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Biomedical subjects

M Izawa

Publications and source records attributed to M Izawa.

At least 55 records · Page 3Linked to original sources
PubMed1998

Expression of the apoptosis-related genes, caspase-1, caspase-3, DNA fragmentation factor, and apoptotic protease activating factor-1, in human granulosa cells.

OBJECTIVE: To investigate protein and messenger RNA expression products for a subset of apoptosis-related genes in human granulosa cells. DESIGN: In vitro experiment. SETTING: Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts. PATIENT(S): Women undergoing oocyte retrieval for IVF after ovulation induction with gonadotropins. INTERVENTION(S): Granulosa cells were isolated from follicular aspirates after oocyte removal. MAIN OUTCOME MEASURE(S): Reverse transcription polymerase chain reaction and Western blotting for apoptosis-related gene products. RESULT(S): The expression of caspase-1, caspase-3, DNA fragmentation factor, and apoptotic protease activating factor-1, which are intermediate molecules in phylogenetically conserved apoptotic pathways, was demonstrated in granulosa cells from patients undergoing IVF. Moreover, proforms, but not activated enzymes, for both caspase-1 and caspase-3 were observed. CONCLUSION(S): Granulosa cells from patients undergoing IVF have intrinsic apoptotic machinery that could be activated for tissue remodeling.

Apoptosis↗
PubMed1998

Identification of a transcript predicting an alternative form of sulfated glycoprotein-2 (clusterin) in rat tissues.

As a clue to understand the role of androgen in rat seminal vesicle, we have isolated several cDNA clones of androgen-responsive mRNAs. During further characterizations of these cDNAs were in progress, a distinct cDNA clone designated p116 was isolated. The sequence study suggested the expression of a transcript predicting an alternative form of Sulfated Glycoprotein-2 (SGP-2)/clusterin. RT-PCR demonstrated the androgen-dependent expression of p116 transcript in the seminal vesicles. The transcript was also detectable in the ventral prostate, the liver and the thymus. Since SGP-2 has been suggested as a classical molecular marker of apoptosis, p116 transcript newly identified in the present study might provide a useful probe to further understand the roles of SGP-2 and its related proteins during the course of apoptotic process not only in male accessory sex organs, but also in many types of cells undergoing apoptosis.

Alternative Splicing↗
PubMed1998

Prophylactic effect of papaverine prolonged-release pellets on cerebral vasospasm in dogs.

OBJECTIVE: A drug delivery system using copoly(lactic/glycolic acid) was developed for the intracranial administration of papaverine. A rod-shaped implant prepared by a heat compression method was tested to determine its efficacy in preventing cerebral vasospasm in dogs. METHODS: Sixteen dogs were randomly assigned to one of two groups, i.e., placebo or papaverine. Control angiography was performed, followed by right craniectomy and the induction of subarachnoid hemorrhage by the placement of a clot in the Sylvian fissure. Two pellets, containing either 25 mg of papaverine or no papaverine, were placed in the cistern. In in vitro studies, 56% of the actual papaverine loading was released in the first 4 days and 78% within 8 days. On Day 7, angiography was repeated and the animals were killed. A similar experiment using low-dose pellets containing 5 mg of papaverine, half of which was released within 7 days, was performed with 16 mongrel dogs. RESULTS: There were significant differences between the papaverine- and placebo-treated groups in the reductions of vessel diameters of the internal carotid, middle cerebral, and anterior cerebral arteries on the clot side. The mean concentration of papaverine in the clot was 4.5 x 10(-4) mol/L. The low-dose pellet failed to prevent cerebral vasospasm, although the mean concentration of papaverine in the clot was 2.3 x 10(-5) mol/L. CONCLUSION: A prolonged-release preparation of papaverine that could be implanted intracranially at the time of surgery prevented vasospasm significantly while maintaining an appropriate concentration of papaverine in the cistern.

Animals↗
PubMed1998

Mechanism of hypocholesterolemic action of S-8921 in rats: S-8921 inhibits ileal bile acid absorption.

