Biomedical subjects

H Yokota

Publications and source records attributed to H Yokota.

At least 37 records · Page 2Linked to original sources

PubMed2002

Carcinoembryonic antigen mRNA in abdominal cavity as a useful predictor of peritoneal recurrence of gastric cancer with serosal exposure.

Peritoneal dissemination is the most frequent type of recurrence in patients with gastric cancer with serosal exposure, irrespective of whether they have undergone curative gastrectomy. The purpose of this study was to establish a method to detect micrometastatic cells in the abdominal cavity and predict peritoneal recurrence in patients with such gastric carcinomas. A total of 86 patients with gastric carcinoma, undergoing gastrectomy, were examined. Reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to detect carcinoembryonic antigen (CEA) mRNA in abdominal lavage fluid. Twenty-four cases without serosal exposure were negative, while all 13 cases with macroscopic peritoneal dissemination were positive for CEA mRNA. Among the 49 cases with macroscopic serosal invasion and without peritoneal metastasis, cancer cells were detected in 27 cases with RT-PCR while in only 6 cases with conventional cytology. All cytologically-positive cases were also positive for CEA mRNA. Among the 27 CEA-positive cases, 15 patients (56%) relapsed with peritoneal metastasis within 12 months after gastrectomy. In contrast, none of the 22 CEA-negative cases had peritoneal recurrence within 16-60 months of observation, whereas in 43 cytologically-negative cases, 10 patients relapsed with peritoneal recurrence. As compared with conventional cytological examination, this method would be clinically more beneficial for detecting free cancer cells in the peritoneal cavity and for predicting peritoneal recurrence in gastric carcinoma with serosal invasion.

Abdominal Cavity↗

PubMed2001

High expression of 92 kDa type IV collagenase (matrix metalloproteinase-9) in canine mammary adenocarcinoma.

The 92 kDa type VI collagenase (matrix metalloproteinase-9 (MMP-9)) activities on zymography assay were found to be 1-6 times higher in benign tumor breast tissues of 12 canines and 4-26 times higher in adenocarcinoma breast tissues of nine canines than that of control tissues, respectively. A full-length canine MMP-9 cDNA was cloned from the adenocarcinoma tissue by reverse transcription-PCR and 5'- and 3'-RACE. The isolated cDNA contained an open reading frame coding for a polypeptide of 704 amino acids. The predicted protein sequence displayed extensive similarity to that of known MMP-9s and contained a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. Western blotting using MMP-9-specific antibodies prepared against the peptide corresponding to Arg(642)-Asp(704) of canine MMP-9 and Northern blotting using a MMP-9-specific cDNA fragment as a probe confirmed that MMP-9 (the 92 kDa protein band) was highly expressed in canine mammary adenocarcinoma tissues. Higher levels of MMP-9 activity were found in the sera of canines with mammary adenocarcinoma. The results indicated that MMP-9 plays an important role in the progression of a canine mammary tumor and that assay of serum MMP-9 is helpful for early diagnosis as progress of adenocarcinoma.

Adenocarcinoma↗

PubMed2001

Effects of CP-99, 994, a tachykinin NK(1) receptor antagonist, on abdominal afferent vagal activity in ferrets: evidence for involvement of NK(1) and 5-HT(3) receptors.

The effect of CP-99, 994, a tachykinin NK(1) receptor antagonist, on abdominal vagal afferent nerve activity in the ferret was investigated. Substance P (1 microg/kg, i.v.) increased vagal afferent activity by 449.0+/-51.9% and this was reduced to 145.9+/-5.7% (p<0.01) by pre-treatment with CP-99, 994 (1 mg/kg, i.v.), and to 149.5+/-1.5% (p<0.001) by granisetron (1 mg/kg, i.v.), a 5-HT(3) receptor antagonist. In addition, the increase in vagal nerve activity induced by 5-HT (25 microg/kg, i.v., 552.0+/-57.0% increase from pre-injection level) was significantly reduced (401.3+/-10.6% increase from pre-injection level, p<0.05) by CP-99, 994 (100 microg/kg, i.v.). These results provide evidence for an involvement of peripheral NK(1) and 5-HT(3) receptors in substance P-induced vagal afferent activation. While the functional consequences (if any) of such peripheral effects were not investigated, they could contribute either directly (e.g. by blockade of receptors on vagal afferents) or indirectly (e.g. modulation of 5-HT release or reduction of local inflammatory response) to the antiemetic effects of CP-99, 994 against cisplatin and other emetic agents acting primarily via the vagus.

