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Biomedical subjects

S Watanabe

Publications and source records attributed to S Watanabe.

At least 325 records · Page 18Linked to original sources

Conditions that affect acquisition of developmental competence by mouse oocytes in vitro: FSH, insulin, glucose and ascorbic acid.

The simplest unit required for the support of oocyte growth and development is the oocyte-granulosa cell complex. Therefore, a culture system was established that utilizes these complexes to assess mechanisms promoting nuclear, cytoplasmic and genomic maturation in mammalian oocytes. Deletion of serum from the culture, results in increased apoptosis in oocyte-associated granulosa cells (OAGCs), however, addition of ascorbic acid (0.5 mM) significantly reduced the level of apoptosis in the OAGCs, although no improvement of oocyte developmental competence was detected. The effects of reducing glucose during oocyte growth were studied since, under some culture conditions, glucose has deleterious effects on early preimplantation development. Reducing the glucose concentration to 1 mM resulted in the production of oocytes with greatly reduced developmental competence. Deleterious effects of FSH plus insulin during oocyte growth in vitro on preimplantation development are reviewed and discussed in terms of the communication of oocytes with inappropriately developing granulosa cells. Evidence that oocytes promote the appropriate differentiation of OAGCs in intact follicles in vivo is also discussed. It is hypothesized that oocytes control the differentiation of these cells, in order to promote intercellular signaling essential for the acquisition of competence to undergo normal embryogenesis.

Animals↗

Dendritic potassium channels in hippocampal pyramidal neurons.

Potassium channels located in the dendrites of hippocampal CA1 pyramidal neurons control the shape and amplitude of back-propagating action potentials, the amplitude of excitatory postsynaptic potentials and dendritic excitability. Non-uniform gradients in the distribution of potassium channels in the dendrites make the dendritic electrical properties markedly different from those found in the soma. For example, the influence of a fast, calcium-dependent potassium current on action potential repolarization is progressively reduced in the first 150 micrometer of the apical dendrites, so that action potentials recorded farther than 200 micrometer from the soma have no fast after-hyperpolarization and are wider than those in the soma. The peak amplitude of back-propagating action potentials is also progressively reduced in the dendrites because of the increasing density of a transient potassium channel with distance from the soma. The activation of this channel can be reduced by the activity of a number of protein kinases as well as by prior depolarization. The depolarization from excitatory postsynaptic potentials (EPSPs) can inactivate these A-type K+ channels and thus lead to an increase in the amplitude of dendritic action potentials, provided the EPSP and the action potentials occur within the appropriate time window. This time window could be in the order of 15 ms and may play a role in long-term potentiation induced by pairing EPSPs and back-propagating action potentials.

Action Potentials↗

2-Methoxy-4-(2-phthalimidinyl)phenylsulfonyl chloride as a fluorescent labeling reagent for determination of phenols in high-performance liquid chromatography and application for determination of urinary phenol and p-cresol.

Highly sensitive fluorescent labeling reagents, 2-(alkyloxy)-4-(2-phthalimidinyl)phenylsulfonyl chlorides (alkyloxy = methoxy, ethoxy, and propoxy; MPS-Cl, EPS-Cl, and PPS-Cl, respectively), for determination of phenols by high-performance liquid chromatography (HPLC) have been developed. These reagents react with phenols in basic medium to produce the corresponding fluorescent sulfonyl esters. The maximum fluorescence wavelengths of the derivatives were 308 nm for excitation and 410 nm for emission. The peaks due to phenol labeled with MPS-Cl, EPS-Cl, and PPS-Cl eluted at 6.3, 8.8, and 12.8 min, respectively, on a reversed-phase column with isocratic elution of methanol/water (2:1, v/v), and the detection limits (signal-to-noise = 3) of the derivatives were 10, 12, and 17 fmol per injection, respectively. Among these reagents, MPS-Cl was advantageous because its derivatives had shorter retention times and higher sensitivities in HPLC. The efficiency of converting phenol to the fluorescent derivative by MPS-Cl was about 100%. When MPS-Cl was applied to the determination of urinary phenol and p-cresol by HPLC using p-ethylphenol as an internal standard, the derivatives were separated at retention times of 6.3, 8.7, and 12.3 min, respectively, under the HPLC conditions described above. The concentrations of phenol and p-cresol in normal human urine were 11.9-293.5 and 8.2-346.1 nmol/mg creatinine, respectively.

