Kayo Yasuda et al. (2003) International Worm Meeting "Mitochondria Aging"

Naoaki Ishii, Akira Akatsuka, Kayo Yasuda, Philip S Hartman, Sataro Goto

[International Worm Meeting, 2003]

Mitochondria are specialized organelles that are intimately responsible for three important processes: i) ATP production, ii) generation of reactive oxygen species (ROS), and iii) regulation of programmed cell death, or apoptosis. In addition, it is believed that cellular damage caused by ROS leads to aging. Electrons are transferred ultimately to oxygen after their passage through four complexes that are anchored to the mitochondrial inner membrane. The proximal ROS is superoxide anion (O2-), which derives from the Q cycle at rates estimated between 0.1 and 2% of the total electron flow. It is known that oxygen is initially converted to superoxide anion by electrons leaked from complex I and mainly from complex III. Recently, we have found that a C.elegans short life span mutant, mev-1, which has a mutation in a subunit (cytochrome b560) of complex II, has several distinct pathophysiologies in mev-1 animals; namely, oxygen hypersensitivity, abnormal accumulation of protein carbonyls and lipofuscin as aging markers, abnormal energy metabolism with acidosis, and ultra-structural abnormalities in mitochondria. We have proved that these phenotypes are caused by over production of reactive oxygen species (ROS) from the complex II. This suggests that ROS production can, under certain conditions, also be caused from complexes other than I and III. To further understand the influence of oxidative stress on mitochondria, we measured the contents of carbonyl protein in several aged mitochondria. We found that the protein carbonyl contents in both mitochondria and cytosol increased with increasing aging. The levels in mitochondria were higher than that in cytosol. We also observed the structures of mitochondria in aged animals with a transmission electron microscope. Morphological abnormalities such as swelling were observed in aged animals. In addition, more severe changes were observed in other organelles. This suggests that mitochondria may be more resistant to oxidative stress than other organelles, even though mitochondria accept more oxidative damage by themselves.

Yasuda H et al. (1990) Worm Breeder's Gazette "Trans-splicing in Tubulin?"

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1990]

At the last worm meeting (CSH, 1989), we reported the nucleotide sequence of the alpha-1 tubulin gene AT3 that was cloned by direct screening of a C. elegans cDNA expression library in a lambda gt11 vector (Bob Barstead), using a tubulin specific monoclonal anti-body 3A5 (provided by M. Fuller). This gene was previously identified by Linda Gremke using a chicken tubulin probe (Don Cleveland). Our clone SQ#TbA77 was cytologically mapped on chromosome I of C. elegans (Alan Coulson and John Sulston, MRC, LMB, UK). As Dr. Tom Blumenthal walked by our poster at the meeting, he pointed out the presence of an eight nucleotide non-coding sequence TTCAGGTT, which corresponds to the acceptor sequence of C. elegans in the trans-splicing reaction of mRNA. This is a rather novel feature for the tubulin gene, as the other alpha or beta tubulin genes, from C. elegans or other metazoans have not been reported to be trans-spliced at the mRNA level. We are currently trying to sequence the mRNA to test if this mRNA is indeed spliced. Figure below shows the physical map of the alpha-1 tubulin gene, and the nucleotide sequence showing the acceptor sequence ( marked by arrowed box), in the non-coding sequence of the tubulin gene. [See Figure 1]

Yasuda H et al. (1990) Worm Breeder's Gazette "An Uniformly distributed Gene-copy cDNA Library"

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1990]

It is worth to obtain many kinds of cDNA molecules, regardless of identified or unidentified genes, in the form of independent clones. One of the useful approach for such a purpose is to construct a cDNA library which represents every gene uniformly, which makes it possible to analyze the clones by random selection. For this purpose,we have applied the genomic DNA hybridization to change the genetic population of cDNA library, for more even representation of the genes. First, we have made a vector-primed cDNA library from C. elegans polyA+ RNA fraction, and converted it to a single stranded form by helper phage infection (Figure). 7mg of the ssDNA was hybridized with 0.14mg of heat denatured C. elegans genomic DNA immobilized on a Nylon membrane filter. After extensive washing of the filter, the remaining ssDNA molecules were eluted with alkali. About 1 g of the ssDNA was recovered. The eluate was neutralized and used directly to transform E. coli host cells, which gave 5x10+E4 transformants. These transformants were found to be almost excluded from the empty clones, namely vector molecule itself with no cDNA, which shared nearly 80% in the original library. we are going to evaluate the new library doing differential hybridization of each clone. [See Figure 1]

