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)     

DIVERSITY OF HALOGENATED SECONDARY METABOLITES IN THE RED ALGA LAURENCIA NIPPONICA (RHODOMELACEAE,CERAMIALES)1,2

Many morphologically similar, but chemically distinct, populations have been found in the marine red alga Laurencia nipponica Yamada (Rhodomelaceae, Ceramiales) growing in Japan. Each chemical type is characterized by a specific end-product of halogenated secondaly metabolite synthesis: chamigrane-type sesquiterpenoids such as prepacifenol and halochamigrene epoxide and C₁₅ bromoethers such as laurencin, laureatin, isoprelaurefucin, epilaurallene, and kumausallene. These seven types of secondary metabolite syntheses remained the same in the wild and under various culture conditions. Because bromoethers and terpenoids are probably synthesized by different metabolic pathways, it is virtually certain that different sets of enzymes participate in their synthesis. Prepacifenol- and laureatin-producing populations were selected as representatives of terpenoid and bromoether groups, respectively. F₁ tetrasporophytes derived from crosses between reciprocal, female and male gametophytes of prepacifenol- and laureatin-producing strains bore both types of metabolites, suggesting that the genes Producing these enzyme systems are encoded by nuclear genomes. The F₁ gametophytes resulting from the reciprocal crosses produced either prepacifenol or laureatin, and the four individuals derived from spore tetrads (a set of tetraspores derived from a single tetrasporangium) produced either prepacifenol or laureatin in a 1:1 ratio, indicating that genes participating in terpenoid synthsis and those participating in bromoether synthesis are on different loci of homologous chromosomes and are segregated at meiosis (tetrasporogenesis). One individual of this interpopulational F₁ gamtophyte produced both parental types of metabolite, perhaps indicating the occurrence of a recombination type. Natural hybrid individuals, including such recombination-type gametophytes, were found in a sympatric locality at which these two chemical types occur. F₁ tetrasporophytes derived from crosses between respective prepacifenol- and laureatin-producing strains and their F₁ gametohytes produced only parental-type metabolite-producing plants. These results indicate that the diverse chemical types can be referred to as races (chemical races).     

LORETA Current Source Density for Duration Mismatch Negativity and Neuropsychological Assessment in Early Schizophrenia

Introduction

Patients with schizophrenia elicit cognitive decline from the early phase of the illness. Mismatch negativity (MMN) has been shown to be associated with cognitive function. We investigated the current source density of duration mismatch negativity (dMMN), by using low-resolution brain electromagnetic tomography (LORETA), and neuropsychological performance in subjects with early schizophrenia.

Methods

Data were obtained from 20 patients meeting DSM-IV criteria for schizophrenia or schizophreniform disorder, and 20 healthy control (HC) subjects. An auditory odd-ball paradigm was used to measure dMMN. Neuropsychological performance was evaluated by the brief assessment of cognition in schizophrenia Japanese version (BACS-J).

Results

Patients showed smaller dMMN amplitudes than those in the HC subjects. LORETA current density for dMMN was significantly lower in patients compared to HC subjects, especially in the temporal lobes. dMMN current density in the frontal lobe was positively correlated with working memory performance in patients.

Conclusions

This is the first study to identify brain regions showing smaller dMMN current density in early schizophrenia. Further, poor working memory was associated with decreased dMMN current density in patients. These results are likely to help understand the neural basis for cognitive impairment of schizophrenia.     

