Biomedical subjects

Shugo Watabe

Publications and source records attributed to Shugo Watabe.

42 records · Page 3Linked to original sources

PubMed2003

Identification of novel tropomyosin 1 genes of pufferfish (Fugu rubripes) on genomic sequences and tissue distribution of their transcripts.

Fugu genome database enabled us to identify two novel tropomyosin 1 (TPM1) genes through in silico data mining and isolation of their corresponding cDNAs in vivo. The duplicate TPM1 genes in Japanese pufferfish Fugu rubripes suggest that additional an ancient segmental duplication or whole genome duplication occurred in fish lineage, which, like many other reported Fugu genes, showed reduction in genomic size in comparison with their human homologue. Computer analysis predicted that the coiled-coil probabilities, that were thought to be the most major function of TPM, were the same between the two TPM1 isoforms. We confirmed that the tissue expression profiles of the two TPM1 genes differed from each other, which implied that changes in expression pattern could fix duplicated TPM1 genes although the two TPM1 isoforms appear to have similar function.

Animals↗

PubMed2003

Molecular cloning and functional characterization of crustapain: a distinct cysteine proteinase with unique substrate specificity from northern shrimp Pandalus borealis.

A cDNA clone encoding a cysteine proteinase of the papain superfamily has been isolated from the hepatopancreas of northern shrimp Pandalus borealis (NsCys). NsCys shares the highest identity of 64% with a cathepsin L-like cysteine proteinase from lobster, and its identity to the well-characterized mammalian cathepsins S, L, and K falls within a narrow range of 54-59%. However, it differs from each of these cathepsins in certain key residues including, for example, the unique occurrence of tryptophan and cysteine residues at the structurally important S2 subsite. Consequently, NsCys produced in Pichia pastoris appears to be distinct in various physicokinetic properties. The recombinant enzyme is active and stable over a wide range of pH values, and its substrate specificity is unusual, as demonstrated by its poor affinity for phenylalanine residues. Instead, it shows the highest specificity for proline residues, a property similar to cathepsin K. Unlike cathepsin K, however, NsCys cleaves valine residues more efficiently than leucine. Similar results were obtained with the natural peptide substrate glucagon. The shrimp proteinase is further distinguished by its potent collagenolytic activity, resulting in a cleavage pattern reminiscent of bacterial collagenase. To distinguish such unique structural and enzymatic properties, we propose the trivial name "crustapain" for the shrimp proteinase, indicating that it is a papain-like cysteine proteinase from a crustacean species.

Amino Acid Sequence↗

PubMed2002

Changes of carp FoF1-ATPase in association with temperature acclimation.

Previously we have shown, using two-dimensional electrophoresis, that mitochondrial ATP synthase (F(o)F(1)-ATPase) beta-subunit is the 55-kDa protein increased in cold-acclimated carp Cyprinus carpio (Kikuchi K, Itoi S, and Watabe S. Fisheries Sci 65: 629-636, 1999). To clarify the coordinate expression in various subunits of carp F(o)F(1)-ATPase with temperature acclimation, we examined the differences in mRNA levels of mitochondrial proteins encoded by both nuclear and mitochondrial genes in fast muscle of carp acclimated to 10 and 30 degrees C. The mRNA levels of nuclear genes per unit weight of total RNA were nearly twofold higher in the 10 degrees C- than 30 degrees C-acclimated carp. However, the transcripts of mitochondrial genes for the 10 degrees C-acclimated carp in terms of the same comparing unit were six to seven times as much as those for the 30 degrees C-acclimated carp. The F(o)F(1)-ATPase activities measured at 10, 25, and 30 degrees C were nearly twofold higher for the cold-acclimated fish than their warm-acclimated counterparts. Such quantitative and qualitative changes in carp F(o)F(1)-ATPase may contribute to extra ATP production required to compensate for energy balance at suboptimal temperatures.

Acclimatization↗

PubMed2002

Differential scanning calorimetry and circular dichroism spectrometry of walleye pollack myosin and light meromyosin.

