Alterations by saponins of passive Ca2+ permeability and Na+-Ca2+ exchange activity of canine cardiac sarcolemmal vesicles

Saponins can both permeabilize cell plasma membranes and cause positive inotropic effects in isolated cardiac muscles. Different saponins vary in their relative abilities to cause each effect suggesting that different mechanisms of action may be involved. To investigate this possibility, we have compared the effects of seven different saponins on the passive Ca2+ permeability and Na+-Ca2+ exchange activity of isolated canine cardiac sarcolemmal membranes. Saponins having hemolytic activity reversibly increased the passive efflux of Ca2+ from sarcolemmal vesicles preloaded with 45Ca2+ with the following order of potency: echinoside-A greater than echinoside-B greater than holothurin-A greater than holothurin-B greater than sakuraso-saponin. Ginsenoside-Rd and desacyl-jego-saponin, which lack hemolytic activity, had no significant effect on this variable. The saponins also stimulated Na+-Ca2+ exchange activity measured as Na+-dependent Ca2+ uptake by sarcolemmal vesicles. Ginsenoside-Rd and desacyl-jego-seponin, which did not affect passive Ca2+ permeability, stimulated the uptake, while in contrast, echinoside-A and -B only slightly increased or decreased this latter variable. Thus, the abilities of these compounds to enhance Na+-Ca2+ exchange activity seem to be inversely related to their abilities to increase the Ca2+ permeability. Effects by the echinosides on Na+-Ca2+ exchange may be masked by the loss of Ca2+ from the vesicles due to the increased permeability. These results suggest that the saponins interact with membrane constituent(s) that can influence the passive Ca2+ permeability and the Na+-Ca2+ exchange activity of cardiac sarcolemmal membranes.     

DNA primase-DNA polymerase alpha assembly from mouse FM3A cells. Purification of constituting enzymes, reconstitution, and analysis of RNA priming as coupled to DNA synthesis

The mouse DNA primase-DNA polymerase alpha complex can be resolved with buffer containing 50% ethylene glycol (Suzuki, M., Enomoto, T., Hanaoka, F., and Yamada, M. (1985) J. Biochem. (Tokyo) 98, 581-584). The dissociated primase and DNA polymerase alpha have been purified sufficiently that there was no cross-contamination with each other. By the use of thus isolated DNA primase and DNA polymerase alpha in addition to DNA primase-DNA polymerase alpha complex, we have studied primer RNA synthesis and DNA elongation separately as well as the coupled reaction of the initiation and elongation of DNA chains. In the absence of deoxyribonucleoside triphosphates, the isolated primase synthesized oligoribonucleotides of an apparent length of 7-11 nucleotides (monomeric oligomer) and multiples of a modal length of 9-10 nucleotides (multimeric oligomer) and fd phage single-stranded circular DNA. Monomeric and dimeric oligomers were synthesized processively, and trimeric and larger oligomers were produced by repeated cycles of processive synthesis. The primase complexed with DNA polymerase alpha mainly synthesized monomeric and a small amount of dimeric oligomers. In the presence of deoxyribonucleoside triphosphates at concentrations above 10 microM, the DNA primase-DNA polymerase alpha complex exclusively synthesized monomeric oligomers only, which were utilized as primers for DNA synthesis. On the other hand, the products synthesized by the isolated primase were qualitatively unchanged as compared with those synthesized in the absence of DNA precursors. When the synthesis of oligomers by the isolated primase was coupled with DNA elongation by the addition of the primase-free DNA polymerase alpha, the synthesis of dimeric oligomers was inhibited as a result of efficient DNA elongation from monomeric oligomers.     

Effect of the strandedness of cofactor DNA on the activities of DNA-dependent ATPases B and C3 from FM3A cells

The requirements of cofactor DNA for DNA-dependent ATPases B and C3 were analyzed in detail. ATPase B and C3 required the presence of a polynucleotide for their activities. Among the DNAs tested, ATPase B showed a preference for poly(dT) as its cofactor. The other deoxyhomopolymers, except poly(dG) and heat-denatured DNA also were effective. The alternating polydeoxyribonucleotide, poly[d(A-T)] had an efficiency 23% that of heat-denatured DNA. Unlike ATPase B, ATPase C3 showed almost no activity with deoxyhomopolymers. The most effective cofactor for ATPase C3 so far tested is poly[d(A-T)]. Relatively high activity was obtained with heat-denatured DNA. The high activity of ATPase B with poly(dT) was reduced by the addition of poly(dA). The addition of noncomplementary homopolymers did not affect enzyme activity. ATPase C3 activity in the presence of 10 microM poly(dT) increased gradually with concentrations of poly(dA) up to 20 microM, after which it decreased. Almost no increase in activity was observed when noncomplementary homopolymers were added. The relatively high activity of ATPase C3 with heat-denatured DNA was suggested by its high sensitivity to ethidium bromide to be due to the double-stranded region in the heat-denatured DNA formed by self-annealing.     