Quantitative analysis of cereulide, an emetic toxin of Bacillus cereus, by using rat liver mitochondria

An emetic toxin cereulide, produced by Bacillus cereus, causes emetic food poisonings, but a method for quantitative measurement of cereulide has not been well established. A current detection method is a bioassay method using the HEp-2 cell vacuolation test, but it was unable to measure an accurate concentration. We established a quantitative assay for cereulide based on its mitochondrial respiratory uncoupling activity. The oxygen consumption in a reaction medium containing rat liver mitochondria was rapid in the presence of cereulide. Thus uncoupling effect of cereulide on mitochondrial respiration was similar to those of uncouplers 2,4-dinitrophenol (DNP), carbonylcyanide m-chlorophenylhydrazone (CCCP), and valinomycin. This method gave constant results over a wide range of cereulide concentrations, ranging from 0.05 to 100 microg/ml. The minimum cereulide concentration to detect uncoupled oxygen consumption was 50 ng/ml and increased dose-dependently to the maximum level. Semi-log relationship between the oxygen consumption rate and the cereulide concentration enables this method to quantify cereulide. The results of this method were highly reproducible as compared with the HEp-2 cell vacuolation test and were in good agreement with those of the HEp-2 cell vacuolation test. The enterotoxin of B. cereus or Staphylococcus aureus did not show any effect on the oxygen consumption, indicating this method is specific for the identification of cereulide as a causative agent of emetic food poisonings.     

Specific terminal labeling of DNA molecules

We report a simple method to directly label or modify a specific terminus of linear DNA molecules. The method is based upon our finding that a presumably triple-stranded structure by RecA-mediated formation at the terminus formed with deoxyoligonucleotides, whose sequence is complementary to the 5' terminus of one of the strands of a double-stranded DNA molecule, is quite stable and can serve as a template for DNA polymerase reaction, with the nucleotides being incorporated by an exchange reaction. This novel type of nucleotide incorporation has made it possible to label a specific terminus of target double-stranded DNA molecules by a direct means (without amplification) regardless of its molecular size, a procedure previously unavailable. As an application, we show that large DNA molecules can be fixed to a solid support in a specific orientation, thus being utilized for various analytical purposes of DNA molecules.     

Anti-predator defence and the complexity-stability relationship of food webs

The mechanism for maintaining complex food webs has been a central issue in ecology because theory often predicts that complexity (higher the species richness, more the interactions) destabilizes food webs. Although it has been proposed that prey anti-predator defence may affect the stability of prey-predator dynamics, such studies assumed a limited and relatively simpler variation in the food-web structure. Here, using mathematical models, I report that food-web flexibility arising from prey anti-predator defence enhances community-level stability (community persistence and robustness) in more complex systems and even changes the complexity-stability relationship. The model analysis shows that adaptive predator-specific defence enhances community-level stability under a wide range of food-web complexity levels and topologies, while generalized defence does not. Furthermore, while increasing food-web complexity has minor or negative effects on community-level stability in the absence of defence adaptation, or in the presence of generalized defence, in the presence of predator-specific defence, the connectance-stability relationship may become unimodal. Increasing species richness, in contrast, always lowers community-level stability. The emergence of a positive connectance-stability relationship however necessitates food-web compartmentalization, high defence efficiency and low defence cost, suggesting that it only occurs under a restricted condition.     

Development of human pancreas

The developmental sequence of human pancreatic secretory proteins has not previously been studied in detail. We applied immunohistochemistry to study 20 fetal and neonatal pancreas' (8th to 39th gestational weeks) using antisera against the following pancreatic secretory proteins: pancreatic secretory trypsin inhibitor (PSTI), serine proteinases (trypsin, chymotrypsin, and elastase I), and amylase. PSTI was first detected in developing buds of the pancreas during the 8th gestational week, and proteinases were observed in acinar cells during the 14th week of gestation. Immunoreactivity for both PSTI and proteinases was found in most acinar cells soon after their appearance. Immunoreactivity for amylase could not be detected in fetal or neonatal pancreas tissue. PSTI was also found in developing islets during the 14th gestational week, but the number of immunoreactive cells had decreased by term. Cells positive for serine proteinases were occasionally in contact with islets in second-trimester fetuses. In discussing these results, we give particular attention to the nonparallel appearance of secretory products in the fetal pancreas, and the significance of cells immunoreactive for secretory proteins in endocrine islets.