Abdomen↗

PubMed2001

BeF(3)(-) acts as a phosphate analog in proteins phosphorylated on aspartate: structure of a BeF(3)(-) complex with phosphoserine phosphatase.

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF(3)(-), which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-A resolution the x-ray crystal structure of the complex of BeF(3)(-) with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF(3)(-) is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg(2+), and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF(3)(-) x PSP and BeF(3)(-) x CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF(3)(-) for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

Aspartic Acid↗

PubMed2001

Detection of abasic sites on individual DNA molecules using atomic force microscopy.

We have developed an atomic force microscopy-based method for detecting abasic sites (AP sites) on individual DNA molecules. By using uracil and uracil DNA glycosylase, we first prepared a 250-bp DNA template consisting of two AP sites at specific locations. We then detected the AP sites by marking them with biotinylated aldehyde-reactive probes and monomeric avidin. We demonstrate here that (i) the location of monomeric avidin bound on a single DNA molecule was detectable by atomic force microscopy; (ii) the observed location of avidin was in good agreement to the predicted AP sites at a few nanometer resolution; and (iii) by end-labeling the 5'-terminus of one DNA strand, the AP sites were determined without directional ambiguity. The technique described here will provide a sensitive way of locating AP sites and contribute to screen DNA damages from individual molecules.

DNA↗

PubMed2001

cDNA cloning and expression of a bovine phenol UDP-glucuronosyltransferase, BovUGT1A6.

A full-length cDNA encoding a phenol UDP-glucuronosyltransferase was isolated by plaque hybridization, RT-PCR and 5'-RACE from a cDNA library prepared from the bovine liver. The deduced amino acid sequence (529 amino acid residues) has A signal sequence (23 amino acid residues) at the amino terminus and a transmembrane-anchoring domain (17 amino acid residues) at the carboxyl terminus. The encoded protein has a potential asparagine-linked glycosylation site (Asn291). The cloned cDNA was named bovUGT1A6 on the basis of the amino acid similarity. BovUGT1A6 cloned in the pAAH5 expression vector was transformed into Saccharomyces cerevisiea AH22 cells to obtain an active 54-kDa bovUGT1A6 enzyme. The expressed enzyme represented UDP-glucuronosyltransferase activities toward 1-naphthol and 4-methylumbelliferone, confirming that the isolated cDNA is an isoform of bovine phenol UDP-glucuronosyltransferase. Microsomal UDP-glucuronosyltransferase activity toward 1-naphthol in the bovine kidney cortex was found to be higher than that in the liver and other organs, and mRNA of bovUGT1A6 was more strongly detected in the kidney on Northern blotting analysis. These results suggest that the bovine kidney, which strongly expresses bovUGT1A6, is a significant organ for xenobiotics glucuronidation.

Amino Acid Sequence↗

PubMed2001

Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution.

