Respiratory stimulation and generation of superoxide radicals in Pseudomonas aeruginosa by fungal naphthoquinones

The mechanism of action of antimicrobial naphthoquinones from the fungus Fusarium was studied by using Pseudomonas aeruginosa. Bostricoidin, methyl ether fusarubin, and fusarubin stimulated the oxygen consumption of bacterial cells and induced cyanide-insensitive oxygen consumption. These activities of the tested compounds were also observed in bacterial membrane preparations in a dose-dependent manner. Naphthoquinones stimulated the generation of superoxide anion and hydrogen peroxide. The naphthoquinone effectively acted as the electron acceptors for bacterial diaphorase, which could explain the antibacterial activity of Fusarium naphthoquinones since electron acceptors lead to the stimulation of respiratory activity and the generation of oxygen radical species. Received: 12 July 1996 / Accepted: 31 October 1996     

REEXAMINATION OF PHYLOGENETIC RELATIONSHIPS WITHIN THE COLONIAL VOLVOCALES (CHLOROPHYTA): AN ANALYSIS OF atpB AND rbcL GENE SEQUENCES

The chloroplast-encoded atp B gene was sequenced from 33 strains representing 28 species of the colonial Volvocales (the Volvocaceae and its relatives) to reexamine phylogenetic relationships as previously deduced by morphological data and rbc L gene sequence data.1128 base pairs in the coding regions of the atp B gene were analyzed by MP, NJ, and ML analyses. Although supported with relatively low bootstrap values (75% and 65% in the NJ and ML analyses, respectively), three anisogamous/oogamous volvocacean genera— Eudorina, Pleodorina, and Volvox, excluding the section Volvox (= Euvolvox, illegitimate name), constituted a large monophyletic group (Eudorina group). Outside the Eudorina group, a robust lineage composed of three species of Volvox sect. Volvox was resolved as in the rbc L gene trees, rejecting the hypothesis of the previous cladistic analysis based on morphological data that the genus Volvox is monophyletic. In addition, the NJ and ML trees suggested that Eudorina is a nonmonophyletic genus as inferred from the morphological data and rbc L gene sequences. Although phylogenetic status of the genus Gonium is ambiguous in the rbc L gene trees and the paraphyly of this genus is resolved in the cladistic analysis based on morphological data, the atp B gene sequence data suggest monophyly of Gonium with relatively low bootstrap values (56–61%) in the NJ and ML trees. On the basis of the combined sequence data (2256 base pairs) from atp B and rbc L genes, Gonium was resolved as a robust monophyletic genus in the NJ and ML trees (with 68–86% bootstrap values), and Eudorina elegans Ehrenberg represented a paraphyletic species positioned most basally within the Eudorina group. However, phylogenetic status and relationships of the families of the colonial Volvocales were still almost ambiguous even in the combined analysis.     

RELATIONSHIP BETWEEN PRESENCE OF A MOTHER CELL WALL AND SPECIATION IN THE UNICELLULAR MICROALGA NANNOCHLORIS (CHLOROPHYTA)1

The cell division mechanisms of seven strains from six species of Nannochloris Naumann were analyzed and compared with those of three species of Chlorella Beijerinck and Trebouxia erici Ahmadjian using differential interference microscopy and fluorescence microscopy. Nannochloris bacillaris Naumann divides by binary fission and N. coccoides Naumann divides by budding. Distinct triangular spaces or mother cell walls were found in the dividing autosporangia of the other five strains from four species of Nannochloris, three species of Chlorella, and T. erici. In an attempt to infer an evolutionary relationship between nonautosporic and autosporic species of Nannochloris, we constructed a phylogenetic tree of the actin genes using seven strains from six species of Nannochloris, three species of Chlorella, and T. erici. Nannochloris species were polyphyletic in the Trebouxiophyceae group. Two nonautosporic species of N. bacillaris and N. coccoides were monophyletic and positioned distally. Moreover, to determine their phylogenetic position within the Trebouxiophyceae, we constructed phylogenetic tree of 18S rRNA genes adding other species of Trebouxiophyceae. Nannochloris species were polyphyletic in the Trebouxiophyceae and appeared in two different lineages, a Chlorella–Nannochloris group and a Trebouxia–Choricystis group. The nonautosporic species, N. bacillaris and N. coccoides, and three autosporic species of Nannochloris belonged to the Chlorella–Nannochloris group. Nannochloris bacillaris and N. coccoides were also monophyletic and positioned distally in the phylogenetic tree of 18S rRNA genes. These results suggest that autosporulation is the ancestral mode of cell division in Nannochloris and that nonautosporulative mechanisms, such as binary fission and budding, evolved secondarily.     

