Biomedical subjects

Shugo Watabe

Publications and source records attributed to Shugo Watabe.

At least 19 recordsLinked to original sources

PubMed2007

Fast skeletal muscle myosin heavy chain gene cluster of medaka Oryzias latipes enrolled in temperature adaptation.

To disclose mechanisms involved in temperature acclimation of fish muscle, we subjected eurythermal fish of medaka Oryzias latipes to cloning of myosin heavy chain genes (MYHs). We cloned cDNAs encoding fast skeletal muscle myosin heavy chain (MYH) isoforms from cDNA libraries of medaka acclimated to 10 and 30 degrees C and observed that different MYH cDNA clones are expressed in the two temperature-acclimated fish. Subsequently, we isolated several overlapping MYH contigs by shotgun cloning strategy from a medaka genomic library. Contig assembly of the complete medaka MYH (mMYH) locus of 219 kbp revealed a cluster of tandemly arrayed 11 mMYHs, in which eight genes are actually transcribed, with the remaining three being pseudogenes. Expression analysis of the transcribed genes revealed that two genes were each highly expressed in medaka acclimated to 10 and 30 degrees C, whereas comparatively lower expression levels of the three genes were exclusively observed in medaka acclimated to 30 degrees C. cDNAs of the remaining genes were too underrepresented in the libraries to determine the expression levels, and the transcripts could only be obtained by reverse transcription-polymerase chain reaction. Deduced amino acid sequences in the loop 1 and loop 2 regions of mMYHs were highly variable, suggesting that these isoforms were functionally different. The present findings consolidate our knowledge on teleost MYH multigene family and would provide further insight into the mechanisms by which expressions of individual MYH molecules are fine-tuned with environmental temperature fluctuations with further functional analysis of the genes concerned.

Adaptation, Physiological↗

PubMed2006

Temperature-dependent growth rates and gene expression patterns of various medaka Oryzias latipes cell lines derived from different populations.

Medaka Oryzias latipes has several geographically and genetically distinct populations. We examined temperature acclimation response in various medaka cell lines derived from different populations. Measurement of cell growth at various temperatures suggested that 15 degrees Celsius was the permissive growth temperature in all cell lines from the Northern Japanese and East Korean populations, but not in those from the Southern Japanese population and medaka-related species Oryzias celebensis, which inhabits a tropical zone. RT-PCR for 102 temperature-responsive genes, previously reported in other species, revealed that the accumulated mRNA level of a gene encoding HSP47 was lower at 25 degrees Celsius than at 33 degrees Celsius, and vice versa for 12 genes including IkappaBalpha and Rab-1c, in OLHNI-1 cell line from the Northern Japanese population. Further analysis by real-time PCR demonstrated that the accumulated mRNA levels of IkappaBalpha and Rab-1c in OLHNI-1 and OLSOK-e7 cell lines from the East Korean population were increased when the culture temperature was shifted from 33 to 15 degrees Celsius, but not in OLHdrR-e3 cell line from the Southern Japanese population. Since IkappaBalpha and Rab-1c are related to the NFkappaB cascade and endoplasmic reticulum-to-Golgi transport, respectively, it is inferred that immune responses and intracellular transport are possibly critical to temperature adaptation for medaka.

Animals↗

PubMed2006

Increased levels of mitochondrial gene transcripts in the thermally selected rainbow trout (Oncorhynchus mykiss) strain during embryonic development.

To investigate molecular mechanisms involved in thermal resistance of rainbow trout, Oncorhynchus mykiss, embryos from thermally selected strain in various developmental stages were treated at 22 degrees C for 30 min and subsequently developed at 12 degrees C using the Donaldson strain as a reference. The embryos were evaluated for their hatching rate along with the ratio of embryos having an abnormal appearance and subjected to mRNA arbitrarily primed reverse transcription-polymerase chain reaction (RAP RT-PCR). One of the genes dominantly expressed in the thermally selected strain (COX II) coded for cytochrome c oxidase subunit II. Northern blot analysis revealed that the accumulated levels of COX II transcripts were more abundant in embryos and unfertilized eggs from the thermally selected strain than those from the Donaldson strain. Furthermore, the differential expression patterns of the ATPase 6-8 gene were similar to those of the COX II gene, whereas the ATP synthase beta-subunit gene showed no significant differences between the two strains.