BACKGROUND: D-Serine is a co-agonist of the N-methyl-D-aspartate subtype of glutamate receptors, a major neurotransmitter receptor family in mammalian nervous systems. D-Serine is converted from L-serine, 90% of which is the product of the enzyme phosphoserine phosphatase (PSP). PSP from M. jannaschii (MJ) shares significant sequence homology with human PSP. PSPs and P-type ATPases are members of the haloacid dehalogenase (HAD)-like hydrolase family, and all members share three conserved sequence motifs. PSP and P-type ATPases utilize a common mechanism that involves Mg(2+)-dependent phosphorylation and autodephosphorylation at an aspartyl side chain in the active site. The strong resemblance in sequence and mechanism implies structural similarity among these enzymes. RESULTS: The PSP crystal structure resembles the NAD(P) binding Rossmann fold with a large insertion of a four-helix-bundle domain and a beta hairpin. Three known conserved sequence motifs are arranged next to each other in space and outline the active site. A phosphate and a magnesium ion are bound to the active site. The active site is within a closed environment between the core alpha/beta domain and the four-helix-bundle domain. CONCLUSIONS: The crystal structure of MJ PSP was determined at 1.8 A resolution. Critical residues were assigned based on the active site structure and ligand binding geometry. The PSP structure is in a closed conformation that may resemble the phosphoserine bound state or the state after autodephosphorylation. Compared to a P-type ATPase (Ca(2+)-ATPase) structure, which is in an open state, this PSP structure appears also to be a good model for the closed conformation of P-type ATPase.

Amino Acid Sequence↗

PubMed2001

Birth of a healthy baby following vitrification of human blastocysts.

OBJECTIVE: To assess vitrification of human blastocysts. DESIGN: Retrospective study of blastocyst vitrification. SETTING: A private clinic. PATIENT(S): Twenty couples with different types of infertility. INTERVENTION(S): Blastocysts were frozen with rapid vitrification and then transferred after thawing. We vitrified blastocysts using a modification of Ishimori's vitrification solution of ethylene glycol and dimethyl sulfoxide (VSED). MAIN OUTCOME MEASURE(S): After thawing, survival was defined by the embryo's development morphology after 6 hours or overnight culture. RESULT(S): Eighteen of 20 patients underwent treatment. Of 45 vitrified blastocysts, 36 survived, for a survival rate of 80% (36 of 45). The implantation rate was 21.9% (7 of 32), and the pregnancy rate (per embryo transfer cycle) was 33.3% (6 of 18). One of the pregnancies resulted in the delivery of a healthy baby. CONCLUSION(S): Supernumerary embryos were grown in culture to blastocysts, and the survival rate of vitrified-thawed blastocysts was the same as that for slow freezing of early stage embryos. Blastocyst vitrification should prove effective for clinical treatment. The present results strongly suggest that this rapid and successful vitrification procedure will replace conventional cryopreservation in the future.

Blastocyst↗

PubMed2001

Birth of healthy twins from in vitro development of human refrozen embryos.

OBJECTIVE: To avoid the risk of ovarian hyperstimulation syndrome and promote pregnancy rate by freezing pronuclear embryos at the oocyte retrieval and using developed refrozen supernumerary embryos in the event of failed pregnancies. DESIGN: A case study. SETTING: Private IVF center. PATIENT(S): Healthy infertile woman undergoing IVF treatment. INTERVENTION(S): Slowly frozen and rapidly refrozen embryos at varying stages of embryonic development were transferred. MAIN OUTCOME MEASURE(S): Survival rate of refrozen and thawed embryos; safety of refrozen embryo transference. RESULT(S): Fertilized pronuclear embryos that were frozen, thawed, and developed to morula embryos were subsequently vitrified, rethawed, and cultured until blastocyst formation before transfer. Healthy fraternal twins were born by caesarian section. The result of transfer was the novel finding of a successful delivery after the use of refrozen embryos. CONCLUSION(S): The transfer of refrozen supernumerary embryos resulted in successful pregnancy and delivery. We will continue to investigate the safety and efficacy of the elective freezing of all embryos as well as their refreezing so that supernumerary embryos can be reused to avoid ovarian hyperstimulation syndrome or in cases of repeated failed pregnancies.

Cryopreservation↗

PubMed2001

Modulation of synaptic transmission in hippocampal CA1 neurons by a novel neurotoxin (beta-pompilidotoxin) derived from wasp venom.