The mechanism of the hypocholesterolemic action of S-8921, methyl 1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)- 4-hydroxy-6,7,8-trimethoxy-2-naphthoate, was examined in rats. In diet-induced hypercholesterolemic rats, 2 weeks oral administration of S-8921 dose- and time-dependently decreased plasma cholesterol level in the daily dose range of 0.1 to 10 mg/kg. Results with the dual-isotope plasma ratio method indicated that S-8921 inhibits cholesterol absorption from the intestine and enhances its elimination from the body. The in situ loop method showed that S-8921 does not inhibit the absorption of cholesterol from rat jejunum, clearly inhibits active absorption of taurocholic acid (TCA) and glycocholic acid (GCA) from rat ileum and does not inhibit passive absorption of cholic acid (CA) from the rat jejunum. In rat ileal brush-border membrane vesicles, S-8921 inhibited the sodium-dependent uptake of TCA in a concentration-dependent manner with IC50 of 2.1 microM, not the Na(+)-dependent D-glucose and L-alanine uptake. These results suggest that S-8921 is a potent, selective inhibitor of the Na(+)-dependent bile acid transport system in the ileal mucosal cell brush-border membrane, and this inhibition is the mechanism by which this drug decreases intestinal bile acid reabsorption to result in a significant decrease of plasma cholesterol.

Animals↗
PubMed1998

[Efficacy of nicardipine prolonged-release pellet on cerebral vasospasm in dogs].

A drug delivery system using copoly (lactic/glycolic acid) was developed for the intrathecal administration of nicardipine. This system was tested to determine its efficacy in preventing cerebral vasospasm in dogs. A rod-shaped implant (1-mm diameter, 10-mm long and which contained approximately 10% of nicardipine) was prepared by a heat compression method. In in vitro studies, 8% of the actual nicardipine loaded was released during day 1, 17% within day 2, 62% within day 4, and 96% within day 10. In in vivo studies, 12 dogs were assigned to two groups: the placebo group and the group treated with nicardipine. Angiography before the experiment was performed followed by right craniectomy and induction of subarachnoid hemorrhage by the placement of a clot in the Sylvian fissure. Eight pellets, either containing 8 mg of nicardipine or without nicardipine were placed in the cistern. On day 7, the angiography was repeated and cerebrospinal fluid was removed from the cisterna magna. The animals were then sacrificed, and the brain and blood clot were taken out. Cerebral vessels were measured three times with a calibrated optical micrometer, and the mean value was obtained. Average reduction of vessel diameters on the clot side in the placebo and nicardipine groups were -37% and -10% on the internal carotid (IC), -48% and -3% on the middle cerebral (MC), and -28% and -1% on the anterior cerebral artery (AC), respectively. There were significant differences in vessel diameters of IC (p < 0.05), MC (p < 0.005), and AC (p < 0.05) between these groups. No vasospasm was found in the left side. The nicardipine concentration in the clot was determined by high performance liquid chromatography. The mean concentration of nicardipine in the clot was 1.5 x 10(-4)M, at which concentration sufficient relaxation is evoked in vitro. The pellets did not last beyond day 7. Regarding the histological findings, there was a marked difference in vessel diameters between the placebo and the nicardipine treated groups. The arteries were surrounded with red blood cells and inflammatory cells. There were no specific changes related to the pellets and no sign of meningoencephalitis. A prolonged-release preparation of nicardipine that can be implanted intracranially at the time of surgery prevented vasospasm significantly in dogs. These results suggest that this new drug delivery system might be put into effect favorably in patients suffering from vasospasm due to subarachnoid hemorrhage.

Animals↗
PubMed1998

[Usefulness of positron emission tomography to STA-MCA anastomosis for the case with cerebral infarction].

The ischemic area surrounding the cerebral infarction in the eloquent area was salvaged by STA-MCA bypass surgery. Both the misery perfusion area evaluated by positron emission tomography (PET) using the [15O] gas inhalation steady-state method and clinical symptoms improved within a year after surgery. To confirm the ischemic area and select the suitable recipient artery was important for successful bypass surgery, because only an artery covering the ischemic area is expected to have low resistance. In this case a large ischemic area with disturbed, vasodilatation by Diamox was detected on the SPECT. However, PET clearly exposed the localized misery perfusion area in the overestimated ischemic area by SPECT. We describe our experience and discuss the technique and efficacy of PET for STA-MCA anastomosis surgery.

Cerebral Arteries↗
PubMed1998

[Spontaneous resolution of idiopathic spinal subdural hematoma: case report].

