Studies on structures and expressions of highly repetitive sequences in the genomes of higher eukaryotes

Total DNAs isolated from several higher animals were transcribed in a HeLa cell extract. Transcription of the total DNAs from rat, mouse and hamster gives a discrete product of 6S RNA (about 180 nucleotides) in addtion to a smeared backgroud, whereas transcription from chick, calf and human DNAs gives a discrete 5S RNA (about 120 nucleotides). In view of the sensitivity to α-amanitin, these transcripts are produced by RNA polymerase III. Fingerprint analyses demonstrated that the 6S RNA species trancribed from rodent DNAs have conserved sequences. We compared the sequences of oligonucleotides of the 6S RNA produced from the mouse total DNA with those of a cloned mouse DNA fragment containing a representative type 2 Alu sequence (B2) which was characterized by Kominami et al. (1983). This result demonstrated that the 6S RNA transcribed from mouse total DNA was in fact the transcript of type 2 Alu sequences. The composition of the oligonucleotides of the 6S RNA transcribed from mouse total DNA seems to reflect precisely that of the consensus sequence of the type 2 Alu family proposed by Krayev et al. (1982). Fingerprints of in vitro transcripts of rat and hamster total DNAs showed nearly identical patterns to that of mouse DNA, confirming that rodents have the repetitive sequence with same length and simlar compositions of oligonucletides. Thus total DNA transcription provides a novel aproach to the detection and analysis of type 2 Alu sequences (Sakamoto et al. 1984).Thesis--University of Tsukuba, D.Sc.(A), no. 335, 1986. 3. 2

Fatty-acid metabolism is involved in stress-resistance mechanisms of Caenorhabditis elegans

Fatty acids are the major components of the phospholipid bilayer and are involved in several functions of cell membrane. We previously reported that fatty-acid metabolism is involved in the regulation of DAF-2/insulin signal in Caenorhabditis elegans. In this study, we investigate the role of fatty-acid metabolism in stress resistance with respect to daf-16 in nematode. We found that fatty-acid metabolism regulates heat, osmotic, and oxidative-stress resistance in C. elegans. RNA interference (RNAi) of fat-6, fat-7, and elo-2 enhanced heat resistance but decreased oxidative-stress tolerance. RNAi of fat-2 strongly increased osmotic-stress resistance, whereas nhr-49-RNAi remarkably reduced osmotic and oxidative-stress tolerance. In daf-16 mutants (mgDf50), RNAi of fat-2 and fat-7 increased viability under osmotic stress, while RNAi of fat-6, fat-7, and elo-2 enhanced heat resistance. Exposure of saturated fatty acids to RNAi worms of fat-1-, fat-7-, and nhr-49 increased osmotic resistance. On the other hand, polyunsaturated fatty acids (PUFAs) reduced osmotic-stress tolerance in fat-2-RNAi worms, whereas PUFAs enhanced it in nhr-49-RNAi worms. Heat-stress resistance in fat-6- and fat-7-RNAi worms was suppressed by oleic acid.These results suggest that stress-resistance mechanisms are regulated by fatty-acid metabolism with or without DAF-16 activity

Killed Bifidobacterium longum enhanced stress tolerance and prolonged life span of Caenorhabditis elegans via DAF-16

Probiotics are bacteria among the intestinal flora that are beneficial for human health. Bifidobacterium longum (BL) is a prototypical probiotic that is widely used in yogurt making, supplements and others. Although various physiological effects of BL have been reported, those associated with longevity and anti-ageing still remain elusive. Here we aimed to elucidate the physiological effects of killed BL (BR-108) on stress tolerance and longevity of Caenorhabditis elegans and their mechanisms. Worms fed killed BL in addition to Escherichia coli (OP50) displayed reduced body length in a BL dose-dependent manner. When compared with those fed E. coli alone, these worms had a higher survival rate following heat stress at 35°C and hydrogen peroxide-induced oxidative stress. A general decrease in motility was observed over time in all worms; however, killed BL-fed ageing worms displayed increased movement and longer life span than those fed E. coli alone. However, the longevity effect was suppressed in sir-2.1, daf-16 and skn-1-deficient worms. Killed BL induced DAF-16 nuclear localisation and increased the expression of the DAF-16 target gene hsp-12.6. These results revealed that the physiological effects of killed BL in C. elegans were mediated by DAF-16 activation. These findings contradict previous observations with different Bifidobacterium and Lactobacillus strains, which showed the role for SKN-1 independently of DAF-16

(−) Epigallocatechin gallate suppresses the differentiation of 3T3-L1 preadipocytes through transcription factors FoxO1 and SREBP1c

