Characterization of spirochetes isolated from ticks (Ixodes tanuki, Ixodes turdus, and Ixodes columnae) and comparison of the sequences with those of Borrelia burgdorferi sensu lato strains.

Ixodes persulcatus serves as a tick vector for Borrelia garinii and Borrelia afzelii in Japan; however, unidentified spirochetes have been isolated from other species of ticks. In this study, 13 isolates from ticks (6 from Ixodes tanuki, 6 from Ixodes turdus, and 1 from Ixodes columnae) and 3 isolates from voles (Clethrionomys rufocanus) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rRNA gene restriction fragment length polymorphism, partial sequencing of the outer surface protein C (OspC) gene, whole DNA-DNA hybridization, and 16S rRNA gene sequence comparison. All of the results revealed that these Borrelia strains clearly represent at least two new species. A third is also likely, although additional strains have to be isolated and characterized before a separate species is designated. We designated all isolates of I. tanuki and C. rufocanus as group Hk501 and all isolates of I. turdus as group Ya501. Phylogenetic analysis based on 16S rRNA gene sequences distinguished these Borrelia strains from those belonging to hitherto known Borrelia species. Furthermore, the genomic groups, each with its own tick vectors with enzootic cycles, were quite different from each other and also from those of Lyme disease Borrelia species known to occur in Japan. The results of 16S rRNA gene sequence comparison suggest that the strain Am501 from I. columnae is related to group Hk501, although its level of DNA relatedness is less than 70%.     

RELATIONSHIPS AMONG MORPHOLOGICAL STATUS, STEROID HORMONES, AND POST-THAWING VIABILITY OF FROZEN SPERMATOZOA OF MALE MINKE WHALES (BALAENOPTERA ACUTOROSTRATA)

Spermatozoa from 21 mature minke whales ( Balaenoptera acutorostrata ) taken in the Antarctic Ocean for Japanese research were recovered from vasa deferentia, diluted 1:9 in a Tris-based diluent, and frozen at - 80°C on board the vessel. After a period ranging from 45 to 125 d, the samples were transferred to liquid nitrogen and transported to the laboratory. After thawing at 37°C the motility (percentage of motile spermatozoa), vitality (proportion of live spermatozoa), and sperm concentration were determined for each sample. These values were tested for correlations with morphological measurements (body size, body weight, testis weight) and serum concentrations of progesterone (Pd), estradiol-17β (E2), and testosterone (T). Ten of 21 samples had motile spermatozoa (2%-40%). Although no motile spermatozoa were observed in 1.1 samples, all sperm samples were examined by eosinnigrosin staining and showed vitality levels of 3%44%. It was found that the motility (Y = 0.54) and vitality (r = 0.53) of the spermatozoa were significantly (P < 0.01) correlated with the E₂ levels (8.50 ± 1.80 pg/ml). Serum T levels (0.07 ± 0.02 ngml) were significantly correlated with the E₂ levels (r = 0.58, P < 0.01>, but sperm concentrations were not correlated with either Ea or T levels. The present study demonstrates that spermatozoa of minke whales can be successfully cryopreserved.     

Organic-Solvent-Tolerant Bacterium Which Secretes Organic-Solvent-Stable Lipolytic Enzyme

A bacterial strain which could be grown in a medium containing organic solvents and which could secrete lipolytic enzyme was isolated. The stability of the lipolytic activity of the supernatant of the culture increased significantly in the presence of organic solvents such as toluene, cyclohexane, ethanol, and acetone.     

Characterization of Borrelia Species Isolated from Ixodid Ticks,Ixodes ovatus

Thirty-two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies.     