Sequestration of 3-oxygenated α-ionone derivatives in the male rectal gland of the solanaceous fruit fly, Bactrocera latifrons

The solanaceous fruit fly, Bactrocera latifrons (Hendel) (Diptera: Tephritidae), infests various solanaceous fruits including eggplant and chili peppers. We found that a freshly cut fruit of an eggplant cultivar [ Solanum melongena L. cv. Long Purple Oriental (Solanaceae)] selectively attracted and provoked voracious feeding behavior in adult B. latifrons males (but not in females) in an indoor test. One of the male-specific attractants/feeding stimulants in eggplant was identified as 3-hydroxy-α-ionone. Sexually mature males that fed on the eggplant pulp selectively accumulated a series of 3-oxygenated α-ionone/α-ionol analogs (e.g., 3-oxo-α-ionol and 3-oxo-7,8-dihydro-α-ionol) in the rectal gland, a suspected pheromone reservoir in male flies. Males fed on synthetic 3-hydroxy-α-ionone, 3-oxo-α-ionol, or 3-oxo-α-ionone partially biotransformed the compounds into 3-oxo-α-ionol, 3-oxo-α-ionone, and 3-oxo-7,8-dihydro-α-ionol and sequestered substantial quantities (as high as 5 µg/gland as a mixture) in a similar ratio in the rectal gland within 6 h after ingestion. These results suggest that the rectal sequestrates may serve as a sex pheromone similar to other Bactrocera species that use phenylpropanoid volatiles to attract conspecific females.     

Human ubiquitin-activating enzyme (E1): compensation for heat-labile mouse E1 and its gene localization on the X chromosome.

We have constructed interspecific somatic cell hybrids between a temperature-sensitive (ts) mutant cell line of mouse FM3A cells, ts85, that has a heat-labile ubiquitin-activating enzyme (E1) and a human diploid fibroblast cell line, IMR-90. A hybrid clone that could grow stably at a nonpermissive temperature (39 degrees C) was obtained. Segregation of the hybrid cells at a permissive temperature (33 degrees C) gave rise to temperature-sensitive clones. The electrophoresis of extracted histones and karyotype analysis of the segregants revealed a close correlation of the ability to grow at 39 degrees C, the presence of uH2A (ubiquitin-H2A semihistone) at 39 degrees C, and the presence of the human X chromosome. One of the hybrid clones that could grow at the nonpermissive temperature contained the X chromosome as the only human chromosome. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of affinity-purified E1 showed that this hybrid clone contained both human and mouse type E1. Thus we conclude that the functional gene for human E1 is located on the X chromosome.     

Seasonal changes in spermatogenic cell degeneration in the seminiferous epithelium of adult Japanese macaques (Macaca fuscata fuscata)

The degenerating pattern of spermatogenic cells in the seminiferous tubule of Japanese macaques was studied to clarify a relationship between seasonal changes of reproductive performances and cytological findings in the Japanese macaque. For light microscopy, testis samples were obtained from five adult animals by biopsy in April (nonmating season) and October (mating season). For electron microscopy, specimens from four additional macaques were used. Degenerating cells were found in all steps of spermatogenesis. In stages I to V of the cycle of the seminiferous epithelium, morphologically atypical pachytene spermatocytes were observed in 14.7 and 10.0% of the cells in the nonmating and mating seasons, respectively, although the difference in percentage was not significant. Mature spermatids with atypical features in those stages occupied 59.6% of the cells in the nonmating season, which significantly decreased to 34.1% in the mating season. These results imply that the seasonal change of sperm production is related, at least in part, to the process of degeneration of the spermatogenic cells in this species.     

Quantitative measurement of spastic ankle joint stiffness using a manual device: A preliminary study

Quantitative measurement of ankle joint stiffness following stroke could prove useful in monitoring the progress of a rehabilitation programme. The objective of this study was to design a manual device for use in the clinical setting. Manual measurement of spastic ankle joint stiffness has historically been conducted using hand-held dynamometers or alternative devices, but some difficulties have been reported in controlling the velocity applied to the ankle during the measurement. In this study, a manually operated device was constructed with a footplate, a torquemeter and a potentiometer. It was mechanically designed to rotate around an approximated axis of the ankle joint and to measure ankle joint angular position and its corresponding resistive torque. Two stroke hemiplegic subjects pariticapted in a pilot study. The results suggested that difficulty in controlling the applied velocity might be complemented by presenting torque data as a function of peak angular velocity in each stretching cycle. Moreover, the results demonstrated that the device could potentially apply a wide range of angular velocities and provide potentially useful clinical information. Quantitative data successfully acquired using this method included the approximate ankle angular position, where the velocity-dependent characteristics of stiffness was notably initiated and its corresponding torque and velocity.     

Comparison of properties between human recombinant and placental copper-zinc SOD

The physicochemical properties of purified recombinant human copper-zinc superoxide dismutase (r-hSOD) were compared with those of human placental copper-zinc superoxide dismutase (h-SOD). No differences were found in specific activity, metal contents, amino acid composition, and tryptic peptide map. The spectrophotometric properties including UV, ESR, and CD spectra were also similar. The result of isoelectric gel electrophoresis showed that the difference in isoelectric point (pI) was derived from acetylation of the N-terminal amino acid (alanine) in h-SOD. In SDS-polyacrylamide gel electrophoresis, both SODs showed the same behavior and enzymic activity was retained only under non-reducing conditions. ESR analysis of the denatured enzyme suggested that the high stability was derived from the structure of the active site around copper. Experiments using other metal-substituted SODs (Cu, Co in place of zinc) suggested that zinc contributed to the stability and the unique electrophoretic behavior of the enzyme.