We examined the effects of beta-pompilidotoxin (beta-PMTX), a neurotoxin derived from wasp venom, on synaptic transmission in the mammalian central nervous system (CNS). Using hippocampal slice preparations of rodents, we made both extracellular and intracellular recordings from the CA1 pyramidal neurons in response to stimulation of the Schaffer collateral/commissural fibers. Application of 5-10 microM beta-PMTX enhanced excitatory postsynaptic potentials (EPSPs) but suppressed the fast component of the inhibitory postsynaptic potentials (IPSPs). In the presence of 10 microM bicuculline, beta-PMTX potentiated EPSPs that were composed of both non-NMDA and NMDA receptor-mediated potentials. Potentiation of EPSPs was originated by repetitive firings of the presynaptic axons, causing summation of EPSPs. In the presence of 10 microM CNQX and 50 microM APV, beta-PMTX suppressed GABA(A) receptor-mediated fast IPSPs but retained GABA(B) receptor-mediated slow IPSPs. Our results suggest that beta-PMTX facilitates excitatory synaptic transmission by a presynaptic mechanism and that it causes overexcitation followed by block of the activity of some population of interneurons which regulate the activity of GABA(A) receptors.

2-Amino-5-phosphonovalerate↗

PubMed2001

Adsorption of endotoxins on glass in the presence of cationic proteins.

Endotoxin activity was detected in empty glass tubes where endotoxins were incubated with lysozyme, histone or RNaseA, indicating adsorption of endotoxins on glass in the presence of cationic proteins. In the case of lysozyme, the recovery of spiked endotoxins (90.0%) using polystyrene tubes for incubation was much greater than the recovery (28.5%) using glass tubes, suggesting that lysozyme-mediated adsorption of endotoxins on glass is a major cause of poor recovery of spiked endotoxins in the LAL assay using glass tubes. In contrast, the recovery of spiked endotoxins (64.7%) using polystyrene tubes in the presence of the non-cationic protein BSA was less than the recovery (103.9%) using glass tubes. The difference in endotoxin recovery using glass or polystyrene tubes in the presence of cationic proteins or BSA can be explained by differences in protein adsorption on the tubes. Consequently, care must be exercised in selecting containers used for the LAL assay of proteins which bind to endotoxins.

Adsorption↗

PubMed2001

Messenger-RNA expression of matrix metalloproteinases, tissue inhibitors of metalloproteinases, and transcription factors in rheumatic synovial cells under mechanical stimuli.

In an effort to elucidate the role of mechanical stimuli in rheumatoid arthritis, we determined mRNA levels of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, and three transcription factors (c-fos, ets-1, and ets-2) under two mechanical shearing conditions as well as simulated unloading. Human synovial cell cultures (MH7A and RA99-01), derived from rheumatoid arthritis patients, were grown for 1 h under mechanical stimuli and the transcript level was assayed by the reverse transcription-polymerase-chain reaction procedure. First, gentle shearing, estimated at approximately 1 dyn/cm(2), induced a consistent decrease in mRNA level of MMP-1, MMP-3, MMP-13, and ets-1 and an increase in the transcript level of TIMP-1, TIMP-2, c-fos, and ets-2. Second, intermediate shearing, estimated at approximately 6 dyn/cm(2), elevated the mRNA level of all MMPs, TIMPs, and the three transcription factors. Third, minimum mRNA level of c-fos, ets-1, and ets-2 was achieved under control conditions at rest, gentle shearing, and simulated unloading, respectively. These in vitro results support a stimulus-dependent transcriptional regulation of MMPs, TIMPs, and transcription factors in cell cultures, suggesting a potential role of shear stress in tissue degradation and prevention in rheumatic joints.

Arthritis, Rheumatoid↗

PubMed2001

Altered mRNA level of matrix metalloproteinase-13 in MH7A synovial cells under mechanical loading and unloading.