Adenosine Triphosphatases↗

PubMed2006

Functional analysis on the 5'-flanking region of carp fast skeletal myosin heavy chain genes for their expression at different temperatures.

Two types of the fast skeletal myosin heavy chain (MYH) genes were cloned from a genomic DNA library of carp (Cyprinus carpio L.) and named MYH10 and MYH30, which showed the sequence similarity to the MYH cDNAs predominantly expressed in carp acclimated to 10 and 30 degrees C, respectively. The 5'-flanking region of about 3 kbp in size each from MYH10 and MYH30 contained various cis-elements to bind to transcriptional regulatory factors such as MyoD family and myocyte enhancer factor 2 (MEF2) family members. To localize functional regions responsible for the MYH gene expression in a temperature-dependent manner, a series of deletion constructs were prepared from the 5'-flanking region, inserted upstream the luciferase gene in a commercially available plasmid, and injected into the dorsal fast muscle of carp acclimated to 10 and 30 degrees C. The sequence of -1004 to -995 bp with the transcriptional activity in MYH30 was identified as an MEF2 binding site. While the activity given by a sequence of -921 to -824 bp in MYH10 contained only a GATA box, that of the activity of the -1 kbp construct from MYH10 was markedly higher in carp reared at 10 degrees C than fish reared at 30 degrees C. On the other hand, no temperature-dependent expressional regulation was observed for MYH30 even with the full-length construct of -3 kbp. The DNA fragment of -921 to -824 bp in MYH10 and MEF2 binding site in MYH30 interacted with nuclear proteins extracted from carp fast skeletal muscle as revealed by electrophoretic mobility shift assay. The signal intensity of a complex formed between the DNA fragment of MYH10 and nuclear extracts from the 10 degrees C-acclimated carp were higher than those with extracts from the 30 degrees C-acclimated fish. Although MEF2-binding site in MYH30 could form complex with nuclear extracts from the 30 degrees C-acclimated carp, the same or stronger signals were detected in complex formed with extracts from the 10 degrees C-acclimated fish.

5' Flanking Region↗

PubMed2006

cDNA cloning of myosin heavy chain genes from medaka Oryzias latipes embryos and larvae and their expression patterns during development.

Several sarcomeric myosin heavy chains (MYHs) were cloned from embryos and larvae of medaka Oryzias latipes. Three genes encoding medaka MYHs (mMYHs) predominantly expressed in embryos (mMYH(emb1)) and larvae (mMYH(L1) and mMYH(L2)), all belonged to fast skeletal MYHs, showing spatiotemporally different expression patterns during development. Besides these mMYHs, a few novel mMYHs were cloned from embryos and larvae at hatching. Whereas mMYH(emb2), mMYH(emb3), and mMYH(L3) belonged to fast skeletal MYH, mMYH(C1) and mMYH(C2) did to slow/cardiac MYH. mMYH(emb1) was expressed ahead of mMYH(L1) and mMYH(L2). In situ hybridization analysis demonstrated that the transcripts of mMYH(emb1) and mMYH(C1) were located in the horizontal myoseptum, whereas those of mMYH(L1) and mMYH(L2) in the inner part of myotomes and pharyngeal muscles, and those of mMYH(C2) in the heart rudiment. In silico cloning based on the medaka genome database showed another mMYHs of the slow/cardiac types, mMYH(C3) and mMYH(C4).

Animals↗

PubMed2006

Characterization of cDNA clones encoding two distinct cathepsins with restricted expression pattern in a marine pelagic fish.

