Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP(+) reductase

All oxygenic photosynthetically derived reducing equivalents are utilized by combinations of a single multifuctional electron carrier protein, ferredoxin (Fd), and several Fd-dependent oxidoreductases. We report the first crystal structure of the complex between maize leaf Fd and Fd-NADP(+) oxidoreductase (FNR). The redox centers in the complex--the 2Fe-2S cluster of Fd and flavin adenine dinucleotide (FAD) of FNR--are in close proximity; the shortest distance is 6.0 A. The intermolecular interactions in the complex are mainly electrostatic, occurring through salt bridges, and the interface near the prosthetic groups is hydrophobic. NMR experiments on the complex in solution confirmed the FNR recognition sites on Fd that are identified in the crystal structure. Interestingly, the structures of Fd and FNR in the complex and in the free state differ in several ways. For example, in the active site of FNR, Fd binding induces the formation of a new hydrogen bond between side chains of Glu 312 and Ser 96 of FNR. We propose that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR.     

Generation of human bipedal locomotion by a bio-mimetic neuro-musculo-skeletal model

To emulate the actual neuro-control mechanism of human bipedal locomotion, an anatomically and physiologically based neuro-musculo-skeletal model is developed. The human musculo-skeletal system is constructed as seven rigid links in a sagittal plane, with a total of nine principal muscles. The nervous system consists of an alpha motoneuron and proprioceptors such as a muscle spindle and a Golgi tendon organ for each muscle. At the motoneurons, feedback signals from the proprioceptors are integrated with the signal induced by foot–ground contact and input from the rhythm pattern generator; a muscle activation signal is produced accordingly. Weights of connection in the neural network are optimized using a genetic algorithm, thus maximizing walking distance and minimizing energy consumption. The generated walking pattern is in remarkably good agreement with that of actual human walking, indicating that the locomotory pattern could be generated automatically, according to the musculo-skeletal structures and the connections of the peripheral nervous system, particularly due to the reciprocal innervation in the muscle spindles. Using the proposed model, the flow of sensory-motor information during locomotion is estimated and a possible neuro-control mechanism is discussed. Received: 03 December 1998 / Accepted in revised form: 09 June 2000     

The NADPH thioredoxin reductase C functions as an electron donor to 2-Cys peroxiredoxin in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H₂O₂. These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.     

Identification of a gene, Desiccate, contributing to desiccation resistance in Drosophila melanogaster

Suitable alterations in gene expression are believed to allow animals to survive drastic changes in environmental conditions. Drosophila melanogaster larvae cease eating and exit moist food to search for dry pupation sites after the foraging stage in what is known as the wandering stage. Although the behavioral change from foraging to wandering causes desiccation stress, the mechanism by which Drosophila larvae protect themselves from desiccation remains obscure. Here, we identified a gene, CG14686 (designated as Desiccate (Desi)), whose expression was elevated during the wandering stage. The Desi expression level was reversibly decreased by transferring wandering larvae to wet conditions and increased again by transferring them to dry conditions. Elevation of Desi expression was also observed in foraging larvae when they were placed in dry conditions. Desi encoded a 261-amino acid single-pass transmembrane protein with notable motifs, such as SH2 and PDZ domain-binding motifs and a cAMP-dependent protein kinase phosphorylation motif, in the cytoplasmic region, and its expression was observed mainly in the epidermal cells of the larval integuments. Overexpression of Desi slightly increased the larval resistance to desiccation stress during the second instar. Furthermore, Desi RNAi larvae lost more weight under dry conditions, and subsequently, their mortalities significantly increased compared with control larvae. Under dry conditions, consumption of carbohydrate was much higher in Desi RNAi larvae than control larvae. Based on these results, it is reasonable to conclude that Desi contributes to the resistance of Drosophila larvae to desiccation stress.     

