A Functional Aqp1 Gene Product Localizes on The Contractile Vacuole Complex in Paramecium multimicronucleatum

In a ciliate Paramecium, the presence of water channels on the membrane of contractile vacuole has long been predicted by both morphological and physiological data, however, to date either the biochemical or the molecular biological data have not been provided. In the present study, to examine the presence of aquaporin in Paramecium, we carried out RT-PCR with degenerated primers designed based on the ParameciumDB, and an aquaporin cDNA (aquaporin 1, aqp1) with a full-length ORF encoding 251 amino acids was obtained from Paramecium multimicronucleatum by using RACE. The deduced amino acid sequence of AQP1 had NPA-NPG motifs, and the prediction of protein secondary structure by CNR5000 and hydropathy plot showed the presence of six putative transmembrane domains and five connecting loops. Phylogenetic analysis results showed that the amino acid sequence of AQP1 was close to that of the Super-aquaporin group. The AQP1-GFP fusion protein clearly demonstrated the subcellular localization of AQP1 on the contractile vacuole complex, except for the decorated spongiome membrane. The functional analyses of aqp1 were done by RNA interference-based gene silencing, using an established feeding method. The aqp1 was found to be crucial for the total fluid output of the cell, the function of contractile vacuole membranes.     

Interaction of Benzaldehyde to the Membrane Protein of Escherichia coli

Adenosine-, guanosine-, cytidine- and uridine-5'-di(tri)-phosphate-3'-diphosphates were enzymatically prepared by the use of Streptomyces adephospholyticus ATP nucleotide pyrophospho-kinase (E.C. 2.7.6.4). Their regulatory effects were then investigated on translation of rat liver, rabbit globin and silkworm pupae chorion mRNAs by the wheat germ lysate system in vitro. They were found to exert base-specific and mRNA species-specific stimulatory or inhibitory effects, respectively. In particular, cytidine 5'-diphosphate-3'-diphosphate stimulated chorion mRNA translation 3-fold. The significance and limitations of the findings were discussed in relation to natural occurrence and possible regulatory functions of the nucleotides in eucaryotes.     

Reversible Inhibition of the Photosynthetic Water-Splitting Enzyme System by SO2-Fumigation Assayed by Chlorophyll Fluorescence and EPR Signal in Vivo

The effect of SO₂ fumigation (2 ppm, v/v) on photosynthesisin spinach leaves in vivo was investigated by measuring Chla fluorescence (OIDP transient) and the electron paramagneticresonance (EPR) signal I. SO₂ fumigation raised the I levelto yield the ID dip and suppressed the DP transient before anyvisible damage occurred in the leaf. In SO₂-fumigated leaves,the time course of EPR signal I indicates that reduction ofP700 by white light illumination was inhibited but dark reductionof P700 was not significantly affected. Photosynthetic O₂ evolutionwas also inhibited by SO₂ fumigation. All of these effects werereversible after removal of SO₂. The variable part of the fluorescencein the presence of DCMU was only slightly affected and decreasedas the fumigation time increased. We concluded that SO₂ fumigationreversibly inhibits the photosynthetic water-splitting enzymesystem and it injures the reaction center of PS II in vivo whenthe fumigation time is prolonged. We discussed the role of possible toxicants derived from SO₂within the leaf on the basis of the SO₂ action on Chl a fluorescence. (Received December 8, 1983; Accepted May 7, 1984)     

Replication Patterns of Repetitive DNA Sequences on the W Chromosome Are Altered during Development of the Chick Embryo

A novel method was developed to study developmental changes in the replication pattern of repetitive DNA sequences on the W chromosome (W-DNA) of the female chick embryo. The amount of total nuclear DNA and W-DNA as well as 5-bromodeoxyuridine (BrdU) incorporation was successively measured on the same cells using multiparametric microfluorometry [1]. With this method we first examined the possibility of changes in replication patterns of W-DNA during development. Measurements were conducted on various heterogeneous cell populations obtained from whole embryo on Day 0.4 and Day 1, and from pectoral muscle, neural tube, liver, and oogonium on Day 9. Parameters of W-DNA replication, duration, and timing were found to vary according to the stage of embryonic development. Developmental features of these changes were further studied on specific cell types during their critical developmental processes. In scutate scale dermis, the W-DNA replication duration showed a characteristic lengthening from around 0.45C during Day 5 through Day 7.4 to 0.9C during Day 7.7 through Day 7.9 and shortening to 0.37C during Day 8.1 through Day 12. Transient lengthening in W-DNA replication duration was also observed in erythrocytes; 0.65C → 1.0C → 0.6C during Day 0.9 through Day 2.17. Timing also shifted earlier in accord with changes in the duration. Replication rate of whole genome DNA was monitored by measuring BrdU incorporation on respective cells and found, to a large extent, comparable to that of W-DNA. The data suggest that a link might be operative between replication patterns of genes and the developmental program.     

Dextransucrase as an Enzyme Associating with Alkaline Earth Metal Ions

Dextransucrase (EC. 2.4.1.5) of Leuconostoc mesenteroides was purified from the culture filtrate by precipitation with solid ammonium sulfate in the presence of egg white albumin followed by successively treating with columns of DEAE-cellulose and Bio Gel P-150.

The purified enzyme lost the activity upon dialysis against EDTA, and was reactivated by the addition of alkaline arth metal ions. The best reactivation was brought about by calcium ion. The enzyme inactivated by EDTA was unstable and readily denatured irreversibly. Several other properties of the purified enzyme were also investigated and discussed.