Arrest of cell cycle progression of HeLa cells in the early G1 phase in K+-depleted conditions and its recovery upon addition of insulin and LDL

Cell cycle progression of synchronized HeLa cells was studied by measuring labeling of the nuclei with [³H]thymidine. The progression was arrested in a chemically defined medium in which K⁺ was replaced by Rb⁺ (Rb-CDM) but was restored upon addition of insulin and/or low density lipoprotein (LDL). Cells started DNA synthesis 12 hr after addition of insulin and/or LDL, regardless of the time of arrest, suggesting their arrest early in the G1 phase. After incubation of cells in Rb-CDM containing insulin or LDL singly for 3, 6, or 9 hr, replacement of the medium by that without an addition resulted in marked delay in entry of cells into the S phase, but in its replacement by medium containing both agents, the delay was insignificant. Synthesis of bulk protein, estimated as increase in the cell volume, was not strongly inhibited. From these results we conclude that cell cycle progression of HeLa cells in K^?-depleted CDM is arrested early in the G1 phase and that the arrest is due to lack of some protein(s) required for entry into the S phase that is synthesized in the early G1 phase.     

Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopy.

Pathogenic prion proteins (PrP(Sc)) are thought to be produced by alpha-helical to beta-sheet conformational changes in the normal cellular prion proteins (PrP(C)) located solely in the caveolar compartments. In order to inquire into the possible conformational changes due to the influences of hydrophobic environments within caveolae, the secondary structures of prion protein peptides were studied in various kinds of detergents by CD spectra. The peptides studied were PrP(129-154) and PrP(192-213); the former is supposed to assume beta-sheets and the latter alpha-helices, in PrP(Sc). The secondary structure analyses for the CD spectra revealed that in buffer solutions, both PrP(129-154) and PrP(192-213) mainly adopted random-coils (approximately 60%), followed by beta-sheets (30%-40%). PrP(129-154) showed no changes in the secondary structures even in various kinds of detergents such as octyl-beta-D-glucopyranoside (OG), octy-beta-D-maltopyranoside (OM). sodium dodecyl sulfate (SDS), Zwittergent 3-14 (ZW) and dodecylphosphocholine (DPC). In contrast, PrP(192-213) changed its secondary structure depending on the concentration of the detergents. SDS, ZW, OG and OM increased the alpha-helical content, and decreased the beta-sheet and random-coil contents. DPC also increased the alpha-helical content, but to a lesser extent than did SDS, ZW, OG or OM. These results indicate that PrP(129-154) has a propensity to adopt predominantly beta-sheets. On the other hand, PrP(192-213) has a rather fickle propensity and varies its secondary structure depending on the environmental conditions. It is considered that the hydrophobic environments provided by these detergents may mimic those provided by gangliosides in caveolae, the head groups of which consist of oligosaccharide chains containing sialic acids. It is concluded that PrP(C) could be converted into a nascent PrP(Sc) having a transient PrP(Sc) like structureunder the hydrophobic environments produced by gangliosides.     

Mechanomyographic and electromyographic responses of the triceps surae during maximal voluntary contractions.

The objective of this study was to examine the effect of joint angle on the electromyogram (EMG) and mechanomyogram (MMG) during maximal voluntary contraction (MVC). Eight subjects performed maximal isometric plantar flexor torque productions at varying knee and/or ankle angles. Maximal voluntary torque, EMG, and MMG from the soleus (Sol), medial (MG) and lateral gastrocnemius (LG) muscles were measured at different joint angles. At varying knee angles, the root mean squared (rms) MMG amplitude of the MG and LG increased with knee joint extension from 60 degrees to 180 degrees (full extension) in steps of 30 degrees, whereas that of the Sol was constant. At varying ankle angles, the rms-MMG of all muscles (Sol, MG, and LG) decreased with torque as ankle joint extending from 80 degrees (10 degrees dorsiflexion position) to 120 degrees (30 degrees plantar flexion position) in steps of 10 degrees. In each case, changes in the rms-MMG of the three muscles were almost parallel to those in torque. In contrast, there were no significant differences in the rms-EMG of all muscles among all joint angles. Our data suggest that the MMG amplitudes recorded from individual muscles during MVCs can represent relative torque-angle relationships that cannot be represented by the EMG signals.