POLYOL ACCUMULATION IN IN VITRO SYSTEMS OF BOMBYX EGGS

The mechanism of polyol accumulation in diapausing Bombyx eggs, conversion of [6-¹⁴C] glucose-6-phosphate into polyols and other neutral sugars was investigated in in vitro reaction systems. When a crude homogenate or a press juice of the eggs was incubated with [6-¹⁴C]glucose-6-P, the labelled trehalose, sorbitol and glycerol accumulated in the reaction mixture. In the press juice incubation system of developing eggs at day 1, ¹⁴C-sorbitol was detected in appreciable amounts, but it decreased rapidly with the development of the embryos. When the press juice was prepared from eggs in diapause, the formation of ¹⁴C-sorbitol was 3–5 times greater in eggs at early stages (day 2 to day 4) than in developing eggs.     

Partial Synchronization of Carrot Cell Culture by Auxin Deprivation

A strain of carrot cells (Daucus carota cv. Kintoki) grew exponentially in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D, 1 mg/1) with a doubling time of about 2 days. When those cells were transferred to a medium lacking 2,4-D, they continued to grow at almost the same rate for about a week. When the cells were again transferred to the auxin-free medium, the rate of cell division gradually decreased. After the cell division had ceased, cells were returned to the ordinary 2,4-D medium. A burst of cell divisions occurred after about 2 days. Timing of DNA synthesis and of mitosis suggested that the cells had been arrested at G₁ phase. In a medium containing indoleacetic acid instead of 2,4-D, the auxin was rapidly degraded and the culture was similarly synchronized as in the auxin-omitted medium.     

CRYOPRESERVATION OF SEA URCHIN EMBRYOS AND SPERM

A simple method for preserving live sea urchin embryos and sperm in liquid nitrogen (LN) wasdeveloped through the use of DMSO as a cryoprotective additive. Samples of late embryos in double test tubes were cooled to— I96°C by two-step freezing, first to — 76°C and then by plunging in LN. In the case of fertilized eggs, samples were previously frozen to — 40°C, at which temperature the samples were kept for 15 min; they were then transferred into LN. After preservation in LN for various lengths of time, samples in the double test tubes were thawed in water at 15°C. The post-thaw survival was more than 90% for late embryos, and about 10% for fertilized eggs. Difference in the length of the cryopreservation period did not affect survival. Post-thaw development in cryopreserved embryos often showed abnormalities in structure. Sperm with 1.5 M DMSO was successfully preserved in LN by a similar method to the one used for cryopreservation of late embryos. Fertilizability in cryopreserved sperm was complete, regardless of the length of the preservation period. Nearly all the eggs fertilized by cryopreserved sperm developed to normal plutei.     

Application of Fluorescent Antibody for Detecting Capsular Substances in Staphylococcus aureus

A fluorescent antibody technique was developed for the determination of the capsular-type of strains of Staphylococcus aureus. It compared favourably with the method using serum-soft agar (Yoshida 1972). With the new technique, many populations of encapsulated and unencapsulated strains were investigated. Of 1421 fresh isolates of Staph. aureus , 54 were encapsulated and among these 54·8% and 48·1% were mono- and polyvalent, respectively. Capsular-type antigens A and B were found in 92·5% and 44·4% of strains respectively; capsular-types C and D were found relatively infrequently. In the other group, of unencapsulated strains, capsular-type antigen production was demonstrated in 125 out of 163 strains examined. Mono- and polyvalent capsular-types (A and B antigen producing strains) comprised 77·6% and 22·4%. respectively. In these capsular-types A and B were found in 54·4% and 62·4%, respectively: capsular-type antigen C and D producing strains were again infrequent. These results indicate that a majority of ordinary Staph . aureus strains produce capsular-type antigens although isolation of the encapsulated strains is infrequent.     

CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES FROM SILKWORM EGGS: OCCURRENCE OF TWO CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES, CHANGES IN THEIR ACTIVITIES AND LEVELS OF CYCLIC NUCLEOTIDES DURING DEVELOPMENT OF THE EGGS

Two cyclic nucleotide-dependent protein kinases, designated as protein kinase-I and -II, were obtained from the eggs of the silkworm, Bombyx mori . Protein kinase-I is highly dependent on cGMP, whereas protein kinase-II is dependent on cAMP. In developing non-diapause eggs, the level of cyclic nucleotide-dependent protein kinase activity is quite high but that in the diapause eggs is not. The developmental changes in the two protein kinases and the level of cyclic nucleotides were also studied during the development of the eggs.     