Chromatography, High Pressure Liquid↗

Analysis of signals and functions of the chimeric human granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells and transgenic mice.

Receptors for GM-CSF, IL-3, and IL-5 are composed of two subunits: alpha, which is specific for each cytokine, and betac, which is shared by all. Although the role of betac in signal transduction has been extensively studied, the role of the alpha subunit has remained to be clarified. To analyze the role of the human (h) GM-CSF receptor alpha subunit, we constructed a chimeric receptor subunit composed of extracellular and transmembrane regions of alpha fused with the cytoplasmic region of betac, designated alpha/beta. In BA/F3 cells, chimeric receptor composed of alpha/beta,beta can transduce signals for mitogen-activated protein kinase cascade activation and proliferation in response to hGM-CSF. Although phosphorylation of Jak1 but not of Jak2 occurred with stimulation of hGM-CSF, the dominant-negative Jak2 but not the dominant-negative Jak1 suppresses c-fos promoter activation. To determine whether the chimeric receptor alpha/beta,beta is functional in vivo, we developed transgenic mice expressing the chimeric receptor alpha/beta,beta. Bone marrow cells from the transgenic mice expressing the alpha/beta,beta receptor form not only GM colonies but also various lineages of colonies in response to GM-CSF. In addition, mast cells were produced when bone marrow cells of the transgenic mouse were cultured with hGM-CSF. Thus, it appears that the cytoplasmic region of the alpha subunit is not required for hGM-CSF promoting activities, even in bone marrow cells.

Animals↗

On the mechanism of protection of distal joints after local gene transfer in collagen-induced arthritis.

Considerable interest has been generated by the observation that adenovirus-mediated gene delivery to a single arthritic joint results in suppression of arthritis in distal joints associated with the presence of small numbers of transduced cells in distal joints. It has been proposed that this is mediated by trafficking of transduced cells from the injected to distal joints. There are, however, alternative explanations that have not been explored, including the possibility that transgene protein or infectious virions circulate to distal sites. To investigate these possibilities, a replication-incompetent adenovirus encoding viral IL-10 (vIL-10) was administered to naive mice and to mice with collagen-induced arthritis by intraarticular, periarticular, or intravenous injection. In all cases, the ability to protect distal joints correlated with serum levels of vIL-10 protein. After intraarticular or intravenous injection, vIL-10 cDNA could be detected not only in distal joints, but also in the liver, which is the major target of circulating adenovirus, demonstrating that adenovirus circulating through the bloodstream is taken up by the joint tissue. Periarticular administration of adenovirus, which resulted in lower serum levels of vIL-10, protected only the injected paws and failed to induce trafficking immunoregulatory cells capable of suppressing distal disease. These observations suggest that circulating vIL-10 protein is the major mediator of distal protection. The presence of small numbers of transduced cells at distal sites can be accounted for by transduction of distal synovium after entry of adenovirus virions into the circulation.

Adenoviridae↗

Adjuvant effect of antibodies against von Willebrand Factor, fibrinogen, and fibronectin on staphylokinase-induced thrombolysis as measured using mural thrombi formed in rat mesenteric venules.

