Seasonal growth and profile structure development of Elodea nuttallii (Planch.) St. John in pond Ojaga-Ike,Japan

Phenological and quantitative observations on Elodea nuttallii (Planch.) St. John, an exotic aquatic plant in Japan, were made in a shallow pond throughout 1979. Shoot elongation began in spring (late March) when the bottom water temperature became higher than about 10°C. Elongation ceased when the shoot apices reached the pond surface and vigorous branching then occurred. The community formed a dense canopy, with 40–65% of the shoot biomass in the topmost 30-cm water layer during the growing season. Maximum plant biomass (712 g dry wt. m^?2) was attained in late July, while the peak root biomass occurred around June, coincident with peak flowering. The anchoring roots and stems eventually died, and after September, the population existed as a floating mat of non-anchored leafy short shoots and decaying old branch stems. This mat sank suddenly to the bottom in December, when water temperatures dropped below approximately 10°C, and overwintered there. The ecological significance of the perennial growth habit and the formation of a floating mat is discussed in terms of the adventive spread of this plant, and an estimation of annual net production and P/B quotient is also made.     

Quartz crystal microbalance immunosensors for environmental monitoring

This paper presents discussion of quartz crystal microbalance (QCM) immunosensors for environmental monitoring. Factors limiting the practical application of antibodies to analytical problems are also presented. Among several candidates for the QCM immunosensor device, selected QCM devices and oscillating circuits were tested thoroughly and developed to obtain highly stable and sensitive frequency signals. The biointerface of QCM immunosensor was designed and controlled to immobilize antibody on the QCM surface, to reduce non-specific binding and to suppress denaturation of immobilizing antibody by self-assembled monolayer technique and artificial phospholipid (2-methacryloyloxyethyl phosphorylcholine (MPC)) polymer. MPC polymer as a antibody-stabilizing reagent was added to reduce non-specific binding of the antigen solution and stabilize the immunologic activity of the antibody-immobilized QCM. In addition, it provides examples for detection and quantitation of environmental samples using QCM immunosensors. The analytical results for fly ash extracted samples of dioxins using the QCM immunosensor indicated a good relationship with GC/MS methods. The integrating protocols of the competitive immunoassay and signal-enhancing step are for detecting low molecular analytes with extremely low detection limits using an QCM immunosensor. Furthermore, its detect limitation was extended from 0.1 to 0.01 ng/ml by the signal-enhancing step when the anti-bisphenol-A antibody conjugated MPC polymeric nanoparticles was used. The QCM immunosensor method has demonstrated its effectiveness as an alternative screening method for environmental monitoring because these results were compared with results obtained through environmental monitoring methods such as ELISA and GC/MS.     

Positive Charges of Translocating Polypeptide Chain Retrieve an Upstream Marginal Hydrophobic Segment from the Endoplasmic Reticulum Lumen to the Translocon

Positively charged amino acid residues are well recognized topology determinants of membrane proteins. They contribute to the stop-translocation of a polypeptide translocating through the translocon and to determine the orientation of signal sequences penetrating the membrane. Here we analyzed the function of these positively charged residues during stop-translocation in vitro. Surprisingly, the positive charges facilitated membrane spanning of a marginally hydrophobic segment, even when separated from the hydrophobic segment by 70 residues. In this case, the hydrophobic segment was exposed to the lumen, and then the downstream positive charges triggered the segment to slide back into the membrane. The marginally hydrophobic segment spanned the membrane, but maintained access to the water environment. The positive charges not only fix the hydrophobic segment in the membrane at its flanking position, but also have a much more dynamic action than previously realized.     

Cloning and nucleotide sequence analysis of the Streptococcus sobrinus gtfU gene that produces a highly branched water-soluble glucan

Streptococcus sobrinus has four gtf genes, gtfI, gtfS, gtfT, and gtfU, on the chromosome. These genes correspond respectively to the enzymes GTF-I, GTF-S1, GTF-S2, and GTF-S3. An Escherichia coli MD66 clone that contained the S. sobrinus gtfU gene was characterized. Immunological properties showed that the protein produced by the E. coli MD66 clone was similar to S. sobrinus GTF-S1. Biological properties and a linkage analysis of the glucans by 13C NMR spectrometry revealed that the protein produced by the E. coli MD66 clone was GTF-S1.     

Solvolysis of charged active esters of carboxylic acids with cyclo (l- or d-Glu-l-His)

Cyclic dipeptide cyclo(l- or d-Glu-l-His) carrying an anionic site and a nucleophilic site has been synthesized and used as a catalyst for the solvolysis of cationic esters in aqueous alcohols. In the solvolysis of 3-acyloxy-N-trimethylanilinium iodide (S⁺_n, n = 2 and 10) and Cl^?H₃N⁺(CH₂)₁₁COOPh(NO₂), no efficient nucleophilic catalysis was observed. On the other hand, in the solvolysis of Gly-OPh(NO₂)·HCl, Val-OPh(NO₂)·HCl and Leu-OPh(NO₂)·HCl a very efficient general base-type catalysis by cyclo(l-Glu-l-His) was observed. In particular, with the latter two substrates the catalysis by cyclo(l-Glul-His) was more efficient than that by imidazole, although the catalysis was not enantiomer-selective. The diastereomeric cyclic dipeptide cyclo(d-Glu-l-His) was almost inactive under the same conditions. Confomation of cyclo(l- or d-Glu-l-His) in aqueous solution was investigated and the structure/catalysis relationship is discussed.     

Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding

The sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase beta was investigated by individually changing each amino acid in this region by site-directed mutagenesis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their Km's for primer DNA were about 10-fold higher than that of the wild type, while Km's for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase beta with primer.     

Preparation of virosomes coated with the vesicular stomatitis virus glycoprotein as efficient gene transfer vehicles for animal cells

The vesicular stomatitis virus (VSV) glycoprotein (G) was used to prepare virosomes as a model vehicle of gene transfer to animal cells, for which viral envelope functions (receptor recognition and binding and the pH-dependent membrane-fusion) were expected to work. Plasmid DNA (pEGFP-N1; Clontech) was first encapsulated into liposomes by a method of repeated freezing and thawing of the mixture of DNA and lipids (phosphatidylcholine, phosphatidylserine and cholesterol mixed at a molar ratio of 5: 1: 4). Then, particle size of the liposomes was stepwise reduced to 200 nm or less in diameter by successive filtrations through a series of plastic filters of various pore sizes (10 micro m, 2 micro m, 0.65 micro m, and then 0.45 micro m). Assembly of the VSV G protein-coated liposomes (VSV G-virosomes) was performed by mixing the DNA-encapsulated liposome suspensions with the purified VSV G proteins at pH 5.5, followed by ultracentrifugation in a discontinuous sucrose gradient. The highest gene-transducing activity was detected in a single band formed between 20% and 45% sucrose layers. Negatively stained electron microscopic images showed that the band contained spherical particles of various sizes, ranging from 40 to 140 nm in diameter, that were covered with viral spike projections. The VSV G-virosomes displayed a roughly similar level of gene-transducing activity to that mediated by cationic liposomes (e.g., Lipofectamine), which was blocked either by pretreatment with anti-VSV G antiserum or by addition of 20 m M NH(4) Cl to transfected cultures. From these results, we assume that the virosome-mediated gene-transduction was first achieved by using the whole functions of VSV G protein, and can also be used for further studies of the protein.