Biomedical subjects

Hideki Ohtake

Publications and source records attributed to Hideki Ohtake.

9 recordsLinked to original sources

PubMed2006

Effects of hyperbaric oxygen on gene expressions of procollagen, matrix metalloproteinase and tissue inhibitor of metalloproteinase in injured medial collateral ligament and anterior cruciate ligament.

Animal experiments were performed to investigate whether and how the administration of hyperbaric oxygen (HBO) affects gene expressions of procollagens, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in injured medial collateral ligament (MCL) and anterior cruciate ligament (ACL). In 64 Sprague-Dawley rats, the MCL of the left knee was lacerated at the midsubstance, and the ACL of the left knee was lacerated adjacent to the tibial insertion in another 64 rats. Of these, 32 rats with lacerated MCL and 32 rats with lacerated ACL were housed in individual cages at normal atmospheric pressure (Groups MC and AC, respectively), while the remaining 64 rats were exposed to 100% oxygen at 2.5 atmospheres absolute for 2 h for 5 days a week (Groups MH and AH, respectively). Rats were sacrificed at 3, 7, 14 and 28 days postoperatively. After macroscopic examination, bilateral MCLs were harvested from Groups MC and MH, and bilateral ACLs from Groups AC and AH. Total RNA was extracted from each specimen and gene expressions of type I and type III procollagens, MMP-2, -9 and -3, and TIMP-1 and -2 were estimated using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Macroscopically, lacerated MCL healed by scar tissue formation, the amount of which appeared to be greater in Group MH than in Group MC. In contrast, no lacerated ACLs united, and little, if any, differences were apparent in macroscopic findings between Groups AH and AC. Gene expression of type I procollagen was significantly greater in Group MH than in Group MC at 7 days postoperatively and was also significantly greater in Group AH than in Group AC at 28 days (P<0.05). No significant differences in type III procollagen gene expression were noted between Groups MH and MC or between Groups AH and AC. In addition, no significant differences in gene expressions of MMPs were seen in either ligament, except that gene expression of MMP-13 was significantly lower at 7 days in Group MH than in Group MC (P<0.05). Gene expressions of TIMPs did not differ significantly between Groups MH and MC in each time interval, whereas gene expressions of TIMPs were significantly greater in Group AH than in Group AC at 7, 14 and 28 days for TIMP-1 and at 3, 7 and 14 days for TIMP-2 (P<0.05). RT-PCR results suggested that HBO enhances structural protein synthesis and inhibits degradative processes by enhancing TIMP activities in the lacerated ACL. However, none of the lacerated ACLs united macroscopically despite administration of HBO, indicating that the effect of HBO is insufficient for healing of the injured ACL. If HBO therapy is used as an adjunctive therapy after primary repair of the injured ACL, the success rate of surgery seems likely to be increased.

Animals↗

PubMed2005

Vector-capping: a simple method for preparing a high-quality full-length cDNA library.

Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains neither a PCR process generating mutations nor restriction enzyme treatment causing truncation of cDNA, (iii) the intactness of cDNA is assured due to the presence of an additional dGMP at its 5' end, (iv) approximately 95% of cDNA clones are full-length when cultured cells or fresh tissues are used, (v) several micrograms of total RNA without mRNA purification is sufficient for preparation of a library containing >10(5) independent clones, and (vi) a long-sized full-length cDNA up to 9.5 kbp can be cloned. This method will accelerate comprehensive gene analysis in a variety of eukaryotes.

Animals↗

PubMed2004

Determination of the capped site sequence of mRNA based on the detection of cap-dependent nucleotide addition using an anchor ligation method.