The change in thrombus mass during thrombolytic therapy is thought to be the difference between its growth and its degradation induced by thrombolytic agents. Platelets play a pivotal role in arterial thrombosis and bind to each other and to exposed subendothelial matrices via adhesive proteins such as von Willebrand Factor, fibrinogen, and fibronectin. The aim of the present study was to assess whether administration of antibodies against these adhesive proteins, in conjunction with plasminogen activator, could enhance the degradation of platelet-rich thrombus. Mural platelet-rich thrombi were formed in rat mesenteric venules using He-Ne laser irradiation. Recombinant staphylokinase was infused continuously and polyclonal antibodies against adhesive proteins were given by bolus injection. The thrombolytic process was analysed using computer-enhanced image analysis software. Administration of each of the antibodies enhanced staphylokinase-induced thrombolysis.

Adjuvants, Immunologic↗

Cloning and characterization of a novel adaptor protein, CIN85, that interacts with c-Cbl.

The c-Cbl protooncogene product is a prominent substrate of protein tyrosine kinases and is rapidly tyrosine-phosphorylated upon stimulation of a wide variety of cell-surface receptors. We have identified a novel c-Cbl-interacting protein termed CIN85 with a molecular mass of 85 kDa which shows similarity to adaptor proteins, CMS and CD2AP. CIN85 mRNA is expressed ubiquitously in normal human tissues and cancer cell lines analyzed. CIN85 was basally associated with c-Cbl. For interaction of CIN85 with c-Cbl, the second SH3 domain of CIN85 was shown to serve as a central player. The CIN85-c-Cbl association was enhanced shortly after stimulation of 293 cells with epidermal growth factor (EGF) and gradually diminished to a basal level, which correlated with a tyrosine phosphorylation level of c-Cbl. Our results suggest that CIN85 may play a specific role in the EGF receptor-mediated signaling cascade via its interaction with c-Cbl.

Adaptor Proteins, Signal Transducing↗

Roles of adenosine A(1) and A(2A) receptors in the expression and development of methamphetamine-induced sensitization.

We studied the effects of adenosine A(1) and A(2A) receptor agonists on the expression and development of methamphetamine-induced sensitization in rats. When animals were treated with the adenosine A(1) receptor agonist, N(6)-cyclohexyladenosine (CHA), along with methamphetamine every 3 days with a total of five administrations, the augmentation of hyperlocomotion by methamphetamine re-administration after 7-day withdrawal (methamphetamine challenge administration) was not inhibited. However, when the adenosine A(2A) receptor agonist, 2-p-(2-carboxyethyl) phenethyl-amino-5'-N-ethylcarboxy-amide adenosine (CGS21680), was administered according to the same schedule, the augmentation was significantly inhibited. On the other hand, when CHA or CGS21680 was administered 30 min before methamphetamine challenge, both drugs dose-dependently inhibited the augmentation of hyperlocomotion. These results suggested that both adenosine A(1) and A(2A) receptors play important roles in the expression of methamphetamine-induced sensitization, and that adenosine A(2A) receptors do so in the development of this sensitization.

Adenosine↗

Viral IL-10 and soluble TNF receptor act synergistically to inhibit collagen-induced arthritis following adenovirus-mediated gene transfer.

Viral IL-10 (vIL-10) and soluble TNF receptor (sTNFR) are anti-inflammatory proteins that can suppress collagen-induced arthritis (CIA). These and related proteins have shown efficacy in the treatment of human rheumatoid arthritis; however, neither alone is able to completely suppress disease. Furthermore, they have short half-lives, necessitating frequent administration. To determine the ability of these proteins to act synergistically following gene transfer, arthritis was induced in DBA/1 male mice by immunization with type II collagen on days 0 and 21. Mice were injected i.v. either before disease onset (day 20) or after disease onset (day 28) with 1010 particles of adenovirus encoding vIL-10, a soluble TNF receptor-IgG1 fusion protein (sTNFR-Ig), a combination of both vectors, or a control vector lacking a transgene. Significant synergism was observed with the combination of vIL-10 and sTNFR-Ig, with a substantial reduction in both the incidence and severity of disease as well as inhibition of progression of established disease. sTNFR-Ig alone had no effect on CIA. vIL-10 alone inhibited disease when given before disease onset, but had minimal effect on established disease. Both proteins inhibited spleen cell proliferation and IFN-gamma secretion in response to stimulation with type II collagen, but only vIL-10 reduced the synovial mRNA levels of the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-6. These findings demonstrate that vIL-10 and sTNFR-Ig act synergistically in suppressing CIA and suggest that gene transfer offers a potential therapeutic modality for the treatment of arthritis.