Cathepsin L (EC 3.4.22.15) from aquatic animals are quite stable and active at neutral or alkaline pH values while their mammalian equivalents work at an acidic environment of the lysosomes. To understand the molecular properties at the gene level we employed a PCR-based strategy using degenerate oligonucleotide primers to isolate cathepsin L-like genes from anchovy Engraulis japonicus. As a result, we obtained two closely related genes encoding cathepsins (aCat1 and aCat2) similar to both cathepsins L and S from other organisms. The predicted precursor protein of 324 amino acid residues for genes differed in six residues and contained conserved residues characteristic of cathepsin L-like cysteine proteases. Phylogenetic analyses failed to produce any precise relationships of aCat1 and aCat2 with other cysteine proteases. However, with a bootstrap value less than 50, these two fish cathepsins formed a separate group to that bearing cathepsins L and S of various organisms. Interestingly, unlike mammalian cathepsin L transcripts of aCat1 and aCat2 were almost exclusively detected in the stomach suggesting that the fish homologues are non-lysosomal secretory enzymes present in the extracellular acidic environment of the stomach and that marine teleosts developed digestive cysteine proteases as a result of evolutionary pressure in response to varying dietary conditions.

Amino Acid Sequence↗

PubMed2006

Molecular cloning and mRNA expression analysis of carp embryonic, slow and cardiac myosin heavy chain isoforms.

Three embryonic class II myosin heavy chains (MYHs) were cloned from the common carp (Cyprinus carpio L.), MYHemb1, MYHemb2 and MYHemb3. MYH DNA clones were also isolated from the slow muscle of adult carp acclimated to 10 degrees C (MYHS10) and 30 degrees C (MYHS30). Phylogenetic analysis demonstrated that MYHemb1 and MYHemb2 belonged to the fast skeletal muscle MYH clade. By contrast, the sequence of MYHemb3 was similar to the adult slow muscle isoforms, MYHS10 and MYHS30. MYHemb1 and MYHemb2 transcripts were first detected by northern blot analysis in embryos 61 h post-fertilization (h.p.f.) at the heartbeat stage, with peak expression occurring in 1-month-old juveniles. MYHemb1 continued to be expressed at low levels in 7-month-old juveniles when MYHemb2 was not detectable. MYHemb3 transcripts appeared at almost the same stage as MYHemb1 transcripts did (61 h.p.f.), and these genes showed a similar pattern of expression. Whole mount in situ hybridization analysis revealed that the transcripts of MYHemb1 and MYHemb2 were expressed in the inner part of myotome, whereas MYHemb3 was expressed in the superficial compartment. MYHS10 and MYHS30 mRNAs were first detected at hatching. In adult stages, the expression of slow muscle MYH mRNAs was dependent on acclimation temperature. MYHS10 mRNA was expressed at an acclimation temperature of 10 and 20 degrees C, but not at 30 degrees C. In contrast, MYHS30 mRNA was strongly expressed at all acclimation temperatures. The predominant MYH transcripts found in adult slow muscle and in embryos at hatching were expressed in adult fast muscle at some acclimation temperatures but not others. A MYH DNA clone was isolated from the cardiac muscle of 10 degrees C-acclimated adult fish (MYHcard). MYHcard mRNA was first detected at 61 h.p.f., but strong signals were only observed in the adult myocardium. The present study has therefore revealed a complex pattern of expression of MYH genes in relation to developmental stage, muscle type and acclimation temperature. None of the skeletal muscle MYHs identified so far was strongly expressed during the late juvenile stage, indicating further developmentally regulated members of the MYH II gene family remain to be discovered.

Amino Acid Sequence↗

PubMed2006

Production of low-estrogen goldfish diet for in vivo endocrine disrupter test.

A low-estrogenic diet for goldfish Carassius auatus was produced for an in vivo estrogen activity test, because commercial fish feed has estrogenic activity and may affect the results of estrogen assays. The newly produced diet (FD5) was formulated with defatted rice bran and casein, and did not contain any soybean meal or fish meal. Phytoestrogen contents (genistein, daidzein, equol, and coumestrol) of FD5 were measured by liquid chromatography-mass spectroscopy/mass spectroscopy (LC-MS/MS) and compared with those of the commercial trout diet (TD) and carp diet (CD). The genistein, daidzein, and coumestrol contents of TD and CD were much higher (5-2000 times) than those of FD5, but equol was detected only in FD5. Estrogenic activity of the fish diets was estimated in vitro by the yeast estrogen-screen assay (YES assay). The estrogenic activity was detected in TD and CD, but not in FD5. The in vivo estrogenic activity of the diets was examined by determining the production of vitellogenin in male goldfish. When male goldfish were fed TD or CD, plasma vitellogenin levels increased, but fish that were fed FD5 maintained low vitellogenin levels. These results indicate that FD5 produced in the present study has a low estrogenic activity, and FD5 would be suitable for the in vivo estrogen activity test using goldfish.

