 1H-15N two-dimensional (2D) nuclear magnetic resonance (NMR) experiments, the complete sequence-specific resonance assignment was determined for the colicin E3 immunity protein (84 residues; ImmE3), which binds to colicin E3 and inhibits its RNase activity. First, the fingerprint region of the spectrum was analyzed by homonuclear 1H-1H HOHAHA and NOESY methods. For the identification of overlapping resonances, heteronuclear 1H-15N (HMQC-HOHAHA, HMQC-NOESY) experiments were performed, so that the complete 1H and 15N resonance assignments were provided. Then the secondary structure of ImmE3 was determined by examination of characteristic patterns of sequential backbone proton NOEs in combination with measurement of exchange rates of amide protons and 3JHN alpha coupling constants. From these results, it was concluded that ImmE3 contains a four-stranded antiparallel beta-sheet (residues 2-10, 19-22, 47-49, and 71-79) and a short alpha-helix (residues 31-36).     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Isolation of human retinal genes: recoverin cDNA and gene.

A human retina cDNA library enriched for retina-specific clones was prepared by subtraction with a non-retina population of cDNA in combination with polymerase chain reaction (PCR) amplifications. A highly retina-specific cDNA clone (1190 bp) was obtained through this library encoding a 200 amino acid protein with three calcium binding sites and 87% homology to the bovine photoreceptor protein, recoverin, which has been shown to mediate the recovery of the dark current after photoactivation, and 58% homology to the calcium-binding chick cone protein, visinin. Analysis of the gene indicated a 9-10 kb single-copy gene with at least three exons and two introns. The three exons contained the entire coding sequence, and all of the calcium-binding EF-hand regions were in putative exon 1. The recoverin gene was mapped to human chromosome 17 by hybridization to a panel of human-rodent hybrid DNAs.     

Cytoprotective effect of TRK-100, a prostacyclin analogue, against chemical injuries in cultured human vascular endothelial cells

We studied the cytoprotective effect of TRK-100, a chemically stable analogue of prostacyclin (PGI2), in the cultured human endothelial cells from umbilical vein. TRK-100 (10 and 100 nM) stimulated significantly proliferation of endothelial cells but did not affect PGI2 production in endothelial cells. Exposure of cultured endothelial cells to homocysteine (2.5 mM) or glucose (50 mM) caused concentration-dependent cytotoxicity, as evidenced by a decrease in number of viable cells. When endothelial cells were treated with TRK-100 simultaneously or prior to, but not after, exposure to injury substances, decreases in viable cell were significantly suppressed. The protective effect of TRK-100 against homocysteine-induced cytotoxicity also appeared in endothelial cells treated with acetylsalicylic acid, suggesting that endogenous PGI2 did not involve in the protective effect of TRK-100.     

A peculiar relationship between genetic diversity and adaptability in invasive exotic species: bluegill sunfish as a model species

A peculiar relationship exists between population genetics and invasion biology. Introduced populations often suffer a depletion of genetic variation, but they can persist and adapt to new environments. Here, we show that this relationship is observed in bluegill sunfish (Lepomis macrochirus), an invasive exotic fish in Japan. Genetic analysis using selectively neutral genetic markers reconfirmed that the bluegill introduced into Japan from the United States in 1960 had a single origin with only 15 founders. The analysis also shows that in the process of range expansion, the introduced bluegills experienced severe depletion of genetic diversity due to the founder effect and/or genetic drift. Despite such a decline in genetic diversity, the bluegill populations exhibited a divergent feeding morphology in response to the colonized environments. Such a morphological divergence can facilitate prey exploitation, thereby causing a greater negative impact on native prey resources. Further, in a trophically polymorphic bluegill population in Lake Biwa, physiological characteristics and genetic structures of the intestinal bacterial communities were associated with the difference in diet among the trophic morphs in the host bluegill population. This empirical evidence suggests that despite the severe decline in genetic diversity, the introduced bluegill populations rapidly adapted to the new environment and formed diverse functional relationships with the native bacterial community. Thus, these findings suggest that genetic variation at selectively neutral markers does not always predict adaptability and invasiveness in introduced populations.     

Synthesis and absolute configuration of hormone alpha1

An important biological event in phytopathogens of the genus Phytophthora is sexual reproduction, which is conducted by two mating types, A1 and A2. A factor known as hormone alpha1 is secreted by the A1 mating type and induces the formation of sexual spores (oospores) in the A2 mating type. Here we describe the asymmetric synthesis and assignment of the absolute configuration of hormone alpha1 by oospore-inducing assays of the synthesized isomers.     

Computationally assisted screening and design of cell-interactive peptides by a cell-based assay using peptide arrays and a fuzzy neural network algorithm

We developed a method of effective peptide screening that combines experiments and computational analysis. The method is based on the concept that screening efficiency can be enhanced from even limited data by use of a model derived from computational analysis that serves as a guide to screening and combining the model with subsequent repeated experiments. Here we focus on cell-adhesion peptides as a model application of this peptide-screening strategy. Cell-adhesion peptides were screened by use of a cell-based assay of a peptide array. Starting with the screening data obtained from a limited, random 5-mer library (643 sequences), a rule regarding structural characteristics of cell-adhesion peptides was extracted by fuzzy neural network (FNN) analysis. According to this rule, peptides with unfavored residues in certain positions that led to inefficient binding were eliminated from the random sequences. In the restricted, second random library (273 sequences), the yield of cell-adhesion peptides having an adhesion rate more than 1.5-fold to that of the basal array support was significantly high (31%) compared with the unrestricted random library (20%). In the restricted third library (50 sequences), the yield of cell-adhesion peptides increased to 84%. We conclude that a repeated cycle of experiments screening limited numbers of peptides can be assisted by the rule-extracting feature of FNN.     

Allochthonous prey subsidies provide an asymmetric growth benefit to invasive bluegills over native cyprinids under the competitive conditions in a pond

Asymmetry in the competition abilities between invasive and native consumers can potentially influence the colonization success by invasive species. We tested whether a subsidy of allochthonous prey enhanced an asymmetric competition between invasive bluegill (Lepomis macrochirus) and two native cyprinid fish, that is, stone moroko (Pseudorasbora parva) and tamoroko (Gnathopogon elongatus elongatus). A field experiment was conducted using enclosures wherein the strength of interspecific competition and the presence/absence of allochthonous prey were manipulated. The experiment revealed that allochthonous prey alleviated the limitation of fish growths caused by a severe competition for aquatic prey resources. However, the importance of allochthonous prey differed considerably between invasive bluegill and the two native cyprinids. Individual bluegills grew faster when the allochthonous prey was supplied, whereas no difference in growth was observed in the two cyprinids whether or not allochthonous prey was supplied. Interestingly, the importance of allochthonous prey on the total amount of bluegill growth varied depending on the numerical abundance of native cyprinid competitors, and this importance increased when the native cyprinids were abundant. These findings indicated that allochthonous prey provides an asymmetric growth benefit to invasive bluegills over the two native cyprinids by alleviating asymmetrically the competition strength in a Japanese pond, especially under the conditions of severe interspecific resource competition and a limitation in the utilization of in situ prey resources.