Adenoviridae↗

Cyclosporine-A enhances choline acetyltransferase immunoreactivity in the septal region of adult rats.

Cyclosporine-A (CsA) is the primary anti-rejection drug used for organ and neural transplantation therapy. In addition to its immunosuppressive action, CsA has been recently shown to exert neuroprotective and neurotrophic effects in the central nervous system when able to cross the blood-brain barrier. Postulated mechanisms for these CsA-induced beneficial effects include the drug's powerful inhibition of the calcium-dependent phosphatase calcineurin (CN) and blockade of the assembly of the mitochondrial permeability transition pore. We report here, for the first time, that adult Wistar rats treated with CsA (10 mg/kg per day, i.p. for 9 days) displayed significantly reduced septal CN expression in combination with enhanced levels of septal choline acetyltransferase (ChAT) immunoreactivity as compared to controls. The observed enhancement of septal ChAT immunoreactivity suggests potential therapeutic utility of CsA for brain disorders characterized by alterations of the cholinergic system.

Animals↗

Analysis of mechanisms involved in the prevention of gamma irradiation-induced apoptosis by hGM-CSF.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) induces proliferation and sustains viability of the mouse interleukin (IL)-3 dependent lymphoid cell line BA/F3 expressing the hGM-CSF receptor. Caspase-3 like enzyme activity and DNA fragmentation were augmented by depletion of this factor from the cell, and exposure to gamma irradiation accelerated kinetics of these events. Anti gamma irradiation-induced apoptosis occurred through various mutant GM-CSF receptors and only the box1 region was essential while the C terminal region, including tyrosine residues which are required for MAPK cascade activation, was dispensable. Consistent with this notion, the addition of PD98059 had no effect on this activity thereby indicating that activation of MAPK is not essential for the activity. As expected, gamma irradiation increased p53 protein and bax mRNA levels and the presence of hGM-CSF dramatically modulated bax/bcl-X(L) ratio. The PI-3K specific inhibitor wortmannin did not affect hGM-CSF dependent anti gamma irradiation induced apoptosis nor bcl-X(L) induction, thus bcl-X(L) but not PI-3K pathway seems to be involved in hGM-CSF dependent anti gamma irradiation-induced apoptosis. It is well documented that the boxl region is essential for GM-CSF dependent activation of JAK2 and JAK2 specific inhibitor AG490 suppressed anti gamma, irradiation-induced apoptosis by hGM-CSF. An artificial JAK2 activating molecule in which extracellular and the transmembrane of beta(c) fused with whole JAK2 can sustain BA/F3 cells survival and proliferation mIL-3 independently, but these cells are susceptible to gamma irradiation. Furthermore GyrB/Jak2, which can activate STAT5 but not the MAPK cascade nor survival of BA/F3 cells, also could not prevent gamma irradiation-induced apoptosis. Although JAK2 is essential for hGM-CSF dependent anti gamma irradiation-induced apoptosis, it appeared that JAK2 does not seem sufficient for the activity.

Animals↗

Palladium-catalyzed borylation of aryl halides or triflates with dialkoxyborane: A novel and facile synthetic route to arylboronates

A direct borylation of aryl halides or triflates with dialkoxyborane was investigated. The coupling reaction of pinacolborane with aryl halides or triflates in the presence of a catalytic amount of PdCl(2)(dppf) together with a base provided arylboronates in high yields. The product distributions were strongly dependent on the base employed, and the tertiary amine, especially Et(3)N, was effective for the selective formation of the boron-carbon bond. The reaction conditions were so mild that arylboronates having a variety of functional groups such as carbonyl, cyano, and nitro groups were readily prepared.