Animals↗

PubMed2005

Rheological properties of fast skeletal myosin rod and light meromyosin from walleye pollack and white croaker: contribution of myosin fragments to thermal gel formation.

Myosin rod and light meromyosin (LMM) of walleye pollack and white croaker were examined for their rheological properties by measuring dynamic viscoelastic parameters. Rods from walleye pollack and white croaker increased their storage moduli (G') in the ranges of 29-43 degrees C and 31-38 degrees C, respectively, in temperature sweep analysis. Walleye pollack LMM showed no peak of G' upon heating, whereas the white croaker counterpart exhibited a single sharp peak of G' at 35 degrees C. Loss modulus (G") showed similar temperature-dependent changes for the two fish species as the case of G', irrespective of rod and LMM, although G" values were lower than those of G'. Thus, rheological properties of rod and LMM were different between walleye pollack and white croaker. Taken together with data previously reported for myosin, it was considered that both myosin rods from walleye pollack and white croaker are attributed to thermal gel formation of myosin in a low-temperature range, though in a species-specific manner.

Animals↗

PubMed2005

A genomic approach to reveal novel genes associated with myotube formation in the model teleost, Takifugu rubripes.

Little is known about the transcriptional networks that regulate myotube production in vertebrates. In the present study, we have used a genomic approach to discover novel genes associated with myotube formation in fast muscle of the tiger puffer fish, Takifugu rubripes. The number of fast muscle fibers per myotome increased until 1.2 kg body mass, and subsequent growth was by fiber hypertrophy alone. Forward and reverse subtracted cDNA libraries were prepared from a 180-g (myotube +) and a 3.4-kg (myotube -) fish, and 1,452 expressed sequence tags (ESTs) were obtained. After these ESTs were grouped into nonredundant clusters and housekeeping and structural genes were eliminated, 57 genes were selected and quantitative PCR was used to investigate their expression levels in different tissues from independent groups of myotube(-) and myotube(+) fish acclimated to the same environmental conditions and diet. Eleven novel genes were found to be consistently differentially expressed, but only four showed appropriate tissue-specific expression. These four genes were upregulated 5-25 times in fast muscle of myotube(-) relative to myotube(+) growth stages, while their expression remained unchanged in the other tissues studied. The novel genes identified, which are also present in other vertebrate genomes, may play a role in inhibiting myotube formation in vertebrate muscle.

Animals↗

PubMed2005

The complete genomic sequence of the carp fast skeletal myosin heavy chain gene.

We have determined the complete DNA nucleotide sequence of the carp Cyprinus carpio fast skeletal myosin heavy chain (MYH) gene. Introns and exons were predicted by comparison with the corresponding carp MYH cDNAs previously reported. The gene encoded the entire mRNA transcript and contained 5958 nucleotides (nt) including 77 nt 5'-untranslated region, 5796 nt coding region for 1931 amino acids, and 85 nt 3'-untranslated region. The coding region was split by 38 introns and the complete gene contained 11,385 nt. This integration of the carp fast skeletal MYH gene was comparable to those of the rat and chicken embryonic MYH genes, which have 41 and 40 exons, respectively. However, the entire gene size of carp MYH was about half those of rat and chicken due to much smaller size of carp introns. We have also demonstrated that this carp MYH gene belonged to so-called intermediate type in a multigene family of carp fast skeletal muscle MYH in comparison of its nucleotide and deduced amino acid sequences to those of carp MYH cDNAs reported previously.

3' Untranslated Regions↗

PubMed2005

Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells.

