Purification and Properties of Sweet Potato NADPH-Cytochrome c (P-450) Reductase

NADPH-cytochrome c reductase, strictly NADPH-cytochrome P-450reductase, was purified by chromatography through DEAE-cellulose,2',5'-ADP-Sepharose, and Sephadex G-100 columns after solubilizationfrom microsomes from Ceratocystis fimbriata-infected sweet potatoroot tissue with Emulgen 913. The enzyme existed in three formsafter solubilization which migrated to positions correspondingto molecular weights of 81,000, 75,000 and 72,000 on an SDS-polyacrylamidegel. Trypsin treatment of the enzyme species with the largestpolypeptide yielded the species with the smallest one. Aftersucrose density gradient centrifugation of the pellet fractionobtained by centrifugation at 100,000?g of the crude extract,the enzyme species with the largest polypeptide was presentin the particulate fractions, whereas that with the smallestone was only found at the top of the gradient. We conclude thatthe enzyme species with the largest polypeptide is in an intact,amphipathic form, whereas that with the smallest one, and probablyalso the other species, is its hydrophilic domain produced byan endogenous protease(s). The K_m values of the enzyme in theintact form for NADPH and cytochrome c were 7.7 and 2.3 µM,respectively. ¹ Present address: Laboratory of Food Hygienics, Faculty ofAgriculture, Kagawa University, Miki-cho, Kida-gun, Kagawa 761-07,Japan. (Received September 6, 1984; Accepted December 27, 1984)     

Isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato

Optimum conditions for the isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato (Ipomoea batatas) were investigated. Growth phase of callus and the ratio of callus-enzyme solution affected the yield of protoplasts. Composition of the medium and protoplast density were examined for protoplast culture.Small colonies were regenerated from the protoplasts. Upon transfer to light a high amount of anthocyanin was accumulated in these colonies.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino)-ethanesulfonic acid     

Occurrence of anthocyanoplasts in cell suspension cultures of sweet potato

Intensely pigmented and spherical vesicles (anthocyanoplasts) were found in anthocyanin-containing cells of sweet potato (Ipomoea batatas) suspension cultures. Anthocyanin synthesis began to first occur 24–48 h after exposure to light, and then numerous small red vesicles were detected under a microscope. The frequency of anthocyanoplast-containing cells rapidly increased to finally about 80% of the total cultured cells after 5 days of irradiation. Fully developed anthocyanoplasts reached 10–15 m in diameter. On the other hand, neither anthocyanin synthesis nor development of anthocyanoplasts was induced in the dark-cultured cells. 2,4-D also inhibited anthocyanin synthesis and development of these vesicles. The results suggest that anthocyanoplasts might be a site of anthocyanin synthesis and/or accumulation.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid     

Influence of overwinteringDaphnia on spring zooplankton communities: An experimental study

To evaluate the influence of overwintering individuals of zooplankton on spring zooplankton communities, the dynamics of zooplankton communities with or without overwintering individuals were observed in experimental ponds from fall to spring. An insecticide, carbaryl, was used to regulate the overwintering individuals. In ponds which received insecticide applications in November or January, all cladoceran and rotiferan species were eliminated by the treatments and did not reappear until late March or early April, even when the chemical disappeared rapidly. The low water temperature may delayed the establishment of the populations from resting eggs. In these ponds, populations of various cladoceran and rotiferan species, which seemed to be originated from resting eggs, were built up in the spring. In control ponds,Daphnia ambigua orD. longispina overwintered as juveniles and adults and established a large spring population earlier than other cladocerans and rotifers overwintering as resting eggs. The latter zooplankters did not increase in the spring probably because their growth was suppressed by the precedingDaphnia species through competition. In nature, even if the number of overwintering individuals is small, they may have a potential to build up a large population earlier than the individuals hatching from resting eggs. As a result, the species which have overwintered as individuals seem to predominate in the spring and have a large influence on the spring zooplankton community.     