The sequence analysis of the 5' ends of cDNAs prepared using the anchor ligation method has revealed that most of the full-length cDNAs have an additional dGMP at their 5' end that is absent in the corresponding genome sequence. Using model RNA transcripts with cap analogues possessing 7-methylguanosine and adenosine, the base of the added nucleotide has been shown to be complementary to the base of the cap analogue, suggesting that the cDNAs possessing an additional dGMP are derived from intact mRNAs with the cap structure. On the other hand, cap-free RNA did not produce cDNA with an extra dGMP. These findings suggest that we can determine whether or not the cDNA starts from the capped site sequence of mRNA based on the presence or absence of an additional dGMP at the 5' end of the cDNA synthesized using the anchor ligation method. This approach will be useful to determine the capped site sequence of mRNA, thus, to identify transcription start sites.

DNA, Complementary↗

PubMed2004

Identification of 36 kDa phosphoprotein in fibrous sheath of hamster spermatozoa.

In our previous studies (Fujinoki et al., 2001, 2003), we reported that two types of 36 kDa proteins, designated 36K-A protein and 36K-B protein, obtained from hamster sperm flagella, are associated with motility activation and phosphorylated in a cAMP-dependent manner at serine residues. In the present experiments, we focused on the hamster (Mesocricetus auratus) 36K-A protein, which was analyzed by peptide mass finger printing and amino acid sequencing. The results suggest that 36K-A protein is a pyruvate dehydrogenase E1 component beta subunit lacking the N-terminal 30 amino acids. Moreover, our results suggest that 36 K-A protein is localized in the fibrous sheath of the principal piece of hamster spermatazoa.

Amino Acid Sequence↗

PubMed2004

Effect of hyperbaric oxygen on the ligament healing process in rats.

Animal experiments were done to investigate whether administration of hyperbaric oxygen promotes scar tissue formation, increases expression of the Type I procollagen gene, and improves the tensile properties of healing ligament. In 76 Sprague-Dawley rats, a 2-mm segment of the medial collateral ligament was removed. Thirty-eight rats were exposed to hyperbaric oxygen at 2.5 atmospheres absolute for 2 hours 5 days per week (Group H), whereas the remaining rats were exposed to room air (Group C). The animals were sacrificed at 3, 7, 14, and 28 days postoperatively. In situ hybridization histochemistry was done to examine the Type I procollagen gene expression in healing ligaments in 40 rats, whereas a tensile failure test was done in the remaining rats. The amount of scar tissue was greater in Group H than in Group C. Type I procollagen gene expression at 7 or 14 days was significantly greater in Group H than in Group C. The ultimate load and stiffness in Group H were significantly greater than in Group C at 14 days. Administration of hyperbaric oxygen promotes scar tissue formation and increases Type I procollagen gene expression in healing ligaments. These effects are associated with the improvement of their tensile properties.

Analysis of Variance↗

PubMed2003

Characterization of a cAMP-dependent protein kinase catalytic subunit from rainbow trout spermatozoa.

The cyclic AMP-dependent phosphorylation of proteins is essential for the initiation of sperm motility in salmonid fishes. This study isolated cDNA for the catalytic subunit of a cAMP-dependent protein kinase (PKA-C) from rainbow trout testis. The deduced amino acid sequence shows 75-80% identity to sequences previously reported in other organisms. However, the N-terminal regions of PKA-C from the testis as well as ovary in the trout appear slightly shorter than those from other tissues, suggesting that small PKA-C might be specific to germ cells. An immunofluorescence study using polyclonal antibody against trout testis PKA-C shows that it localizes along sperm flagellum. Furthermore, immunoelectron microscopy revealed that PKA-C is anchored to the outer arm dynein of flagellar axonemes. These results suggest that PKA-C is involved in regulating the flagellar motility of sperm via phosphorylation of a subunit of the outer arm dynein.

Amino Acid Sequence↗

PubMed2003

Classification of DNA-binding mode of antitumor and antiviral agents by the electrochemiluminescence of ruthenium complex.