We isolated multiple HSPs from rainbow trout Oncorhynchus mykiss RTG-2 cells and quantitatively compared their mRNA levels between unstressed and heat-shocked cells using real-time RT-PCR analysis. Consequently, we isolated nine cDNAs encoding HSPs from heat-shocked RTG-2 cells, namely, Hsp90betaa, Hsp90betab, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. Quantitative RT-PCR analyses, in which Hsp70b isolated previously was included, showed that the mRNA accumulation levels of Hsp70a, Hsp70b, Hsc70a, Hsc70b, and Hsp47 were significantly increased after heat shock, and the increased levels of two Hsp70s, Hsp70a, and Hsp70b, were most conspicuous. In the case of Hsc70s, the increased level of Hsc70b was more remarkable than that of Hsc70a. These results demonstrate the importance of a comprehensive expression analysis of HSPs for better understanding of the cellular stress response in fish, especially in tetraploid species such as rainbow trout.

Animals↗

PubMed2005

Characterization of the pufferfish Takifugu rubripes apolipoprotein multigene family.

We have characterized the apolipoprotein multigene family of the pufferfish Takifugu rubripes. The pufferfish mainly contains 28-kDa, 27-kDa, and 14-kDa apolipoproteins in its plasma and was designated apo-28 kDa, apo-27 kDa, and apo-14 kDa, respectively. N-terminal amino acid sequencing revealed that pufferfish apo-28 kDa and apo-27 kDa have an identical amino acid sequence except an additional propeptide in the former; and both are homologues of apoA-I from other animals. The sequence of pufferfish apo-14 kDa is homologous to that of eel apo-14 kDa previously reported, both being apparently specific to fish. In silico screening, using the publicly available Fugu genome database confirmed the pufferfish apoA-I and apo-14 kDa genes. The database further contained the genes encoding four types of apoA-IV, one apoC-II and two types of apoE. Thus, pufferfish contains nine genes encoding apolipoprotein multigene family. Two apoA-IV and one apoE genes were tandemly arrayed and located on one scaffold. Thus two sets of these genes formed two gene clusters. The apoC-II and apo-14 kDa genes are also located on a single scaffold. apoA-I and apo-14 kDa gene transcripts were mainly expressed in liver and less abundantly in brain. The transcripts of the former gene were also observed in intestine. In contrast, the transcripts encoding four apoA-IVs, one apoC-II, and two apoEs were mainly expressed in intestine. These structural details of pufferfish apolipoproteins and tissue distribution of their gene transcripts provide a novel evidence for better understanding of evolutionary relationships of apolipoprotein multigene family.

Amino Acid Sequence↗

PubMed2005

Twitchin as a regulator of catch contraction in molluscan smooth muscle.

Molluscan catch muscle can maintain tension for a long time with little energy consumption. This unique phenomenon is regulated by phosphorylation and dephosphorylation of twitchin, a member of the titin/connectin family. The catch state is induced by a decrease of intracellular Ca2+ after the active contraction and is terminated by the phosphorylation of twitchin by the cAMP-dependent protein kinase (PKA). Twitchin, from the well-known catch muscle, the anterior byssus retractor muscle (ABRM) of the mollusc Mytilus, incorporates three phosphates into two major sites D1 and D2, and some minor sites. Dephosphorylation is required for re-entering the catch state. Myosin, actin and twitchin are essential players in the mechanism responsible for catch during which force is maintained while myosin cross-bridge cycling is very slow. Dephosphorylation of twitchin allows it to bind to F-actin, whereas phosphorylation decreases the affinity of the two proteins. Twitchin has been also been shown to be a thick filament-binding protein. These findings raise the possibility that twitchin regulates the myosin cross-bridge cycle and force output by interacting with both actin and myosin resulting in a structure that connects thick and thin filaments in a phosphorylation-dependent manner.

Animals↗

PubMed2005

Identification of three isoforms for mitochondrial adenine nucleotide translocator in the pufferfish Takifugu rubripes.

Three adenine nucleotide translocator (ANT) genes were identified through in silico data mining of the Fugu genome database along with isolation of their corresponding cDNAs in vivo from the pufferfish (Takifugu rubripes). As a result of phylogenetic analysis, the ANT gene on scaffold_254 corresponded to mammalian ANT1, whereas both of those on scaffold_6 and scaffold_598 to mammalian ANT3. The ANT gene encoded by scaffold_6 was expressed ubiquitously in various tissues, whereas the ANT genes encoded by scaffold_254 and scaffold_598 were predominantly expressed in skeletal muscle and heart, respectively.

