Control of Late Development of Dictyostelium discoideum by Proteins Functioning at Early Stages

We examined two mutants of D. discoideum which are temperature-sensitive for development. At the nonpermissive temperature one mutant becomes arrested in development during the transition from the finger to the migrating slug. Temperature-shift experiment indicates that the temperature-sensitive period begins at considerably earlier tip-forming stage. The other mutant becomes arrested at the Mexican hat stage and the temperature-sensitive period coinsided with this stage. The analysis of protein synthesis by two-dimensional gels, however, showed specific changes at the nonpermissive temperature at an earlier finger-forming stage.
These results indicate the presence of a control of late development by proteins at early stages.     

Isolation of the Antheridiogen of Anemia phyllitidis

The antheridiogen (antheridium-inducing substance) of the fern species Anemia phyllitidis has been obtained in pure form based on the isolation procedure described below. Pure antheridiogen is active to a dilution of 10 μg/l in antheridium formation and 0.3 μg/l in dark-germination. Its molecular formula is C₁₉H₂₂O₅.     

Anesthetic effects of a three-drugs mixture —comparison of administrative routes and antagonistic effects of atipamezole in mice—

The anesthetic mixture of medetomidine (MED), midazolam (MID) and butorphanol (BUT) produced anesthetic duration of around 40 minutes (min) in ICR mice. We reported that this anesthetic mixture produced almost the same anesthetic effects in both male and female BALB/c and C57BL/6J strains. Intraperitoneal (IP) administration of drugs has been widely used in mice. However, various injectable routes of the anesthetic mixture may cause different anesthetic effects. First, we examined effects of the anesthetic mixture by subcutaneous (SC) and intravenous (IV) injection compared to IP injection. After injection of the anesthetic mixture, administration of atipamezole (ATI) induced mice recovery from anesthesia. Secondly, we examined how different dosage and optimum injection timing of ATI affected mice recovery from anesthesia. We used an anesthetic score to measure anesthetic duration and a pulse oximeter to monitor vital signs under anesthesia. Usually, drugs from SC injection work more weakly than IP or IV injection. However, we found no significant differences of anesthetic duration among the three different injection routes. Antagonistic effects of ATI (0.3 mg/kg and 1.5 mg/kg) worked equally when administered at 30 min after injection of the anesthetic mixture. Antagonistic effects of ATI (1.5 mg/kg) were stronger than ATI (0.3 mg/kg) at 10 min after injection of the anesthetic mixture. The anesthetic mixture is a useful drug to induce nearly the same anesthetic effects by different injection routes and has an antagonist of ATI which helps mice quickly recover from anesthesia. These results may contribute to the welfare of laboratory animals.     

ATTEMPTS TO CULTURE ISOLATED URODELAN BLASTOMERES THAT CONTINUE TO DIVIDE WITHOUT AGGREGATING*

The blastomeres isolated from urodelan blastulae continued to divide without aggregating of daughter cells when inoculated with Ca²⁺-free neutral Holtfreter solution into glass culture dishes coated with agar. When standard Holtfreter solution with pH 8.2 was used as a culture medium, Ca²⁺ content from 1/40 to 1/20 of the original strength was essential for the purpose of the present observations; other divalent cations such as Mg²⁺, Ba²⁺ or Mn²⁺ replaced Ca²⁺. Under these experimental conditions, cell pedigrees were obtained during the incubation period. The greatest number of cell divisions so far observed in vitro was 8 for Hynobius lichenatus, and 9 for Cynops pyrrhogaster. Some related observations on the behavior of isolated blastomeres are also presented.     

Biochemical assessment of evolution and taxonomy of the morphologically poorly diverged geckos, Gekko yakuensis and G. hokouensis (Reptilia: Squamata) in Japan, with special reference to their occasional hybridization

We assessed the validity of two gekkonid species, Gekko yakuensis and G. hokouensis, in southern Japan. We first assigned all 398 specimens into 18 samples merely on the basis of localities. By conducting significance test for deviations of genotype frequencies from Hardy‐Weinberg at 11 allozyme loci, we checked the reproductive unity of constituents in each of those local samples, and where necessary, rearranged them into subsamples on the basis of genetic markers so that we recognized minimum reproductively cohesive units. We then compared allele frequencies among all samples and subsamples examined. Results clearly indicated that all but two can be classified into two groups that can be discriminated from each other by remarkable allele frequency differences at four diagnostic loci, and by large genetic distances even between sympatric subsamples. Observations of morphological features of the samples and subsamples confirmed that the two groups correspond to G. yakuensis and G. hokouensis, supporting validities of these two species. Allele frequency comparisons, however, also revealed that the remaining two samples, both from southern Kyushu, possessed ‘marker alleles' of both G. yakuensis and G. hokouensis at all four diagnostic loci. These samples thus were considered to represent populations that have been derived through hybridization of the two species. Detailed analyses for genetic structures demonstrated that all hybrid genotypes in the two samples are post‐Fi generations with only one individual resulting from the back‐cross with a pure line population of G. yakuensis. This finding negates the possibility that the hybrid populations are maintained by a constant supply of newly produced Fj hybrids, but suggests that the hybrid genotypes constitute stable breeding populations. This implies that the genealogical independence of G. yakuensis and G. hokouensis in several other sympatric areas has been maintained by operations of some isolation mechanisms at a pre‐mating phase. Investigations of the morphological variation in each sample or subsample revealed that although the two species can be externally largely discriminated from each other by slight modifications of the currently used diagnoses, it is difficult to detect their hybrids based solely on the morphological features.     

Pattern of sperm storage and migration in the reproductive tract of the swallowtail butterfly Papilio xuthus: cryptic female choice after second mating

Females of the swallowtail butterfly Papilio xuthus L. (Lepidoptera: Papilionidae) mate multiply during their life span and use the spermatophores transferred to increase their longevity as well as fecundity. Sperm from different males may be stored in the sperm storage organs (bursa copulatrix and spermatheca). To clarify the pattern of sperm storage and migration in the reproductive tract, mated females are dissected after various intervals subsequent to the first mating, and the type and activity of sperm in the spermatheca are observed. When virgin females are mated with virgin males, the females store sperm in the spermatheca for more than 10 days. Sperm displacement is found in females that are remated 7 days after the first mating. Immediately after remating, these females flush out the sperm of the first male from the spermatheca before sperm migration of the second male has started. However, females receiving a small spermatophore at the second mating show little sperm displacement, and the sperm derived from the small spermatophore might not be able to enter the spermatheca. Females appear to use spermatophore size to monitor male quality.     

Variation of Alkaloid Production in Nicotiana rustica Callus Cultures

Callus cultures have been established from the seed, root and leaf of Nicotiana rustica L. var. brasilia in a synthetic medium containing 1 μM 2,4-D and μM kinetin. These callus tissues behaved similarly not only in growth and organogenesis but also in nicotine production. The nicotine contents of callus cultures, which were in the order of 0.25–0.58% of dry weight during a few passages subsequent to callus induction, rapidly decreased to trace amounts in succeeding subcultures in association with the decline of the root-regenerating activity. On the other hand, free cells prepared from a callus tissue in the third passage developed into individual clones showing wide differences in growth and nicotine production. One of these clones gave rise to a relatively stable strain which is capable of producing nicotine at a high rate (0.29% of dry weight) in the absence of organization. The significance of these findings is discussed in connection with some results which have been reported for other callus cultures of Nicotiana species.