The thermodynamic properties of myosin and its C-terminal fragment, light meromyosin (LMM), from walleye pollack, a typical cold-water fish efficiently utilized on an industrial scale, were analyzed by using differential scanning calorimetry (DSC) and circular dichroism (CD) spectrometry. Recombinant walleye pollack LMM expressed in Escherichia coli was also subjected to DSC and CD measurements for reference. The two proteins prepared from frozen surimi showed three endothermic peaks, the transition temperatures (T(m)) of which were quite similar, although overall DSC patterns differed considerably from one another. Their alpha-helical contents determined by CD were low compared to values reported before for other species. On the other hand, recombinant LMM gave four endothermic peaks at 27.4, 30.8, 36.5, and 43.4 degrees C in DSC and showed an alpha-helical content of approximately 80%. The peak at 27.4 degrees C could not be observed in walleye pollack LMM prepared from frozen surimi and thus was possibly attributed to its C terminus, because this extreme C-terminal region is supposedly truncated during preparation of LMM by tryptic digestion.

Amino Acid Sequence↗

PubMed2002

Accumulation of H+ in vacuoles induced by a marine peptide toxin, theonellamide F, in rat embryonic 3Y1 fibroblasts.

The effects of theonellamide F, a marine bicyclic peptide, on vacuolar formation in cultured cells were studied. Theonellamide F induced large vacuoles in 6 types of mammalian cells. The vacuoles induced by theonellamide F in 3Y1 cells accumulated acridine orange, a fluorescent probe indicating the presence of an acidic organelle. Their disappearance following treatment with bafilomycin A1 suggests that these vacuoles contain vacuolar ATPase to maintain an acidic internal milieu, and this is similar to those induced by Helicobacter pylori toxin VacA. The vacuoles induced by theonellamide F were not significantly decreased in size or number by nocodazole treatment, and the localization of a small GTPase, rab7, did not always correspond to the outline of the vacuoles. These results suggest that the molecular mode of action of vacuolar formation by theonellamides may differ from that by VacA and can be considered unique.

Journal Article↗

PubMed2002

Temperature plasticity of contractile proteins in fish muscle.

Three myosin heavy chain isoforms with different actin-activated Mg(2+)-ATPase activities were found in the fast skeletal muscle from carp (Cyprinus carpio) acclimated to 10 and 30 degrees C. The composition of three types of myosin heavy chain was dependent on acclimation temperature, demonstrating the presence of temperature-specific myosin isoforms in carp. Subsequently, the temperature-dependence of the sliding velocity of fluorescent F-actin in myosins isolated from 10 degrees C- and 30 degrees C-acclimated carp was measured. At 8 degrees C, the filament velocity was three times higher for myosin from 10 degrees C- than from 30 degrees C-acclimated fish. Activation energies (E(a)) for the sliding velocity of F-actin were 63 and 111 kJ mol(-1) for myosins from 10 degrees C- and 30 degrees C-acclimated fish, respectively. Activation energy for actin-activated Mg(2+)-ATPase activity was 0.46 kJ mol(-1) in myosin from 10 degrees C-acclimated fish and 0.54 kJ mol(-1) in myosin from 30 degrees C-acclimated fish. The inactivation rate constant (K(D)) of Ca(2+)-ATPase was 7.5x10(-4)s(-1) at 30 degrees C for myosin from 10 degrees C-acclimated fish, which was approximately twice that for myosin from 30 degrees C-acclimated fish. It is suggested that these differences in thermostability reflect a more flexible structure of the myosin molecule in cold-acclimated carp, which results in a reduced activation enthalpy for contraction and, hence, a higher sliding velocity at low temperatures. Structural analysis of cDNAs encoding the carp myosin heavy chain demonstrated striking differences in two surface loops of myosin subfragment-1 (S1), loops 1 and 2, between the 10 degrees C and 30 degrees C types, which were predominantly expressed in carp acclimated to 10 degrees C and 30 degrees C, respectively. Chimeric myosins composed of Dictyostelium discoideum myosin backbones with loop sequences of carp S1 heavy chain isoforms demonstrated that the diversity of the loop 2 sequence of carp S1 affected the V(max) of actin-activated Mg(2+)-ATPase activity.

Animals↗