Yasuda K et al. (2000) Japanese Worm Meeting "The role of Cyt-1 in mitochondrial function and metabolism in C. elegans"

Ishii N, Yasuda K, Ookubo T, Tsuda M, Senoo-Matsuda N

[Japanese Worm Meeting, 2000]

The mev-1 (kn1) mutant of C. elegans is hypersensitive to oxygen. Recently, we have shown that mev-1 encodes a subunit of the enzyme succinate CoQ oxidoreductase cytochrome b (Cyt-1), which is a component of complex II of the mitochondrial electron transport chain. We have also shown that Cyt-1 plays a key role in the regulation of aging in C. elegans. To further elucidate the effects of the Cyt-1 mutation on oxygen hypersensitivity, we examined mitochondria morphology, the defense system against oxidative stress and the changes in energy metabolism in mev-1 mutants. Electron microscopy revealed that, in young adult, mev-1 mitochondria had morphological abnormalities, particularly at high oxygen concentrations. The level of reduced glutathione (GSH) as a low molecular weight antioxidant was significantly decreased in mev-1 mutants. The function of the complex II is limited to electron transport but remains the conversion from succinate to fumarate in TCA cycle. We found that ATP production was reduced and lactate/pyruvate (L/P) ratio was increased in mev-1 mutants. These results suggest that Cyt-1 has critical roles in the defense system against oxidative stress which is concerned with energy metabolism.

Yasuda K et al. (1997) International C. elegans Meeting "Protein carbonyl with aging of daf mutants"

Yasuda K, Fujiwara Y, Adachi H, Ishii N

[International C. elegans Meeting, 1997]

A dauer-constitutive mutant, daf-2, have been shown to extend adult life span more than two-fold of wild type. The mutation of daf-2 gene enhance resistance to a variety of environmental stresses!@and expression of an antioxidant enzyme, superoxide dismutase. The increased life span is enhanced by a daf-12 mutation and suppressed by a daf-16 mutation. Protein carbonyl has been reported as a specific indicator of oxidative damage to increase with increasing aging. The Protein carbonyl contents in these mutants were measured to know a correlation between life spans and oxidative stress. The life-spans of these mutants were in the order of daf-2;daf-16 < wild type < daf-2 < daf2;daf-12. These protein carbonyl contents were increasing with increasing aging, however, in contrast with this result, these were in the order of daf-2;daf-16 > wild type > daf2 > daf2;daf-12 as a mirror image of these life spans. These results provide that oxidative damage is one of the major causal factors for determination of life-spans in C.elegans.

Yasuda M et al. (1998) Oncogene "Regulation of apoptosis by a Caenorhabditis elegans BNIP3 homolog."

Dsaeipper C, Yasuda M, Gong XL, Chinnadurai G

[Oncogene, 1998]

We have identified a C. elegans protein, ceBNIP3, homologous to the human BCL-2/EIB-19K interacting BCL-2 family pro-apoptotic protein BNIP3. In transiently transfected mammalian cells, ceBNIP3 complexes with CED-9, the worm homolog of BCL-2. CeBNIP3 also efficiently heterodimerizes with the cell death protease proCED-3 by direct binding via the prodomain. Transfection of ceBNIP3 and CED-3 results in enhanced proteolytic processing of the CED-3 zymogen and in cooperative induction of apoptosis. Coexpression of CED-9 suppresses the cooperative cell death induced by ceBNIP3 and CED-3. In cells coexpressing CED-9, ceBNIP3 and CED-3, all three proteins exist as a ternary complex suggesting that CED-9 may suppress cooperative apoptosis induced by CED-3 and ceBNIP3 by simultaneous complex formation with CED-3 and ceBNIP3. Our results suggest that ceBNIP3 may be a novel component of the C. elegans apoptosis paradigm and may initiate apoptosis by recruiting CED-3 to mitochondria and other cytoplasmic membranes.