In an effort to elucidate the interplay between mechanical load and proteolytic gene expression in arthritic tissue degradation, we investigated cellular morphology and mRNA levels of matrix metalloproteinase-13 (MMP-13) genes under mechanical stress in human MH7A synovial cells. The cells were isolated from the knee joint of a rheumatoid arthritis patient. Using a reverse transcription-polymerase chain reaction procedure, we found that loading by an oscillatory shaker transiently decreased the level of MMP-13 mRNA and unloading by a clinostat increased its mRNA level. The unloaded cells appeared to be rounded and displayed a poorly developed track of peripheral fibers, whereas the cells under loading tended to align to the shear flow and were elongated. We also found that altering the oscillatory direction of mechanical loads contributed to a further reduction in mRNA expression of MMP-13. Our results demonstrate the role of mechanical loading and unloading in the transcriptional regulation of MMP-13 in synovial cells, and suggest the potential value of physical therapy for arthritic joints.

Arthritis, Rheumatoid↗

PubMed2001

The application of three-dimensional internal structure microscopy in the observation of mare ovary.

The ovary of the mare has a unique structure which differs totally from that of other mammals. However, because of its relatively large size, conventional histological techniques were unsuitable for the observation of the internal structure of the whole ovary. Three-dimensional internal structure microscopy (3D-ISM) consists of a cryotome-CCD camera-laser disc recorder-PC-based control system coupled with a graphic workstation. The internal structure of the ovary is observed by processing over more than 1,000 stored images of serially sliced surfaces of each frozen equine ovary. The 3D reconstruction was done using the full-coloured, volume-rendering method. The relationship between the localization of medulla, cortex and ovulation fossa was clarified. The ovulation fossa is localized in the centre of the ovary and is surrounded by a broad ovarian cortex. A trace of ovulation was observed only at the ovulation fossa. Medulla are localized in narrow peripheral areas. The phenomenon of the competition to occupy the cortical area ahead of the ovulation fossa by developing secondary follicles was visualized. Spatial localization of various sized follicles was identified from 3D-reconstructed images. In this study, it has been clarified that application or this novel computerized technique can clarify the anatomical arrangements of the equine ovary and the complex mechanism of equine follicular development.

Animals↗

PubMed2001

Solution nuclear magnetic resonance structure of a protein disulfide oxidoreductase from Methanococcus jannaschii.

The solution structure of the protein disulfide oxidoreductase Mj0307 in the reduced form has been solved by nuclear magnetic resonance. The secondary and tertiary structure of this protein from the archaebacterium Methanococcus jannaschii is similar to the structures that have been solved for the glutaredoxin proteins from Escherichia coli, although Mj0307 also shows features that are characteristic of thioredoxin proteins. Some aspects of Mj0307's unique behavior can be explained by comparing structure-based sequence alignments with mesophilic bacterial and eukaryotic glutaredoxin and thioredoxin proteins. It is proposed that Mj0307, and similar archaebacterial proteins, may be most closely related to the mesophilic bacterial NrdH proteins. Together these proteins may form a unique subgroup within the family of protein disulfide oxidoreductases.

Amino Acid Sequence↗

PubMed2001

Relationship between serum TNF activity and insulin resistance in dairy cows affected with naturally occurring fatty liver.

To clarity the relationship between tumor necrosis factor (TNF) and insulin resistance in dairy cows affected with fatty liver, naturally occurring cases were investigated. The affected cows were classified into following three groups according to histopathologic findings of the liver: mild fat droplet deposition (group 1; n=11), severe fat droplet deposition (group 2; n=10), and cloudy swelling (group 3; n=8). Serum TNF activities in Group 2 (8.67 +/- 2.16 U/ml) and Group 3 (11.65 +/- 1.92 U/ml) were significantly higher than that in Group 1 (3.57 +/- 0.81 U/ml) (p<0.05). The insulin-tolerance tests showed that the insulin-stimulated glucose disposal rates (GDR) in Group 2 (27.6 +/- 7.8%) and Group 3 (15.8 +/- 9.1%) were significantly lower than that in Group 1 (41.7 +/- 9.8%). There was a significant negative correlation between serum TNF activity and GDR in affected cows (r=-0.56, p<0.01). These results indicate that serum TNF activity is correlated with insulin resistance in cows with fatty liver.