Journal Article↗

Tetrodotoxin-sensitive persistent current boosts the depolarization of retinal amacrine cells in goldfish.

To light illumination retinal amacrine cells respond with graded depolarization accompanied by a spike discharge. It has been assumed that the graded depolarization is produced solely by the excitatory synaptic inputs from bipolar cells. Here we demonstrate that a tetrodotoxin (TTX)-sensitive persistent current also contributes to the graded depolarization. This current was isolated in amacrine cells of the goldfish retinal slice preparations by the whole-cell patch clamp technique. The activation threshold of the persistent current was about -50 mV, approximately 10 mV more positive than the membrane potential in the dark. Therefore, it is highly likely that the TTX-sensitive persistent current is a booster of the excitatory postsynaptic potential in amacrine cells.

Action Potentials↗

Formation and stability of the dispersed particles composed of retinoic acid, sesame oil and phosphatidylcholine.

All trans-retinoic acid (RA) was dispersed by sonication with soybean phosphatidylcholine (PC). The particle size in the dispersion was increased to 240 nm up to the RA mol fraction range (X(RA)) of 0.4. At X(RA)=0.5, the RA/PC mixture was difficult to disperse and the macroscopic oil/water phase separation was observed. On the other hand, by the addition of sesame oil (SO) to RA (molar ratio of RA:SO=1:1), stable aqueous dispersions (diameter: 40-80 nm) were obtained in the mol fraction range RA and SO mixture (X(M)) of 0.1-0.8. In order to clarify these dispersal mechanism, the dispersed particles were characterized and the interaction among RA, SO and PC was investigated using several physicochemical techniques. The trapped aqueous volume inside the RA/PC particles was determined using the aqueous space marker, calcein and it was increased with the addition of RA into small unilamellar vesicles of PC. On the other hand, that of RA/SO/PC particles was decreased remarkably with increase in X(M) and the decline in the fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the PC membrane by the addition of the quencher CuSO(4). These results indicate that the interaction of RA with PC bilayers and the structure of RA/PC mixture will be changed by the addition of SO.

Algorithms↗

Dietary docosahexaenoic acid ameliorates, but rapeseed oil and safflower oil accelerate renal injury in stroke-prone spontaneously hypertensive rats as compared with soybean oil, which is associated with expression for renal transforming growth factor-beta, fibronectin and renin.

We have noted that n-3 fatty acid-rich oils, such as fish oil, perilla oil and flaxseed oil as well as ethyl docosahexaenoate (DHA) prolonged the survival time of stroke-prone spontaneously hypertensive rats (SHRSP) rats by approximately 10% as compared with linoleate (n-6)-rich safflower oil. Rapeseed oil with a relatively low n-6/n-3 ratio unusually shortened the survival time by approximately 40%, suggesting the presence of minor components unfavorable to SHRSP rats. This study examined the effects of dietary oils and DHA on renal injury and gene expression related to renal injury in SHRSP rats. Rats fed rapeseed oil- and safflower oil-supplemented diets developed more severe proteinuria than those fed soybean oil-supplemented diet used as a control, but there were no significant differences in blood pressure. In contrast, the DHA-supplemented diet inhibited the development of proteinuria and suppressed hypertension. The mRNA levels for renal TGF-beta, fibronectin and renin were higher in the rapeseed oil and safflower oil groups after 9 weeks of feeding of the experimental diet than in the soybean oil and DHA groups. The fatty acid composition of kidney phospholipids was markedly affected by these diets. These results indicate that the renal injury observed in the groups fed safflower oil with a high n-6/n-3 ratio and rapeseed oil with presumed minor components is accompanied by increased expression of the TGF-beta, renin and fibronectin genes, and that dietary DHA suppresses renal injury and gene expression as compared with soybean oil.

Animals↗

Viability of Q-wave infarcted myocardium with restored positive and persistent negative T waves after optimal revascularization compared with dobutamine stress echocardiography.