Kayo Yasuda et al. (2005) International Worm Meeting "Age-related change of mitochondrial structure and function in C.elegans"

Philip Hartman, Kayo Yasuda, Naoaki Ishii, Hitoshi Suda, Takamasa Ishii

[International Worm Meeting, 2005]

Mitochondria are specialized organelles that are intimately responsible for three important processes: i) ATP production, ii) generation of reactive oxygen species (ROS), and iii) regulation of programmed cell death, or apoptosis. In these processes, it is believed that cellular damage caused by ROS leads to aging. Electrons are transferred ultimately to oxygen after their passage through four complexes that are anchored to the mitochondrial inner membrane. It is known that, in addition to its role as a terminal electron acceptor, oxygen can be converted to superoxide (O2 - ) by electron leaked from complexes I and mainly complex III. Such endogenously generated ROS can readily attack a wide variety of cellular entities, resulting in damage that compromises cell integrity and function. To explore the role of mitochondrial function in aging C. elegans, we accessed mitochondrial structure and function by measuring the following: i) the level of carbonylated mitochondrial proteins as a marker of oxidative damage; ii) enzyme activities of complexes I and II; iii) oxygen consumption rate; and iv) mitochondrial morphology as detected using transmission electron microscopy. The levels of carbonyl proteins increased with aging in mitochondria but not the cytoplasm. In contrast, enzyme activities of complex I and II and the rates of oxygen consumption decreased with aging. These results suggest that the highly active metabolic state in the mitochondria of young animals leads to over production ROS, which, owing to their proximity, primarily damage mitochondria rather than other cellular constituents. Morphological abnormalities such as swelling and enlargements were also noted under the electron microscope. These could lead to the mitochondrial fusions that were observed in older animals. It has been hypothesized that mitochondrial fusion is a defense against mitochondrial damage. However, even with this defense system, damaged mitochondria likely accumulate and result in cellular and organismal aging.

Yasuda H et al. (1988) Worm Breeder's Gazette "a tubulin tale from Toyohashi: two genes one polypedtide."

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1988]

Previously we have described our efforts to clone tubulin genes by direct screening of C. elegans expression libraries using nerve specific anti-tubulin antibodies(WBG 10, 1:59, 1987). For alpha- tubulin genes, 14 lambda gt11 clones were identified with a monoclonal antibody 3A5 (kindly given by M. Fuller), which is specific for alpha- tubulin and stains C. elegans neurons. Two of the 14 clones were subcloned in pUC 118, and sequenced by dideoxy method of Sanger and Coulson. One of these clones SQ#TbA77 (insert size 1.0 Kb) contained a DNA which is 79% homologous to the chicken alpha-tubulin DNA sequence; and its amino acid sequence deduced from DNA sequence is 88% homologous to the C-terminal region of the chicken alpha-tubulin. The second putative clone on sequencing revealed DNA that has no homology with tubulin DNA. Similarly, we have identified 15 lambda gt11 clones using E6B6, ( kind gift of T. Arai) a monoclonal antibody which is specific for alpha-tubulin and also stains C. elegans nervous system. Three of the 15 clones were subcloned and sequenced as stated above. One of these SQ#TbB03 has a sequence 76% homologous to the chicken tubulin DNA. We compared the nucleotide sequence of SQ#TbB03 with Nw#B8, the only other -tubulin cloned and sequenced in C. elegans (L. Gremke, Ph.D. Thesis,1987). The two nucleotide sequences and the deduced amino acid sequence is given below. It is quite remarkable that the two -tubulin genes have 100% homology at the level of amino acid sequence. Currently, we are trying to get the complete DNA sequence data of the tubulins identified by SQ#TbA77 and SQ#TbB03. [See Figure 1]

