Kikukatsu Ito

Isolation of two distinct cold-inducible cDNAs encoding plant uncoupling proteins from the spadix of skunk cabbage (Symplocarpus foetidus)

To know the involvement of mitochondrial uncoupling protein (UCP) in thermogenesis in plants, cDNA cloning of UCP-like gene was performed using cDNA library prepared from the spadix of skunk cabbage (Symplocarpus foetidus). Two novel cDNAs (SfUCPa and SfUCPb) encoding uncoupling proteins were isolated. The SfUCPa cDNA contained an open reading frame of 303 amino acids (predicted molecular weight 32.6 kDa) while the SfUCPb cDNA encoded 268 amino acids (predicted molecular weight 29.0 kDa). While the SfUCPA protein had six transmembrane domains and three mitochondrial energy-transfer protein signatures that are characteristic of all known UCPs, SfUCPB lacks the fifth transmembrane domain and the third energy-transfer protein signature, suggesting an altered topology in the inner membrane of the mitochondria. The expression of SfUCPa and SfUCPb mRNAs was detected only in the spadix but not in the leaf. Further, the extents of the expression of SfUCPa and SfUCPb mRNAs were induced by low-temperature treatment. These data suggest that SfUCPA and SfUCPB may have an important role in thermogenesis in the spadix of skunk cabbage.

Functional Coexpression of the Mitochondrial Alternative Oxidase and Uncoupling Protein Underlies Thermoregulation in the Thermogenic Florets of Skunk Cabbage

Two distinct mitochondrial energy dissipating systems, alternative oxidase (AOX) and uncoupling protein (UCP), have been implicated as crucial components of thermogenesis in plants and animals, respectively. To further clarify the physiological roles of AOX and UCP during homeothermic heat production in the thermogenic skunk cabbage (Symplocarpus renifolius), we identified the thermogenic cells and performed expression and functional analyses of these genes in this organism. Thermographic analysis combined with in situ hybridization revealed that the putative thermogenic cells surround the stamens in the florets of skunk cabbage and coexpress transcripts for SrAOX, encoding Symplocarpus AOX, and SrUCPb, encoding a novel UCP that lacks a fifth transmembrane segment. Mitochondria isolated from the thermogenic florets exhibited substantial linoleic acid (LA)-inducible uncoupling activities. Moreover, our results demonstrate that LA is capable of inhibiting the mitochondrial AOX pathway, whereas the proportion of pyruvate-stimulated AOX capacity was not significantly affected by LA. Intriguingly, the protein expression levels for SrAOX and SrUCPb were unaffected even when the ambient air temperatures increased from 10.3°C to 23.1°C or from 8.3°C to 24.9°C. Thus, our results suggest that functional coexpression of AOX and UCP underlies the molecular basis of heat production, and that posttranslational modifications of these proteins play a crucial role in regulating homeothermic heat production under conditions of natural ambient temperature fluctuations in skunk cabbage.

Taxonomic status of the Japanese triploid forms of Fasciola : comparison of mitochondrial ND1 and COI sequences with F. hepatica and F. gigantica

Because it is difficult to identify morphologically the Japanese forms of Fasciola, additional taxonomic criteria are required. In order to clarify the genetic relationships between Fasciola hepatica, Fasciola gigantica, and the Japanese triploid forms of Fasciola, we compared nucleotide sequences of mitochondrial NADH dehydrogenase subunit 1 (ND1) and cytochrome c oxidase subunit I (COI) genes. Intraspecific variation in the ND1 and COI sequences was low, except for 1 specimen of F. gigantica in the ND1 sequence. The ND1 and COI sequences of Japanese triploid forms of Fasciola were nearly identical to those found in F. gigantica but were different from those of F. hepatica. Thus, the Japanese triploid forms of Fasciola are thought to be categorized as F. gigantica.

Developmental appearance of transcription factors that regulate liver-specific expression of the aldolase B gene.

The region from -202 to -1 of the rat aldolase B (AldB) promoter directs tissue-specific transcription in vitro. Deletion of the half of this promoter distal to its origin reduced its ability for tissue-specific expression. Two protein-binding sites in this distal element have been identified and characterized. One bound a factor named AIF-A, which is probably identical to previously characterized liver-specific factors HNF-1 and APF. The other, containing a CCAAT motif, bound a factor named AIF-B. These two factors were considerably enriched in liver cells compared with other cells not expressing AldB. In the liver, these two factors increased prior to the activation of the AldB gene during development. Liver-specific in vitro transcription assays indicated that the binding of both factors to their target sequences was required for AldB transcription. These results suggest that AIF-A and AIF-B are positively acting transcription factors that regulate tissue-specific and development stage-specific activation of the AldB gene.

Pyruvate-sensitive AOX exists as a non-covalently associated dimer in the homeothermic spadix of the skunk cabbage, Symplocarpus renifolius

The cyanide‐resistant alternative oxidase (AOX) is a homodimeric protein whose activity can be regulated by the oxidation/reduction state and by α‐keto acids. To further clarify the role of AOX in the skunk cabbage, Symplocarpus renifolius, we have performed expression and functional analyses of the encoding gene. Among the various tissues in the skunk cabbage, SrAOX transcripts were found to be specifically expressed in the thermogenic spadix. Moreover, our data demonstrate that the SrAOX protein exists as a non‐covalently associated dimer in the thermogenic spadix, and is more sensitive to pyruvate than to other carboxylic acids. Our results suggest that the pyruvate‐mediated modification of SrAOX activity plays a significant role in thermoregulation in the skunk cabbage.