Tea catechin is one of the compounds that are closely related to obesity and insulin sensitivity. In order to determine the effect of catechin on adipocyte differentiation, we treated 3T3-L1 preadipocytes with different kinds of catechins. Our results showed that catechins, especially epigallocatechin gallate (EGCG), significantly reduced intracellular lipid accumulation and repressed the activity of glycerol-3-phosphate dehydrogenase, an enzyme involved in lipid synthesis. Furthermore, glucose and fatty acid transport were also suppressed by catechin. We then analyzed the activity of transcription factors—forkhead transcription factor class O1 (FoxO1) and sterol regulatory element-binding protein-1c (SREBP1c)—which are involved in adipocyte differentiation and lipid synthesis, respectively. The transcriptional activities of both these factors significantly decreased by EGCG. Western blot analysis revealed that EGCG induced the insulin signal-mediated phosphorylation of FoxO1 (Thr24, Ser256). These results suggest that EGCG suppresses the differentiation of adipocytes through the inactivation of FoxO1 and SREBP1c

Mouse 3T3-L1 cells acquire resistance against oxidative stress as the adipocytes differentiate via the transcription factor FoxO

Repression of excessive increase and enlargement of adipocytes that is closely associated with obesity is effective in the prevention and treatment of metabolic syndrome. Generally, apoptosis is induced in cells via a wide variety of intracellular or extracellular substances, and recently, it has been suggested that the FoxO subfamily is involved in the induction of apoptosis. We aimed to elucidate the mechanism of FoxO-mediated apoptosis-induction in the adipocytes under the reactive oxygen species (ROS) stimulus. The treatment of differentiated and undifferentiated 3T3-L1 cells with glucose oxidase (GOD), an enzyme that generates H2O2, induced apoptosis and led to the accumulation of 8-OHdG. Apoptosis analysis revealed that GOD treatment induced apoptosis in differentiated 3T3-L1 cells less efficiently than in undifferentiated preadipocytes. GOD remarkably increased the levels of Bad, Bax, and Bim—the genes that are actively involved in cell apoptosis. GOD treatment also increased the expression of FoxO3a mRNA and protein. The introduction of FoxO3a-siRNA into 3T3-L1 cells suppressed the oxidative stress-induced expression of Bim mRNA, as well as the GOD-induced apoptosis. Furthermore, the expression of MnSOD, Cu/ZnSOD, and catalase, as well as of FoxO, increased significantly along with the progression of adipocyte differentiation. These results indicated that ROS-induced apoptosis in undifferentiated 3T3-L1 cells via the expression of FoxO3a, whereas FoxO expression suppressed the ROS-induced apoptosis in differentiated 3T3-L1 cells via the expression of ROS-scavenging enzymes

Linalool odor stimulation improves heat stress tolerance and decreases fat accumulation in nematodes

Aromatherapy uses plant essential oils and fragrant ingredients for relaxation, sleep assistance, and improvement of restlessness related to dementia. Certain aromatic substances increase the life span and stress tolerance of nematodes. We investigated effects of exposure to linalool, a linear chain monoterpenic alcohol that is present in the essential oils of many plants, and its optical isomer, l-linalool, in Caenorhabditis elegans. Nematodes were repelled by the odor of both linalool and l-linalool; however, linalool odor stimulation decreased fat accumulation and increased motility after thermal stress. Analysis of a gene-deficient mutant revealed that the DAF-16 insulin-signaling pathway, which is involved in heat stress tolerance, was enhanced by linalool treatment. Linalool stimulation increased the expression of downstream genes such as sod-3 and hsp-12.6 via DAF-16. We conclude that linalool odor induces a repelling behavior in nematodes, improves heat stress tolerance through the DAF-16 signaling pathway, and affects fat accumulation

Krebs Cycle Intermediates Protective against Oxidative Stress by Modulating the Level of Reactive Oxygen Species in Neuronal HT22 Cells

Krebs cycle intermediates (KCIs) are reported to function as energy substrates in mitochondria and to exert antioxidants effects on the brain. The present study was designed to identify which KCIs are effective neuroprotective compounds against oxidative stress in neuronal cells. Here we found that pyruvate, oxaloacetate, and α-ketoglutarate, but not lactate, citrate, iso-citrate, succinate, fumarate, or malate, protected HT22 cells against hydrogen peroxide-mediated toxicity. These three intermediates reduced the production of hydrogen peroxide-activated reactive oxygen species, measured in terms of 2′,7′-dichlorofluorescein diacetate fluorescence. In contrast, none of the KCIs—used at 1 mM—protected against cell death induced by high concentrations of glutamate—another type of oxidative stress-induced neuronal cell death. Because these protective KCIs did not have any toxic effects (at least up to 10 mM), they have potential use for therapeutic intervention against chronic neurodegenerative diseases

Effects of inorganic mercury and methylmercury on osteoclasts and osteoblasts in the scales of the marine teleost as a model system of bone