Amino Acid Sequence↗

PubMed2005

Accumulation and depuration rates of paralytic shellfish poisoning toxins in the shore crab Telmessus acutidens by feeding toxic mussels under laboratory controlled conditions.

Accumulation and depuration rates of paralytic shellfish poisoning toxins (PSP) in the crab Telmessus acutidens were investigated by feeding toxic and non-toxic mussels under laboratory controlled conditions. The crab accumulated toxins in the hepatopancreas in proportion to the amount of toxic mussels they ingested, and the toxicity in the crab hepatopancreas became 3.2 fold of that in the prey mussels after 20 days of feeding. During depuration, a fast reduction of the total toxicity was observed in the crab, and the retention rate of the toxicity after 5 days depuration with feeding of non-toxic mussels was 45.8+/-18.7%. The reduction of the toxicity was moderated in the later period of depuration, and the retention rates of the total toxicity after 10 and 20 days were 54.1+/-29.8% and 14.5+/-9.0%, respectively. The toxin profiles in the crab and mussel were investigated by high performance liquid chromatography, and reductive conversions of the toxins were observed when the toxins were transferred from the mussel to the crab. Consequently, high concentrations of GTX2 and GTX3, and STX that were not detected in the prey mussels, were found in the crab.

Animals↗

PubMed2005

Genomic sequences encoding two types of medaka hemopexin-like protein Wap65, and their gene expression profiles in embryos.

Medaka genomic BAC clones, which contained two types of medaka hemopexin-like protein gene (Wap65), mWap65-1 and mWap65-2, were screened and their genomic sequences were determined by the shotgun strategy. The exon-intron organizations were highly conserved between both mWap65s and human hemopexin genes. The 5'-flanking regions of mWap65-1 and mWap65-2 contained various putative transcription factor binding sites including elements for developmental regulation. The expression patterns of mWap65s during embryonic development were examined by quantitative real-time PCR, demonstrating that both mWap65 transcripts were observed in early embryonic stages, but their expression patterns were different. Interestingly, in situ hybridization revealed that mWap65-2 transcripts were restricted to liver, whereas mWap65-1 transcripts were detected along the edge of pectoral fin buds and the median fin fold of tail buds in embryos at stage 32. Furthermore, we generated transgenic medaka expressing GFP driven by mWap65-1 and mWap65-2 promoters and observed GFP expression patterns during ontogeny. Although localizations of GFP varied among individuals, embryos uniformly expressed GFP 1 day after injection of mWap65-1-hrGFP and mWap65-2-hrGFP constructs, suggesting that mWap65-1 and mWap65-2 promoters were activated in very early stages. The differences between mWap65-1 and mWap65-2 in their expression profiles indicate their distinct roles during ontogeny.

Animals↗

PubMed2005

Overexpression in Escherichia coli and functional reconstitution of anchovy trypsinogen from the bacterial inclusion body.

We have synthesized and optimized a high-yielding Escherichia coli expression system to produce trypsinogen from anchovy Engraulis japonicus and have developed conditions for its successful refolding. Recombinant anchovy trypsinogen precipitated in E. coli Rosetta (DE3) pLacI strain as inclusion bodies was denatured by 6 M guanidine-HCl followed by refolding with drop wise addition to a large excess of a folding buffer containing 0.5 M non-detergent sulfobetaine (NDSB-251) and a redox potential of oxidized and reduced glutathiones. The folded trypsinogen was autocatalytically activated to its mature form, trypsin, and purified with a MonoQ ion-exchange column. NH2-terminal amino acid sequencings revealed that E. coli efficiently processed NH2-terminal methionine residue from the expressed trypsinogen and that trypsinogen was activated at the correct site to generate active trypsin. The recombinant enzyme showed kinetic properties comparable to those of the native enzyme and demonstrated a typical cleavage preference for arginine over lysine residue against a protein substrate. The optimized expression and folding procedures yielded 12 mg of purified, active trypsin from 1 L of bacterial culture or 45 g wet weight cells, which is quite enough for various analytical and semipreparative purposes.

Amino Acid Sequence↗