Expression of uncoupling protein and alternative oxidase depends on lipid or carbohydrate substrates in thermogenic plants

Thermogenesis, in which cellular respiratory activity is considerably stimulated, requires mitochondrial uncoupling protein (UCP) in mammals and an alternative oxidase (AOX) in plants. Here, we show that the genes for both proteins are expressed in thermogenic plants, but the type correlates with the respiratory substrate. A novel gene termed PsUCPa encoding a variant of UCP was specifically expressed in thermogenic flowers of Philodendron selloum, which uses lipids as substrates. However, a gene termed DvAOX encoding for AOX protein was expressed in thermogenic flowers of Dracunculus vulgaris, which presumably uses carbohydrates as substrates. These findings suggest that cellular metabolism is a major determinant in selective expression of appropriate thermogenic genes in plants.

Effects of floral thermogenesis on pollen function in Asian skunk cabbage Symplocarpus renifolius

The effects of temperature on pollen germination and pollen tube growth rate were measured in vitro in thermogenic skunk cabbage, Symplocarpus renifolius Schott ex Tzvelev, and related to floral temperatures in the field. This species has physiologically thermoregulatory spadices that maintain temperatures near 23°C, even in sub-freezing air. Tests at 8, 13, 18, 23, 28 and 33°C showed sharp optima at 23°C for both variables, and practically no development at 8°C. Thermogenesis is therefore a requirement for fertilization in early spring. The narrow temperature tolerance is probably related to a long period of evolution in flowers that thermoregulate within a narrow range.

Identification of a gene for pyruvate-insensitive mitochondrial alternative oxidase expressed in the thermogenic appendices in Arum maculatum

Heat production in thermogenic plants has been attributed to a large increase in the expression of the alternative oxidase (AOX). AOX acts as an alternative terminal oxidase in the mitochondrial respiratory chain, where it reduces molecular oxygen to water. In contrast to the mitochondrial terminal oxidase, cytochrome c oxidase, AOX is nonprotonmotive and thus allows the dramatic drop in free energy between ubiquinol and oxygen to be dissipated as heat. Using reverse transcription-polymerase chain reaction-based cloning, we reveal that, although at least seven cDNAs for AOX exist (AmAOX1a, -1b, -1c, -1d, -1e, -1f, and -1g) in Arum maculatum, the organ and developmental regulation for each is distinct. In particular, the expression of AmAOX1e transcripts appears to predominate in thermogenic appendices among the seven AmAOXs. Interestingly, the amino acid sequence of AmAOX1e indicates that the ENV element found in almost all other AOX sequences, including AmAOX1a, -1b, -1c, -1d, and -1f, is substituted by QNT. The existence of a QNT motif in AmAOX1e was confirmed by nano-liquid chromatography-tandem mass spectrometry analysis of mitochondrial proteins from thermogenic appendices. Further functional analyses with mitochondria prepared using a yeast heterologous expression system demonstrated that AmAOX1e is insensitive to stimulation by pyruvate. These data suggest that a QNT type of pyruvate-insensitive AOX, AmAOX1e, plays a crucial role in stage- and organ-specific heat production in the appendices of A. maculatum.

Functional expression of plant alternative oxidase decreases antimycin A-induced reactive oxygen species production in human cells.

Alternative oxidase (AOX) plays a pivotal role in cyanide‐resistance respiration in the mitochondria of plants, fungi and some protists. Here we show that AOX from thermogenic skunk cabbage successfully conferred cyanide resistance to human cells. In galactose medium, HeLa cells with mitochondria‐targeted AOX proteins were found to have significantly less reactive oxygen species production in response to antimycin‐A exposure, a specific inhibitor of respiratory complex III. These results suggest that skunk cabbage AOX can be used to create an alternative respiration pathway, which might be important for therapy against various mitochondrial diseases.

Cell adhesion to substratum and activation of tyrosine kinases are essentially required for G1/S phase transition in BALB/c 3T3 fibroblasts

Cell adhesion to substratum and activation of tyrosine kinases are essential for the progression of cell cycle through G1 phase in mammalian cells. The kinetic studies of mouse BALB/c 3T3 fibroblasts showed that serum was no longer required for the progression of G1/S phase transition. In contrast, cell adhesion was essentially required in late G1 phase, especially at the period of G1/S transition. Among the kinase inhibitors used to elucidate the signal transduction caused by cell adhesion, tyrosine kinase inhibitors, genistein and herbimycin A, blocked the G1/S transition most effectively when cells were exposed to the inhibitors at the period of G1/S transition. Cell adhesion was not critically required for cells to undergo DNA synthesis once they had passed the G1/S boundary, and the effects of tyrosine kinase inhibitors on the progression of S phase were also not critical. The expressions of histone H2B and dihydrofolate reductase (DHFR) genes (S phase specific genes) and also the transcription factor E2F-1 gene (an activator of DHFR gene) were suppressed when cells were cultured without adhesion or exposed to the tyrosine kinase inhibitors. These results suggest that cell adhesion to substratum plays an important role in the G1/S phase transition of mouse BALB/c 3T3 fibroblasts through the activation of tyrosine kinases other than growth factor receptor-tyrosine kinases.