A 32-year-old female presented with about a week's history of dull pain in the head and neck pain. On the day of admission, she experienced a sudden onset of severe pain, extending from the occipital to the lower back region and hypesthesia and paresthesia of the right C8-Th3 dermatome. On admission, she was alert but there was a band of hypesthesia and paresthesia at the level of C8-Th3. Cervical X-ray, computed tomography scan (CT) and magnetic resonance images (MRI) of the head revealed no definite abnormalities. The peripheral blood picture was normal, and the bleeding and clotting times were in the normal range. The cerebrospinal fluid (CSF) was bloody and the CSF pressure was 220 mm H2O. An emergent 4-vessel intracranial angiography disclosed no abnormal findings. MRI and myelography revealed an intradural extramedullary lesion extending from the Th2 to the Th5 levels, ventral to the spinal cord. A T1-weighted MR image showed iso-low intensity and a T2-weighted MR image showed low intensity at these lesions. Spinal angiography revealed no abnormal vessels. In one month, spinal compression cleared gradually without surgery and her clinical symptoms disappeared about 40 days after admission. Spontaneous spinal subdural hematoma is uncommon and only 28 cases have been reported. 15 cases have been treated with surgical procedure, but spinal compression cleared spontaneously in this case. This spontaneous recovery is such a rare event that only 3 such cases have been reported in the literature. However, with the prevailing MRI, this disease will be detected more frequently in the future.

Adult↗
PubMed1997

A novel control system for polymerase chain reaction using a RIKEN GS384 thermalcycler.

We have developed a novel high-throughput thermalcycler, the RIKEN GS384, which has a maximum of 1536 wells and whose temperature can be controlled accurately and simultaneously for a very small volume of a reaction mixture. In practice, the reaction is carried out using four 384-well (3.5 mm in diameter) plate formats which can be automatically moved using a robotic arm. To achieve accurate temperature control with high thermo-conductivity, we adopted Teflon-coated aluminum well plates closely sandwiched between silicon sheet-covered lids on top and a graphite sheet below. The lids were kept at a higher temperature (2 to 5 degrees C) than the reaction wells. The temperature of the 1536 sample wells was controlled accurately without temperature variability among the wells or evaporation, even for samples of very small volume (minimum 2 microliters). We also developed a new type of plate format which is similar to the 384-well place in terms of plate size, shape, and material, but which differs in the number (1536) and size (1.6 mm in diameter) of the wells. Since the amplification reactions could be done precisely as well, a total of 6144 reactions can potentially be carried out simultaneously using the GS384 thermalcycler. This is very promising for DNA microfabrication technology. This thermalcycler offers the advantage of high-throughput DNA analysis which should be useful for DNA diagnoses or for the human genome project.

Humans↗
PubMed1997

YSK1, a novel mammalian protein kinase structurally related to Ste20 and SPS1, but is not involved in the known MAPK pathways.

To clarify the upstream regulatory mechanism of mitogen-activated protein kinase (MAPK), we performed the reverse transcriptase-based polymerase chain reaction (RT-PCR) with degenerate primers synthesized based on sequences conserved among the kinase domains of yeast MAPK kinase kinases (MAPKKKs), Stell, Bck1, and Byr2. We isolated several mammalian cDNA fragments that encode kinase subdomains sharing significant sequence homology with yeast MAPKKKs. Subsequent screening of a HeLa cell cDNA library using one of these cDNA fragments as a probe resulted in the isolation of a full-length cDNA that encodes a novel protein kinase. The catalytic domain sequence of this gene product is closely related to those of budding yeast Sps1 and Ste20 protein kinases. Thus, we call this protein YSK1 (Yeast Sps1/Ste20-related Kinase 1). The transcript of YSK1 was detected in a wide range of tissues and cells. Immunoprecipitated YSK1 shows protein kinase activity. Although YSK1 is significantly similar in its kinase domain to kinases of the yeast and mammalian MAPK pathways, the overexpression of YSK1 did not lead to the activation of the ERK (extracellular signal-regulated kinase) pathway, JNK (c-Jun NH2-terminal kinase)/SAPK (stress-activated protein kinase) pathway, or p38/Mpk2 pathway. These results suggest that YSK1 may be involved in the regulation of a novel intracellular signaling pathway.

Amino Acid Sequence↗
PubMed1997

A domain containing the Cdc42/Rac interactive binding (CRIB) region of p65PAK inhibits transcriptional activation and cell transformation mediated by the Ras-Rac pathway.

The molecular bases of the versatile functions of Rho-like GTPases are still unknown. Using luciferase assays with rat 3Y1 cells, we found that Rac1 is integrated downstream of Ras in the TRE (TPA response element) activation pathway. Coexpression of a mutant of p65PAK, PAK/RD, lacking the kinase domain but containing the Cdc42/Rac interactive binding (CRIB) region, suppressed the TRE activation and cell transformation caused by constitutively activated forms of Ras (RasV12) and Rac1 (Rac1V12). PAK/RD is a good tool to investigate the signaling pathways in which Rac and Cdc42 are involved.

Amino Acid Sequence↗
PubMed1997

Involvement of caspases in apoptosis induced by ultraviolet B irradiation in A431 human epithelioid tumor cells.