K Yasuda et al. (1999) International C. elegans Meeting "Effects of temperature and oxygen on life span in mev-1"

N Ishii, K Yasuda, P Hartman

[International C. elegans Meeting, 1999]

Mutations in the mev-1 gene confer hypersensitivity to oxygen and methyl viologen (Ishii et al., 1990. Mutat. Res. 237, 165). mev-1 encodes a subunit of the mitochondrial enzyme succinate dehydrogenase (Ishii et al., 1998. Nature 394, 694). In order to determine the effects of temperature on aging in mev-1 animals, life spans were measured at 15C and 25C in wild type and the mev-1 mutant under three different oxygen concentrations (1%, 21% and 90%). As noted previously, oxygen modestly reduced the life spans of mev-1 animals at 1% and 21%; specifically, mev-1 lived about 80% as long as wild type at these two concentrations. Conversely, 90% oxygen reduced mev-1 lifespan over 50% relative to wild type. Temperature was found to modify this relationship in a subtle but interesting fashion. In wild type, effects of oxygen of life span were not particularly influenced by temperature. This can best be visualized by comparing the ratios of the life spans at 16C versus 25C: wild type lived 18% longer when reared at 16C versus 25C under 1% oxygen. It lived 29% and 11% longer under oxygen concentrations of 21% and 90%, respectively. Conversely, temperature substantially modulated the ability of oxygen to reduce life span in mev-1 . Specifically, mev-1 animals lived 38.2% and 47.4% longer at 16C than at 25C when reared at 1% and 21%, respectively. However, the life span was only 4.6% longer at 16C as opposed to 25C when animals were incubated under 90% oxygen. These data can be explained by supposing that, at lower oxygen concentrations, the metabolic increases imposed by growth at 25C elevated celluar levels of reactive oxygen species (ROS). This effect should be particularly acute in mev-1 animals because most ROS are the consequence of respiration and the mev-1 defect in succinate dehydrogenase likely results in overproduction of ROS. Collectively these data add further substance to the notion that ROS impact aging in both mev-1 and wild-type animals.

Yasuda-Kamatani Y et al. (2002) Gen Comp Endocrinol "Cloning and expression of a cDNA for a putative G protein-coupled receptor from the hepatopancreas of the crayfish, Procambarus clarkii."

Yasuda A, Yasuda-Kamatani Y

[Gen Comp Endocrinol, 2002]

The authors cloned a novel cDNA encoding a putative G protein-coupled receptor (GPCR) from the hepatopancreas of the crayfish, Procambarus clarkii, using a screening approach with synthetic oligonucleotides. The oligonucleotides were designed homologous to the transmembrane spanning domain III of previously cloned receptors from various living organisms. Sequence analysis revealed that one of the positive clones contained a cDNA insertion of 3489 bp representing the mRNA coding for a part of a putative GPCR (termed HP1R). The clone was truncated at the 5[prime prime or minute] end. The long 3[prime prime or minute]-untranslated regions (UTR) of 2446 bp contained three typical AATAAA censensus sequences for mRNA polyadenylation followed by the poly(A) tail at the 3[prime prime or minute] end. To obtain a full-length cDNA clone, rapid amplification of the 5[prime prime or minute] cDNA ends (5[prime prime or minute] RACE) technique was then applied. Sequence analysis revealed that the full-length cDNA clone had an open-reading frame of 1116 bp with a 103-bp 5[prime prime or minute]-UTR. The predicted amino acid sequence of HP1R was 372 residues long. Hydropathicity analysis of HP1R suggested the presence of seven transmembrane domains. The database search revealed that the predicted sequence is most closely related to probable GPCR AH9.1 from Caenorhabditis elegans (27% identity, 48% homology). In addition, HP1R has lower homologies with receptors for somatostatin, opioid, dopamine, adrenalin, and so on. The similarity implied that HP1R is a new member of putative GPCRs whose endogenous ligands were unknown. In addition, RT--PCR analysis suggested that the transcript was expressed predominantly in the hepatopancreas but poorly in the muscle or brain. [copyright]2002 Elsevier Science (USA).