Heptapeptide toxin production during the batch culture of two Microcystis species (Cyanobacteria)

Changes in the content of cyclic heptapeptide hepatotoxins called microcystins were investigated during batch culture of two Microcystis species using high performance liquid chromatography. After adsorption to ODS-silica gel cartridges and elution with methanol, the toxins were analyzed and quantified by HPLC. 35 μg per 100 mg dry cells of microcystin-RR, 34 μg of -YR and 43 μg of -LR were present at the beginning of the exponential growth phase of M. viridis. Microcystin-RR increased markedly towards the end of the exponential phase with the maximum content of 112 μg per 100 mg cells was measured at the late stage of the exponential phase. A remarkable increase of microcystin-YR from 130 μg per 100 mg cells to 1020 μg was observed during the exponential phase of a highly toxic strain of M. aeruginosa. However no clear differences were found in the pattern of change among the three toxins during the growth course.     

Close linkage of MEN2A with RBP3 locus in Japanese kindreds

Summary The gene responsible for multiple endocrine neoplasia type 2A (MEN2A) has recently been assigned to the pericentromeric region of chromsome 10 in European Caucasian kindreds by linkage analysis using a DNA marker, interstitial retinol-binding protein 3 (RBP3). We have found tight linkage between the MEN2A and RBP3 loci in Japanese MEN2A kindreds. The maximum lod score is 5.19 at a recombination fraction of 0.00. This result suggests that mutation of a certain gene close to RBP3 is responsible for MEN2A irrespective of ethnic backgrounds.     

Mother-infant interactions of wild-born,individually-caged cynomolgus monkeys (Macaca fascicularis) during the first 14 weeks of infant life

This study documents age-related changes in the interactions of wild-born cynomolgus macaque mothers and their infants living in individual cages during the first 14 weeks of infant life. Body contact between mother and infant, maternal holding, and infant sucking were found to decrease, and the mothers showed an increased frequency of aggression toward their infants with age. These results were broadly similar to those reported for mother-infant interactions in other macaques living in social groups. Nevertheless, a clear difference between the present cynomolgus macaques and other macaques in social groups was apparent. The cynomolgus macaque mothers tended to permit their infants to move about freely without displaying maternal protectiveness such as restraint or retrieval, unlike other macaque mothers in social groups. Such maternal behaviors might derive from the experience of living in individual cages for many years and the relative safety of living in individual cages. The lack of maternal restraint and retrieval could be responsible for the observed sex differences in behavior: male infants moved more actively, and broke, and made contact with their mothers more frequently than did female infants. Moreover, mothers of female infants held and groomed them more frequently and were less aggressive toward them.     

Mutational analysis of the RNase-like domain in subunit 2 of fission yeast RNA polymerase II

Local sequence similarity exists between the subunit 2 of eukaryotic RNA polymerases II and the barnase-type bacterial RNases. The RNase-like domain from the Rpb2 ofSchizosaccharomyces pombe was expressed inEscherichia coli as a GST fusion protein and examined for its RNase activity. When the GST fusion protein was incubated in vitro with³²P-labeled RNA, the RNA degradation activity was less than 0.1%, if any, of the level of synthetic barnase. In order to check the in vivo function of this region, we constructed two mutantrpb2 alleles,rpb2 ^E357A andrpb2 ^H3a6L , each carrying a single amino acid substitution at the site correponding to one of the three essential amino acid residues forming the catalytic site in barnase (mutation of barnase at the corresponding sites results in complete loss of RNase activity) and five other mutantrpb2 alleles, each carrying a single mutation at various positions within the RNase-like domain but outside the putative catalytic site for RNase activity. When these mutantrpb2 alleles were expressed in anrpb2-disruptedS. pombe strain, all the mutants grew as well as the wild-type parent and did not show any clear defective phenotypes. These results suggest either that the RNase-like domain in Rpb2 does not function as an RNase in vivo or that the RNase activity of this domain, if present at all, is not essential for cell growth.