To clarify the clinical difference in viability of myocardium with negative and positive T waves in Q-wave anterior or anteroseptal myocardial infarction, we performed low-dose dobutamine stress echocardiography in 17 patients with negative T waves and in 13 patients with positive T waves with optimal revascularization of infarct-related arteries in the chronic phase of infarction. At baseline the wall motion score (WMS) of the negative and positive T groups was 25.8 +/- 3.0 and 22.3 +/- 2.2 points (p <0.05), respectively. At peak stress WMS in each group was 27.2 +/- 4.2 and 19.8 +/- 2.4 points (p <0.0001), respectively. With dobutamine stress WMS in the positive T group was more decreased than that of the negative T group (p <0.0001). We conclude that the restored positive T waves in Q-wave myocardial infarction indicate a significantly greater amount of viable myocardium than the negative T waves, showing better regional wall motion improvement with low-dose dobutamine stress.

Aged↗

Indications for bilateral modified radical neck dissection in patients with papillary carcinoma of the thyroid.

HYPOTHESES: After subtotal thyroidectomy with modified radical neck dissection of the affected side, nodal recurrence at the contralateral cervical side indicates a poor prognosis for patients with papillary thyroid cancer. Bilateral modified radical neck dissection is beneficial for patients at high risk for contralateral nodal recurrence. DESIGN AND SETTING: Retrospective study of patients with papillary cancer who were treated surgically from January 1, 1970, through December 31, 1995, at the Noguchi Thyroid Clinic and Hospital Foundation, Beppu, Japan. PATIENTS: Patients (N = 1776) had primary tumors greater than 10 mm in maximum diameter and underwent thyroidectomy and ipsilateral modified radical neck dissection with curative intent. RESULTS: Thirty-two patients (1.8%) developed contralateral lymph node metastases during the mean follow-up period of 12.1 years. The risk factors for contralateral nodal recurrence were male sex, large primary tumor, tumor extension over the isthmus, extracapsular adhesion or invasion to surrounding tissues, and the presence of gross nodal metastasis at initial surgery. These patients had a greater number of distant metastases (31.1% vs 0.7%; P<.001) and a lower 10-year survival rate (83.7% vs 99.3%; P<.001) than patients without nodal recurrence. CONCLUSION: Bilateral modified radical neck dissection should be considered for patients with papillary carcinoma who show risk factors for contralateral nodal recurrence, as it could prevent a second operation and may improve their outcome.

Adult↗

Biliary excretion of TT virus (TTV).

A novel DNA virus (TT virus; TTV) was isolated from a patient with post-transfusion hepatitis of unknown etiology. If TTV replicates in the liver, TTV may appear in the bile. In the present study, to clarify whether fecal-oral infection occur via biliary excretion, the presence of TTV DNA was assessed in paired serum and bile samples collected from 28 patients with obstructive jaundice without parenchymal liver disease. TTV DNA was detected by polymerase chain reaction (PCR) using semi-nested primers, and quantified by Real Time Detection PCR (RTD-PCR). The nucleotide sequence of isolates TTV DNAs was also determined and the sequences were compared between serum and bile samples. Among 28 patients, 7 were positive for TTV DNA in both samples, and 3 and 2 were positive in serum and bile respectively. Of 7 patients positive for TTV DNA in both samples, the TTV DNA titer was higher in serum of 4 patients and in bile of 1 patient. Among 7 patients positive for TTV DNA in serum and bile, 6 had the same sequence in both samples. Multiple distinct types of TTV DNA clones were isolated from serum in 2 patients and from bile in 4 patients. In conclusion, TTV DNA is detected frequently in bile from patients with obstructive jaundice, suggesting a fecal-oral route of infection and high prevalence of asymptomatic TTV carriers. TTV DNA was detected only in serum from some patients, suggesting that replication of TTV may occur in other organs as well as in the liver.

Aged↗