Inducible Formation of Glutamate Dehydrogenase in Rice Plant Roots by the Addition of Ammonia to the Media

The activity of glutamate dehydrogenase (l-glutamate: NAD oxidoreductase, EC 1.4.1.2.; GDH) of rice plants changes in response to the nitrogen source supplied to the culture solution. The activity of NADH-GDH(aminating) in roots is rapidly increased by the addition of ammonia, whereas the activity in shoots is much less affected by nitrogen supply. The activity increased with increasing concentration of ammonia at least up to 14.3 mM. In roots GDH activity was found in both the mitochondrial and soluble fractions. The increase of NADH-GDH activity caused by the ammonia treatment occurs mainly in the latter fraction. The new band with GDH activity was detected on the zymogram of polyacrylamide gel electrophoresis and this inducible enzyme is active with both NAD and NADP. On the other hand, the constitutive enzyme activity active with NAD is also increased by the ammonia treatment. The increase of enzyme activity is prevented by the addition of cycloheximide or chloramphenicol to culture medium. The incorporation of ¹⁴C-leucine(U) into GDH proteins was also studied using polyacrylamide gel electrophoresis. Higher radioactivity was found in induced samples than in non-induced ones. These results show that the increase of GDH activity in roots by ammonia treatment seems to depend on de novo protein synthesis.     

Bombyx acid cysteine proteinase

Summary

Three kinds of yolk proteins (vitellin, egg-specific protein and 30 k-proteins) are found in silkmoth eggs and have been well characterized. Essentially these proteins are considered to be amino acid reserves for developing embryos. Since at an early stage of egg development the cysteine proteinase accounts for the majority of the total proteinase activity, it may be involved in the degradation of yolk proteins. The enzyme is stored in the eggs as an inactive pro-form, indicating that the activation of the enzyme might be one of the key steps in yolk protein degradation. To investigate at the molecular level how yolk proteins degradation takes place, we have studied Bombyx acid cysteine proteinase (BCP) during an early period of embryonic development. We summarize how proteinases are regulated and are involved in the degradation of Bombyx yolk proteins during embryogenesis. These will be discussed mainly in light of recent results obtained from eggs of the silkmoth, Bombyx mori.     

Fourteen polymorphic microsatellite loci in the field gudgeon,Gnathopogon elongatus elongatus

We isolated and characterized 14 polymorphic microsatellite loci for the field gudgeon, Gnathopogon elongatus elongatus, a popular freshwater species in streams including agricultural canals in Japan. The number of observed alleles for each locus ranged from 9 to 24, and the values of observed and expected heterozygosities ranged from 0.750 to 1.000 and from 0.832 to 0.953, respectively. These microsatellite markers will be useful for studies of population genetic structure and genetic variability of the field gudgeon.     

Isolation and characterization of 10 microsatellite loci in the paper wasp Polistes rothneyi (Hymenoptera; Vespidae)

We developed 10 polymorphic microsatellite loci for the Japanese paper wasp Polistes rothneyi using the magnetic particle method. Eight of the 10 loci tested were highly polymorphic, having four to eight alleles in P. rothneyi with an expected heterozygosity of 0.60–0.75. They also appeared to be applicable to other related species such as Polistes jokahamae. These loci can be used to study parameters concerning genetic relatedness such as worker reproduction and kin structure.     

Monitoring ovarian cycle and conception by fecal progesterone analysis in sika deer

The ovarian cycle and conception of sika deer were studied to reveal factors responsible for delayed conception. Concentration of progesterone in feces from 12 female Hokkaido sika deer (Cervus nippon yesoensis Heude, 1884) was measured during the mating season in 2000. The cyclic pattern of fecal progesterone synchronized with estrous symptoms, which could hence be interpreted as indicating ovarian cycle. All observed females ovulated by 14 October. However, during the early mating season, females did not permit copulation at ovulation, and the length of luteal phase following ovulation without estrus was 9.8±4.6 days (5–24days). Most females conceived at the first copulation, which were confirmed by progesterone profiles that was sustained at a high level after the copulation. This indicates the presence of a functional corpus luteum, a state of pregnancy. Thus, some females had repeated ovulation without copulation several times, creating a 3–4week variation in the timing of conception. But some females conceived very late in the mating season after the repetition of ovulation and copulation.