First finding of urease-positive thermophilic strains of Campylobacter in river water in the Far East, namely, in Japan and their phenotypic and genotypic characterization

Two strains of urease-positive thermophilic Campylobacter (UPTC), CF89–12 and CF89–14, which were identified as UPTC by biochemical characterization, were found for the first time in river water in the Far East, namely, in Japan. The biochemical characteristics were identical to those of strains described previously by Bolton and colleagues. Furthermore, these two strains were positive for arylsulphatase. Consequently, it was demonstrated that UPTC may possibly be differentiated phenotypically from Campylobacter lari by the arylsulphatase test, as well as urease and nalidixic acid tests. Analysis by pulsed-field gel electrophoresis (PFGE) after digestion with Apa I, Sal I and Sma I, which were found to produce distributions of DNA fragments to be suitable for analysis of the genomic DNA from the thermophilic Campylobacter , respectively, demonstrated that these three restriction enzymes produced distributions of a relatively limited number of genomic DNA fragments and also demonstrated that the PFGE profiles obtained with the three restriction enzymes were indistinguishable between the two strains, respectively. The PFGE analysis and conventional fixed-field agarose gel electrophoresis suggested that the both genomes were approximately 1862 kb in length. Even though the two isolates of UPTC were isolated from water in different rivers in Japan, the results suggested that a single strain. as opposed to two distinct strains, was isolated. PFGE profiles after digestion with Sal I and Sma I, respectively, were also demonstrated to be distinctly different among strains isolated in Japan and previously in Europe. This is the first example of the isolation of UPTC from natural sources in countries other than those in Europe.     

SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

Mitochondrial DNA sequences of various species of the genus Equus with special reference to the phylogenetic relationship between Przewalskii's wild horse and domestic horse

The noncoding region between tRNA^Pro and the large conserved sequence block is the most variable region in the mammalian mitochondrial DNA D-loop region. This variable region (ca. 270 bp) of four species of Equus, including Mongolian and Japanese native domestic horses as well as Przewalskii's (or Mongolian) wild horse, were sequenced. These data were compared with our recently published Thoroughbred horse mitochondrial DNA sequences. The evolutionary rate of this region among the four species of Equus was estimated to be 2–4 × 10^–8 per site per year. Phylogenetic trees of Equus species demonstrate that Przewalskii's wild horse is within the genetic variation among the domestic horse. This suggests that the chromosome number change (probably increase) of the Przewalskii's wild horse occurred rather recently.Correspondence to: N. Ishida     

Enhancement of aromatic amino acid-nucleic acid base stacking interaction by metal coordination to base: fluorescence study on a tryptophan-Pt(II)-guanine ternary complex

In order to investigate the effect of the Pt(II) ion on the stacking interaction between tryptophan and a guanine base, the quenching of Trp fluorescence was monitored for some systems in the absence and presence of the metal ion, and the association constants were obtained by the analysis of Eadie-Hofstee plots. All spectral data suggested that the stacking interaction is enhanced by the Pt(II) coordination to the guanine N7 atom. The result indicates the importance of the metal ion as a bookmark in the specific recognition of a nucleic acid base by an aromatic amino acid residue.     

Localization of the antigen for the monoclonal antibody ②9F11-B-E4 interspecific cross-reaction in the freshwater triclads

The localization of the antigen for monoclonal antibody 9F11-B-E₄ was clarified by immuno-electron microscopy. The antigens were localized on the mitochondria and Golgi bodies in the male germ cells and on the secretory granules of various glands cells in the penis bulb and subepidermal parenchymal tissue of Phagocata vivida. The results of the interspecific cross-reaction tests with seven other freshwater triclads showed that these secretory granules are species-specific. A positive interspecific reaction was showed with Dugesia (family Dugesiidae), but not with Polycelis within the same family Planariidae which suggests the position of Phagocata within the Planariidae needs to be reassesed.     