The DNA-binding mode of antitumor and antiviral agents has been evaluated by electrochemiluminescence (ECL) of tris(1,10-phenanthroline)-ruthenium complex (Ru(phen)(3)(2+)) in the presence of oxalate ion in pH 7.3 Tris buffer solution. An emission of Ru(phen)(3)(2+) was observed repeatedly with a voltage above 1000mV subjected to a potential sweep from 0 to 1250mV. The addition of lambdaDNA into the solution containing 1 micro M of Ru(phen)(3)(2+) caused the decrease in the ECL intensity, which became half at a DNA concentration of 20 micro M. This is due to the binding of Delta-type of Ru(phen)(3)(2+) with DNA in the major groove of DNA. When the various concentrations of the drug were added to the solution containing 1& micro M Ru(phen)(3)(2+), the ECL intensity was not affected by the concentration of the drug in the absence of DNA. In the presence of DNA (10 micro M), however, two ECL emission patterns were observed when the concentration of the drug was varied. The pattern that the ECL intensity increased with increasing the drug concentration was observed for cisplatin, daunomycin, and DC92-B. This may have resulted from the DNA binding of the drug with a major groove site, where Ru(phen)(3)(2+) should bind. Ru(phen)(3)(2+) nonbinding to DNA might exist in the bulk solution and exhibits ECL emission. The drug exhibiting the drug-concentration-dependent ECL is classified as a drug with a major groove binding character. The addition of drugs, such as mitomycin C and duocarmycin SA, did not cause a change in the ECL intensity even in the presence of DNA. This result indicates that these drugs bind to DNA with minor groove binding. Since similar trends were observed for actinomycin D, distamycin A, doxorubicin, and chromomycin A3; these drugs are also considered as minor groove binding agents. All these results demonstrate that the DNA-binding mode of the drug can be evaluated easily by utilizing the ECL of Ru(phen)(3)(2+), which is used as the sensing probe.

Antineoplastic Agents↗

PubMed2003

Identification of 36-kDa flagellar phosphoproteins associated with hamster sperm motility.

In our previous paper [M. Fujinoki et al. (2001) BIOMED: Res. 22, 45-58], we reported that two types of 36-kDa protein, which were designated as 36K-A protein and 36K-B protein, obtained from hamster sperm flagella were phosphorylated at serine residues associated with the regulation of motility activation. In the present experiments, it was suggested that these two types of 36-kDa protein were phosphorylated in a cAMP-dependent manner associated with motility activation of hamster spermatozoa. Because the 36K-B protein was the most intensely phosphorylated in a cAMP-dependent manner, attempts were made to further characterize it. The 36K-B protein was assumed to be localized in the middle piece. The localization of the 36K-B protein was the same as that of the 36-kDa protein reported in our previous paper [Y. Si et al. (1999) Mol. Reprod. Dev. 52, 328-334]. In order to identify the 36K-B protein, it was analyzed by peptide mass finger printing and amino acid sequencing. The results suggested that the 36K-B protein was a pyruvate dehydrogenase E1 component beta subunit and a component of the mitochondrial sheath of the middle piece.

Amino Acid Sequence↗

PubMed2003

Identification of the 58-kDa phosphoprotein associated with motility initiation of hamster spermatozoa.

In our previous paper [M. Fujinoki et al. (2001) BIOMED: Res. 22, 45-58], we reported that the 58-kDa protein obtained from hamster sperm flagella was phosphorylated at serine residues in association with the start of motility. In the present experiments, we identified and localized the 58-kDa protein. The 58-kDa protein was assumed to exist in the acrosomal region domain of the sperm head and the whole sperm flagellum. In particular, a large amount of 58-kDa protein was localized in the equatorial segment of the acrosomal region domain of the sperm head and the middle piece of the sperm flagellum. In the next step, the 58-kDa protein was identified by peptide mass finger printing and LC-MS/MS analysis. The results suggested that the 58-kDa protein was ATP synthase H(+) transporting F1 beta, which is one of the mitochondrial components. Therefore, it is likely that the 58-kDa protein is associated with ATP production in the mitochondrial sheath in the middle piece of the sperm flagellum, and H(+) transport in the sperm head and the sperm flagellum except for the middle piece, since ATP synthase also acts as an H(+) pump.

Amino Acid Sequence↗