Uncertainty of knee joint muscle activity during knee joint torque exertion: the significance of controlling adjacent joint torque.

In the single-joint torque exertion task, which has been widely used to control muscle activity, only the relevant joint torque is specified. However, the neglect of the neighboring joint could make the procedure unreliable, considering our previous result that even monoarticular muscle activity level is indefinite without specifying the adjacent joint torque. Here we examined the amount of hip joint torque generated with knee joint torque and its influence on the activity of the knee joint muscles. Twelve healthy subjects were requested to exert various levels of isometric knee joint torque. The knee and hip joint torques were obtained by using a custom-made device. Because no information about hip joint torque was provided to the subjects, the hip joint torque measured here was a secondary one associated with the task. The amount of hip joint torque varied among subjects, indicating that they adopted various strategies to achieve the task. In some subjects, there was a considerable internal variability in the hip joint torque. Such variability was not negligible, because the knee joint muscle activity level with respect to the knee joint torque, as quantified by surface electromyography (EMG), changed significantly when the subjects were requested to change the strategy. This change occurred in a very systematic manner: in the case of the knee extension, as the hip flexion torque was larger, the activity of mono- and biarticular knee extensors decreased and increased, respectively. These results indicate that the conventional single knee joint torque exertion has the drawback that the intersubject and/or intertrial variability is inevitable in the relative contribution among mono- and biarticular muscles because of the uncertainty of the hip joint torque. We discuss that the viewpoint that both joint torques need to be considered will bring insights into various controversial problems such as the shape of the EMG-force relationship, neural factors that help determine the effect of muscle strength training, and so on.     

TAXONOMIC REEXAMINATION OF 17 SPECIES OF NITELLA SUBGENUS TIEFFALLENIA (CHARALES,CHAROPHYCEAE) BASED ON INTERNAL MORPHOLOGY OF THE OOSPORE WALL AND MULTIPLE DNA MARKER SEQUENCES1

In an attempt to reconstruct the natural taxonomic system for Nitella, 17 species of Nitella subgenus Tieffallenia were reexamined using SEM observations of the internal morphology of the oospore wall (IMOW) and phylogenetic analyses of 4553 base pairs from multiple DNA markers (atpB, rbcL, psaB, and ITS‐5.8S rRNA genes). Our SEM observations identified three types of IMOW: homogeneous (HG), weakly spongy (W‐SG), and strongly spongy (S‐SG) types. Based on differences in the IMOW, species with reticulate or tuberculate oospore wall ornamentation in the external morphology of the oospore wall (EMOW) were subdivided into two distinct groups (characterized by the HG or S‐SG types of IMOW, respectively), which were robustly separated from each other in our molecular phylogenetic analyses. In our molecular phylogeny, the subgenus Tieffallenia consisted of four robust monophyletic groups—three clades of the HG type and a spongy (S‐SG and W‐SG) type clade—that were characterized by differences in the IMOW and EMOW. In addition, our SEM observations and sequence data verified the distinct status of five species (N. japonica Allen, N. oligospira A. Braun, N. vieillardii stat. nov., N. imperialis stat. nov., and N. morongii Allen) that R. D. Wood had assigned as infraspecific taxa. Moreover, our SEM observations of the IMOW also suggested that N. megaspora (J. Groves) Sakayama originally identified by LM includes at least two distinct species, characterized by W‐SG and S‐SG types of IMOW, respectively.     

Termite queens have disproportionately more DNA in their fat body cells: reproductive division of labor and endoreduplication

Increases in DNA content caused by endoreduplication are widely observed in the metabolically active tissues of plants and animals. During egg production, insect females synthesize very large amounts of vitellogenin in their fat bodies, and female fat bodies of some insects become polyploid to accelerate vitellogenin production. Social insects have developed reproductive division of labor, wherein queens lay most of the eggs while other individuals have reduced fertility and undertake tasks required for maintaining the colony. Therefore, only queens are engaged in vitellogenin synthesis for egg production in social insects. Here, we show that termite queens have disproportionately more DNA in their fat body cells. Our DNA content analysis using flow cytometry demonstrated that more cells contained 4C‐DNA than 2C‐DNA in the fat bodies of Reticulitermes speratus queens. This high level of endoreduplication was not found in the fat body cells of other castes. This caste‐dependent doubling of DNA content in fat body cells suggests that termites exploit endoreduplication to boost egg production, in conjunction with the development of reproductive division of labor. This study highlights nuclear polyploidization as an adaptive strategy in social insects.     