Effects of some factors on protoplast formation of a microalga,Prototheca zopfii

The effects of experimental factors on protoplast formation of Prototheca zopfii Kru¨ger in 0.85 m NaCl using Macerozyme R-200 were studied based on a fractional factorial experimental design. The rate of protoplast formation was mainly affected by the incubation temperature and the age of algal cells. The optimal condition for the maximum protoplast yield was determined based on a response surface model. These were: mid-logarithmic phase cells and Macerozyme concentration of 4% at a temperature of 35°C.     

Stimulative and inhibitory effects of bacteria on the growth of microalgae

Several examples of stimulative and inhibitoryeffects of bacteria on microalgal growth areintroduced, and the importance of bacteria in algalmass culture is investigated. Diatoms are often usedas live food for planktonic larvae of sea urchin andbivalves. Monodispersed Chaetoceros ceratosporum hasbeen cultivated by using clean, high nutrient content,deep seawater (DSW). However, the growth rate and cellyield of diatoms fluctuated, to relatively largeextent, with the season that DSW was collected. Whensome bacterial strains isolated from DSW were added tothe culture, diatom growth was often stimulated and arelatively constant cell yield was obtained. Anotherdiatom species, C. gracilis, was also stimulated byadding some bacterial strains to cultures. Thepositive effect of bacteria on diatoms was observednot only for planktonic species, but also on attachedspecies. A benthic diatom, Nitzschia sp., wasstimulated by a bacterial film of Alcaligenes on thesurface of the substratum. On the other hand, a strainof Flavobacterium sp. isolated from natural seawaterduring the decline period of an algal bloom had a strongalgicidal effect on the red tide plankton,Gymnodinium mikimotoi. Recent reports demonstratethat many bacterial strains have significantalgicidal effects on many species of red tideplankton. These results indicate that bacterialeffects should be taken into account to obtain stablemass culture of food microalgae.     

X-ray diffraction studies of enkephalins. Crystal structure of [(4''-bromo) Phe4,Leu5]enkephalin.

In order to investigate the structure-activity relationship of [Leu5]- and [Met5]enkephalins, [(4'-bromo)Phe4, Leu5]-, [(4'-bromo)Phe4, Met5]- and [Met5] enkephalins were synthesized and crystallized. The crystal structure of [(4'-bromo) Phe4, Leu5]- enkephalin was determined by X-ray diffraction method using the heavy atom method and refined to R = 0.092 by the least-squares method. The molecule in this crystal took essentially the same type I' beta-turn conformation found in [Leu5]enkephalin [Smith & Griffin (1978) Science 199, 1214-1216). On the other hand, the preliminary three-dimensional Patterson analyses showed that the most probable conformations of [(4'-bromo)Phe4,Met5]- and [Met5]enkephalins are both the dimeric extended forms. Based on these insights, the biologically active conformation of enkephalin was discussed in relation to the mu- and delta-receptors.     

An X-ray study on the interaction between indole ring and pyridine coenzymes: Crystal structure of 1-methyl-3-carbamoylpyridinium: Indole-3-acetic acid (1:1) monohydrate charge-transfer complex

The crystal and molecular structures of the title complex has been determined by X-ray diffraction methods, as a model for tryptophan residues in protein-pyridine coenzyme interactions. The structure was solved by direct methods and was refined by standard methods (final R = 0.073). The light-green crystals consist of alternate layers of indole-3-acetic acid and 1-methyl-3-carbamoylpyridinium molecules piled up to the c-direction, and are stabilized by the crystal water participating in hydrogen bonds in the a- and b-directions. The parallel stackings and interplanar spacing distances between indole and pyridinium rings strongly suggest a II–II¹ charge transfer from the indole ring to the lowest unoccupied orbital of the pyridinium ring in the ground state. Furthermore, this crystal structure provides evidence that quaternization of the N1 position enhances electron-acceptor properties of pyridine. On the other hand, the proton magnetic resonance spectra suggest that the stacking mode between both rings in solution is very similar to the one observed in the crystal structure.