Animals↗

PubMed2001

Life-cycle toxicity of 4-nonylphenol to medaka (Oryzias latipes).

We studied the chronic effects of 4-nonylphenol (4-NP) on reproductive status of medaka (Oryzias latipes) over two generations of continuous exposure. The exposure study of the parental (F0) medaka was begun on embryos within 24 h postfertilization and continued with monitoring through embryological development, hatching, posthatch survival, growth, sexual differentiation, and reproduction under flow-through exposures to mean measured 4-NP concentrations of 4.2, 8.2, 17.7, 51.5, and 183 microg/L for up to 104 d. Eggs spawned from the F0 fish at 102 and 103 d posthatch were also examined for hatchability, survival after hatching, growth, and sexual differentiation until 60 d posthatch. The 183-microg/L treatment significantly reduced the embryo survival and swim-up success of the F0 fish. The cumulative mortality after swim-up of the F0 fish exposed to 17.7 and 51.5 microg/L were significantly higher than the control mortality. No concentration-related effect of 4-NP was observed on the growth of surviving F0 fish at 60 d posthatch. However, the sex ratio estimated from the appearance of their secondary sex characteristics was skewed toward female in the 51.5-microg/L treatment. Additionally, gonadal histology showed that 20% of the fish in the 17.7-microg/L treatment and 40% in the 51.5-microg/L treatment had testis-ova, indicating that 4-NP affects the gonadal development and survival of medaka at similar concentrations in juveniles. The sex ratio of the F0 fish in the 51.5-microg/L treatment was completely skewed toward female; subsequently, the effects on fecundity and fertility in this generation were monitored at mean measured concentrations of 4.2, 8.2, and 17.7 microg/L from 71 to 103 d posthatch. Fecundity was unaffected by any of the treatments examined. The mean fertility in the 17.7-microg/L treatment was reduced to 76% of that in the controls, although no statistically significant differences were determined. Overall, these results indicate that the lowest-observed-effect concentration (LOEC) and no-observed-effect concentration (NOEC) of 4-NP through the life cycle of the F0 medaka were 17.7 and 8.2 microg/L, respectively. In the F1 medaka, no significant effects were observed on hatching success, posthatch mortality, or growth, but sexual differentiation at 60 d posthatch was affected. Induction of testis-ova in the gonads of the F1 fish was observed in both the 8.2- and the 17.7-microg/L concentrations. The results indicate that 4-NP can have significant effects on reproductive potential of medaka at concentrations as low as 17.7 microg/L.

Animals↗

PubMed2001

Spontaneous thoracic spinal cord herniation--case report.

A 54-year-old female presented with spontaneous thoracic spinal cord herniation manifesting as chronic progressive motor weakness in both legs. Spastic paraparesis (4/5) and pathological reflexes such as ankle clonus were noted. She also had mild bladder dysfunction but no bowel dysfunction. She had no sensory disturbance, including tactile and pinprick sense. Magnetic resonance (MR) imaging revealed that the atrophic spinal cord was displaced into the ventral extradural space at the T4-5 intervertebral level with markedly dilated dorsal subarachnoid space. Computed tomography obtained after myelography showed no evidence of intradural spinal arachnoid cyst. She underwent surgical repair of the spinal cord herniation via laminectomy, and spinal cord herniation through the ventral dural defect was confirmed. Postoperative MR imaging revealed improvement of the spinal cord herniation, but her symptoms were not improved. Spontaneous spinal cord herniation is a rare cause of chronic myelopathy, occurring in the upper and mid-thoracic levels, and the spinal cord is usually herniated into the ventral extradural space. Early differential diagnosis from intradural spinal arachnoid cysts is important for a satisfactory outcome.

Female↗