Decrease of hepatic delta-aminolevulinate dehydratase activity in an animal model of fatigue

Fatigue can be defined physiologically as inability to maintain the expected power output. At present, no standard of fatigue are yet available. In order to find biomarkers of fatigue, we investigated the level of delta-aminolevulinic acid (ALA), the first intermediate metabolite in the heme biosynthetic pathway, in the plasma and urine of an animal model of fatigue. To prepare fatigued animals, we kept rats for 5 days in a cage filled with water to a height of 1.5 cm. As a result, the plasma and urinary ALA levels were increased in the fatigued animals as compared with those in the control animals. One day after the rats had been returned to their normal cages, these increased levels were restored to the control ones. We also examined the activity of the enzyme ALA dehydratase (ALAD), which is the second enzyme in the heme biosynthetic pathway, and ALAD gene expression during the fatigue and its recovery sessions. The ALAD activity, as well as its gene expression, in the liver of the fatigued animals was decreased as compared with those of the control animals. Both activity and gene expression of ALAD were recovered to their respective control levels after the rats had been allowed to rest in their normal cages for 1 day. Furthermore, the activity of ALA synthase (ALAS), the rate-limiting enzyme in the heme biosynthesis, in the liver was increased after the fatigue session for 5 days. Although this level of increase in the plasma concentration of ALA may not induce fatigue, increase in plasma and urinary ALA levels can be biomarkers of fatigue.     

Nine color eleven parameter immunophenotyping using three laser flow cytometry.

BACKGROUND: This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in analog hardware, in combination with final software compensation. An application characterizing low frequency murine B cell subpopulations is given. METHODS: The fluorochromes used are: fluorescein (FITC), phycoerythrin (PE), Cy5PE and Cy7PE, excited at 488 nm by an argon laser; Texas Red (TR), allophycocyanin (APC), and Cy7APC excited at 595 nm by a pumped dye laser; and cascade blue (CB) and cascade yellow (CY) excited at 407 nm by a violet-enhanced krypton laser. Custom additions to commercial electronics and an extended optical bench allow the measurement of these nine parameters plus forward and side scatter light signals. RESULTS: We find the use of partial analog compensation reduces the variation in the background staining levels introduced by the compensation process. Novel B cell populations with frequencies below 1% are characterized. CONCLUSIONS: Nine color flow cytometry is capable of providing measurements with high information content. The choice of reagent-dye combinations and the ability to compensate in multi-parameter measurement space are crucial to obtaining satisfactory results.     

Light, nutrients and primary productivity in Lake Biwa: An evaluation of the current ecosystem situation

Simple correlation and multiple regression analyses were performed to examine the relationship between primary productivity and environmental factors in the north basin of Lake Biwa. The primary production rates used in the analyses were estimated monthly or bimonthly during the growing season (April–November) in 1992, 1996 and 1997 with the ¹³C method. Elemental (C, N and P) contents of seston were used to assess nutrient conditions. Analyses revealed that 86% of variance in depth-integrated primary production rates (areal PP) can be explained by changes in light intensity, and sestonic C, N and P concentrations. Water temperature had no effect on areal PP. To assess relative effects of light and nutrients on PP, the P:B ratio was estimated by normalizing PP with sestonic C. The areal P:B ratio correlated most significantly with the sestonic N:P ratio, followed by light intensity. When regression analyses were made at each depth, however, the P:B ratio correlated significantly only with the sestonic N:P ratio at 0 and 1 m depths, while light intensity was also incorporated into the regressions at deeper than 2.5 m. In these regressions, the P:B ratio was negatively correlated with sestonic N:P ratio but positively with light intensity. The results suggest that the primary production rate in this lake was mainly limited by P relative to N supply rates, but was not free from light limitation in a large part of the epilimnion. In Lake Biwa, the vertical water mixing regime as well as the nutrient supply seem to be important in determining the growth and composition of primary producers, since the surface mixing layer extends into 10–15 m depths during most of the growing season.