To evaluate the effects of inorganic mercury (InHg) and methylmercury (MeHg) on bone metabolism in a marine teleost, the activity of tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) as indicators of such activity in osteoclasts and osteoblasts, respectively, were examined in scales of nibbler fish (Girella punctata). We found several lines of scales with nearly the same TRAP and ALP activity levels. Using these scales, we evaluated the influence of InHg and MeHg. TRAP activity in the scales treated with InHg (10-5 and 10-4 M) and MeHg (10-6 to 10-4 M) during 6 hrs of incubation decreased significantly. In contrast, ALP activity decreased after exposure to InHg (10-5 and 10-4 M) and MeHg (10-6 to 10-4 M) for 18 and 36 hrs, although its activity did not change after 6 hrs of incubation. As in enzyme activity 6 hrs after incubation, mRNA expression of TRAP (osteoclastic marker) decreased significantly with InHg and MeHg treatment, while that of collagen (osteoblastic marker) did not change significantly. At 6 hrs after incubation, the mRNA expression of metallothionein, which is a metal-binding protein in osteoblasts, was significantly increased following treatment with InHg or MeHg, suggesting that it may be involved in the protection of osteoblasts against mercury exposure up to 6 hrs after incubation. To our knowledge, this is the first report of the effects of mercury on osteoclasts and osteoblasts using marine teleost scale as a model system of bone. © 2014 Zoological Society of Japan

Static and dynamic hypergravity responses of osteoblasts and osteoclasts in medaka scales

Fish scales are a form of calcified tissue similar to that found in human bone. In medaka scales, we detected both osteoblasts and osteoclasts and subsequently developed a new scale assay system. Using this system, we analyzed the osteoblastic and osteoclastic responses under 2-, 3-, and 4-gravity (G) loading by both centrifugation and vibration. After loading for 10 min, the scales from centrifugal and vibration loading were incubated for 6 and 24 hrs, respectively, after which the osteoblastic and osteoclastic activities were measured. Osteoblastic activity significantly increased under 2- to 4-G loading by both centrifugation and vibration. In contrast, we found that osteoclastic activity significantly decreased under 2- and 3-G loading in response to both centrifugation and vibration. Under 4-G loading, osteoclastic activity also decreased on centrifugation, but significantly increased under 4-G loading by vibration, concomitant with markedly increased osteoblastic activity. Expression of the receptor activator of the NF-αB ligand (RANKL), an activation factor of osteoclasts expressed in osteoblasts, increased significantly under 4-G loading by vibration but was unchanged by centrifugal loading. A protein sequence similar to osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, was found in medaka using our sequence analysis. The ratio of RANKL/OPG-like mRNAs in the vibration-loaded scales was significantly higher than that in the control scales, although there was no difference between centrifugal loaded scales and the control scales. Accordingly, medaka scales provide a useful model by which to analyze bone metabolism in response to physical strain. © 2013 Zoological Society of Japan

Cloning of two members of the calcitonin-family receptors from stingray, Dasyatis akajei: Possible physiological roles of the calcitonin family in osmoregulation

In cartilaginous fish, two cDNAs encoding calcitonin-family receptors were isolated for the first time from the stingray brain. The open reading frame of one receptor cDNA coded a 525-amino acid protein. The amino acid identity of this receptor to human calcitonin-receptor-like receptor (CRLR) is 64.5%, frog CRLR is 64.7%, and flounder CRLR is 61.2% and this was higher than to human calcitonin receptor (CTR) (46.1%), frog CTR (54.7%), and flounder CTR (48.9%). We strongly suggested that this receptor is a ray CRLR based on phylogenetic analysis. In case of the second receptor, amino acid identity among CRLRs (human 50.5%, frog 50.7%, flounder 48.0%) and CTRs (human 43.2%, frog 49.1%, flounder 41.8%) was similar. From phylogenetic analysis of both CRLRs and CTRs, we believe that this receptor is ray CTR. The expression of ray CRLR mRNA was predominantly detected in the nervous system (brain) and vascular system (atrium, ventricle, and gill), which reflects the similar localization of CGRP in the nervous and vascular systems as mammals. It was observed that the second receptor was expressed in several tissues, namely cartilage, brain, pituitary gland, gill, atrium, ventricle, pancreas, spleen, liver, gall bladder, intestine, rectal gland, kidney, testis and ovary. This localization pattern was very similar to flounder CTR. Both receptor mRNAs were strongly expressed in the gill. This suggests that the calcitonin-family members are involved in the osmoregulation of stingray as this fish is known to be euryhaline. When a stingray was transferred to diluted seawater (20% seawater), the expression of both receptors significantly decreased in the gill. Similar results were obtained in the kidney of the stingray. Thus, our cloning and isolation of both receptors in the stingray will be helpful for elucidation of their physiological role(s) such as osmoregulation including calcium metabolism of cartilaginous fish. © 2012 Elsevier B.V