Isolation of mitochondria from Plasmodium falciparum showing dihydroorotate dependent respiration

Using N₂ cavitation, we established a protocol to prepare the active mitochondria from Plasmodium falciparum showing a higher succinate dehydrogenase activity than previously reported and a dihydroorotate-dependent respiration. The fact that fumarate partially inhibited the dihydroorotate dependent respiration suggests that complex II (succinate–ubiquinone reductase/quinol–fumarate reductase) in the erythrocytic stage cells of P. falciparum functions as a quinol–fumarate reductase.     

Characterization of picomolar affinity binding sites for [125I]-human calcitonin gene-related peptide in rat brain and heart

We have characterized picomolar affinity binding sites for human calcitonin gene-related peptide (CGRP) in rat brain and heart (atria and ventricle) membranes. By saturation analysis, apparent dissociation constant (KD) values of high affinity sites for [125I]-human CGRP are 9 approximately 15 pM (brain), 34 pM (ventricle) and 85 pM (atria). Low affinity sites with KD values of about 50 nM are found in rat brain and ventricle, but not in atria. Human and rat CGRP potently inhibited [125I]-human CGRP binding to these high affinity sites with apparent inhibition constant (Ki) values comparable to their KD values. Salmon calcitonin marginally inhibited these binding with Ki values between 0.1 microM and 1 microM. Extremely potent cardiovascular and gastrointestinal actions of CGRP might be mediated through CGRP binding sites with picomolar affinity which are similar to those we characterized in this study.     

Purification of all thirteen polypeptides of bovine heart cytochrome c oxidase from one aliquot of enzyme. Characterization of bovine fetal heart cytochrome c oxidase

A protocol has been worked out for separating all thirteen different polypeptides in the beef heart cytochrome c oxidase complex from a single aliquot of enzyme. This involves an initial separation of polypeptides by gel filtration on a Biogel P-60 column in SDS, a step which purifies subunits CIV and CVIII and gives mixtures of CV + CVI, ASA, AED and STA, as well as CVII, CIX and IHQ. These mixtures are then resolved by reverse-phase high-performance liquid chromatography. The separation procedures have been applied to fetal heart cytochrome c oxidase of gestation between 100 and 200 days. No differences were found in the N-terminal sequences of any of the cytoplasmically made subunits or in the entire sequence of CIX between late fetal and adult forms of the enzyme.     

Mechanism of Benzyladenine-Induced Stimulation of the Synthesis of 5-Aminolevulinic Acid in Greening Cucumber Cotyledons: Benzyladenine Increases Levels of Plastid tRNAGlu

The mechanism of the stimulatory effect of a cytokinin, namely,benzyladenine (BA), on the synthesis of 5-aminolevulinic acid(ALA) in cucumber cotyledons was studied. The rate of synthesisof ALA by plastids isolated from BA-treated cotyledons was twicethat by plastids from untreated controls. Western blot analysisof stromal proteins showed that BA did not affect the levelof glutamyl-tRNA synthetase or of glutamate l-semialdehyde (GSA)aminotransferase. Analysis of free amino acids revealed thatBA did not increase the level of glutamate in the stroma. However,the amount of total plastidic RNA was doubled in BA-treatedcotyledons. Northern blot analysis showed that the level ofplastid tRNA^Glu was increased by treatment with BA to the sameextent as that of another plastid tRNA, reflecting an increasein total plastidic RNA. The rate of formation of glutamyl-tRNAwas also doubled in plastids from BA-treated cotyledons. Theresults indicate that stimulation of the synthesis of ALA byBA is due to an increased level of tRNA^Glu in plastids. (Received June 6, 1993; Accepted November 26, 1993)     

Variation in therbcL sequence ofStegnogramma pozoi subsp.mollissima (Thelypteridaceae) in Japan

Stegnogramma pozoi (Lagasca) K. Iwats. subsp.mollissima (Fischer ex Kunze) K. Iwats. is one of the most common ferns in Japan with a very broad geographical distribution and habitat ranges. We examined the intrasubspecific variation of therbcL gene, and found three types in the subspecies (Atami-1, Atami-2, and Tateyama types). The Atami-1 and 2 types shared three apomorphic nucleotides and their geographical distribution is intermingled. Thus, we considered them to have intraspecific variation. In contrast, the Tateyama type had 4–5 different nucleotides than the Atami types, and showed a more northern distribution. Leaf morphology of the Tateyama type is distinct from that of the Atami types, though the leaf shape in Tateyama type varies considerably and in some cases overlaps with other types. Ferns have fewer morphological features that are systematically significant than flowering plants because they lack complex reproductive organ. It is reasonable to expect that species recognition of ferns based only on leaf morphology can be fine tuned by molecular features. Variation in therbcL sequences might be useful as an indication of significant biological units.     

Complete Sequence and Gene Organization of the Genome of a Hyper-thermophilic Archaebacterium, Pyrococcus horikoshii OT3

The complete sequence of the genome of a hyper-thermophilicarchaebacterium, Pyrococcus horikoshii OT3, has been determinedby assembling the sequences of the physical map-based contigsof fosmid clones and of long polymerase chain reaction (PCR)products which were used for gap-filling. The entire lengthof the genome was 1,738,505 bp. The authenticity of the entiregenome sequence was supported by restriction analysis of longPCR products, which were directly amplified from the genomicDNA. As the potential protein-coding regions, a total of 2061open reading frames (ORFs) were assigned, and by similaritysearch against public databases, 406 (19.7%) were related togenes with putative function and 453 (22.0%) to the sequencesregistered but with unknown function. The remaining 1202 ORFs(58.3%) did not show any significant similarity to the sequencesin the databases. Sequence comparison among the assigned ORFsin the genome provided evidence that a considerable number ofORFs were generated by sequence duplication. By similarity search,11 ORFs were assumed to contain the intein elements. The RNAgenes identified were a single 16S-23S rRNA operon, two 5S rRNAgenes and 46 tRNA genes including two with the intron structure.All the assigned ORFs and RNA coding regions occupied 91.25%of the whole genome. The data presented in this paper are availableon the internet at http://www.nite.go.jp.