CD90 and CD110 correlate with cancer stem cell potentials in human T-acute lymphoblastic leukemia cells

Although cancer stem cells (CSCs) have been recently identified in myeloid leukemia, published data on lymphoid malignancy have been sparse. T-acute lymphoblastic leukemia (T-ALL) is characterized by the abnormal proliferation of T-cell precursors and is generally aggressive. As CD34 is the only positive-selection marker for CSCs in T-ALL, we performed extensive analysis of CD markers in T-ALL cell lines. We found that some of the tested lines consisted of heterogeneous populations of cells with various levels of surface marker expression. In particular, a small subpopulation of CD90 (Thy-1) and CD110 (c-Mpl) were shown to correlate with stem cell properties both in vitro and in transplantation experiments. As these markers are expressed on hematopoietic stem cells, our results suggest that stem cell-like population are enriched in CD90+/CD110+ fraction and they are useful positive-selection markers for the isolation of CSCs in some cases of T-ALL.     

The specific mitochondrial DNA polymorphism found in Klinefelter's syndrome

Hypervariable segments of mitochondrial DNA (mtDNA) (HV1 and HV2) were analyzed in Klinefelter's syndrome and compared to normal population data. One pair of samples consisting of a Japanese mother and affected son with Klinefelter's syndrome (involved in a criminal case), and seven unrelated DNA samples from Caucasian Klinefelter males (two involved in criminal cases and five diagnosed) were collected in Japan and the United States. The diagnosis of Klinefelter's syndrome was established previously by multiplex XY-STR typing detecting two X alleles and one Y allele in the samples. Haplotype analysis of the mtDNA sequence in Klinefelter males was found to be identical, unique, and specific, as it was not found in the normal population. Astonishingly, family data exhibited that the haplotype of the mtDNA in the son was apparently different from the mother's, suggesting that the mtDNA of Klinefelter male would not be inherited from mother to son. Our data indicate that possible interaction of the sex chromosome and the mtDNA exists, and suggests that the specific mtDNA haplotype could cause the abnormal cell to fertilize and reproduce itself.     

Comprehensive molecular phylogeny of the sub-family Dipterocarpoideae (Dipterocarpaceae) based on chloroplast DNA sequences

Dipterocarpoideae, the largest sub-family of well-known plant family Dipterocarpaceae, dominates in South Asian rain forests. Although several previous studies addressed the phylogeny of the Dipterocarpaceae family, relationships among many of its genera from the Dipterocarpoideae sub-family are still not well understood. In particular, little is known about the relationships of the genera Vateriopsis, Stemonoporus, Vateria and inconsistence remains between phylogenetic results and taxonomic classifications of Shorea and Hopea species. We studied molecular phylogeny of the sub-family Dipterocarpoideae using the trnL-trnF spacer, trnL intron and the matK gene sequences of chloroplast DNA (cpDNA). This study is the first comprehensive phylogeny reconstruction for the sub-family Dipterocarpoideae based on cpDNA, as it includes most genera (14) and a large number of species (79) with most species endemic to Sri Lanka, as well as one species from Seychelles and one species from the genus Monotes from Madagascar. Phylogenetic trees were constructed using the Neighbor Joining (NJ) and Maximum Likelihood (ML) methods using combined set of sequences including all three cpDNA regions. The topologies of the NJ and ML trees were to a certain extent, consistent with the current taxonomy of Dipterocarpoideae based on morphology and with previous molecular phylogenies based on cpDNA. Furthermore, our results provided new evidence regarding the relationships of the following genera: Vateriopsis and Stemonoporus and about the validity of the previous morphology based classifications of Shorea species. In addition, the topology of our trees was consistent with the classification of Shorea species proposed by Maury (1978), Maury-Lechon (1979) and Symington (1943). Finally, our results provided evidence for the affinity of the genus Monotes to Asian Dipterocarpoideae rather than to Tiliaceae and indicated that it is a good candidate for outgroup species for future studies of the former sub-family.     

The diversity of mycorrhizal fungi in Japanese Cephalanthera species

The diversity of mycorrhizal fungi among four Japanese Cephalanthera (Orchidaceae) species was examined at a total of seven sites, based on sequence variation in nuclear ribosomal DNA. This is the first report to detect mycorrhizal fungi in C. subaphylla. Two patterns of mycorrhizal associations were confirmed from our results. C. falcata has lower fungal specificity, associating with Russulaceae, Sebacinales and Thelephoraceae. By contrast, the three Cephalanthera species C. erecta, C. subaphylla, and C. longifolia were associated mainly with Thelephoraceae fungi. There is no large difference found in mycobionts between green and albino individuals of C. falcata and no distinct preference of Japanese Cephalanthera species for a specific fungal group of Thelephoraceae.