Six hours after ultraviolet B (UVB) irradiation (11.6 mJ/cm2), the viability of A431 cells decreased, and, at the same time, fragmentation of genomic DNA into nucleosomal units was observed. Z-Asp-CH2-DCB (100 microM), an inhibitor of interleukin-1 beta-converting enzyme (caspase-1) and caspase-1-like proteases, markedly inhibited UVB-induced cell death and DNA fragmentation. Both YVAD-CMK, an inhibitor of caspase-1, and DEVD-CHO, an inhibitor of caspase-3, moderately inhibited the UVB-induced cell death. A combination of YVAD-CMK and DEVD-CHO acted additionally in inhibiting cell death. These observations suggest strongly the cooperative involvement of caspases in the apoptosis induced in A431 cells by UVB.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Current treatment of brain abscess in patients with congenital cyanotic heart disease.

OBJECTIVE: The goal of this study was to define clearly the role of management in patients with cyanotic heart disease and brain abscesses by evaluating retrospectively the factors influencing poor outcome in these patients. METHODS: This study included 62 patients with cyanotic heart disease and brain abscesses diagnosed in the computed tomography era. Basic characteristic parameters (number, size, location, computed tomographic classification and organism type of abscess, convulsion, type of cyanotic heart disease, age distribution, immunocompromised status, pretreatment neurological state, and intraventricular rupture of brain abscess [IVROBA]) and therapeutic parameters (type of antibiotics and duration of administration, steroid medication and therapeutic modalities, aspiration with or without cerebrospinal fluid drainage, total extirpation after aspiration, or primary extirpation and medical treatment) were evaluated as independent predictors of poor outcome (totally disabled state or death) by using univariate and multivariate logistic regression analysis. We also statistically estimated the possible causes of IVROBA and the multiplicity of brain abscess. RESULTS: Although there were no statistically significant correlations between patients with good and poor outcomes in regard to other basic characteristic and therapeutic parameters, patients with poor outcomes were older (P < 0.02), more frequently had IVROBA (P < 0.005), and had a higher frequency of neurological deterioration (P < 0.01) than those with good outcomes. Multiple logistic regression analysis predicted that poor outcome increased the relative risk of IVROBA by a factor of 18.9 (odds rate, 18.9; 95% confidence interval, 1.7-211.6; P < 0.02). More patients with multiple abscesses had positive immunocompromised states than those with single abscesses (P < 0.01). Deep-located abscesses also more frequently had IVROBA (P < 0.005) and abscesses located in the parieto-occipital region ruptured into the occipital horn of the lateral ventricle in a short period (P < 0.02). CONCLUSIONS: Our findings suggest that IVROBA strongly influences poor outcome in patients with cyanotic heart disease. The key to decreasing poor outcomes may be the prevention and management of IVROBA. To reduce operative and anesthetic risk in these patients, abscesses should be managed by less invasive aspiration methods guided by computed tomography. Abscesses larger than 2 cm in diameter, in deep-located or parieto-occipital regions, should be aspirated immediately and repeatedly, mainly using computed tomography-guided methods to decrease intracranial pressure and avoid IVROBA. IVROBA should be aggressively treated by aspiration methods for the abscess coupled with the appropriate intravenous and intrathecal administration of antibiotics while evaluating intracranial pressure pathophysiology.

Adolescent↗
PubMed1997

The stability of truncated epidermal growth factor receptor mRNA is higher than that of the full-length receptor mRNA in rat hepatoma cells.

Truncated and full-length epidermal growth factor (EGF) receptors are produced in rat liver cells. The truncated EGF receptor mRNA is almost identical to the full-length EGF receptor mRNA except for the lack of a 3' region of the full-length receptor mRNA. To understand the stability of rat EGF receptor mRNAs, we analyzed the expression of EGF receptor mRNAs in the hepatoma cell line, AH66 and liver cells. Ten, 7 and 5 kb full-length and 2.7 kb truncated EGF receptor mRNAs were detected in both of them. The half-lives of the 10 and 2.7 kb EGF receptor mRNAs were determined in AH66 cells using a transcriptional inhibitor, 5,6-dichlororibofuranosylbenzimidazole. The half-lives of the 10 and 2.7 kb mRNAs were 1.2 and 11 h, respectively. These results indicated that the truncated mRNA is 4 times more stable than the full-length mRNA in rat cells. As for the stability, the role of a sequence of 3'-untranslated region of the EGF receptor mRNA was discussed.

Animals↗
PubMed1997

Induction of a receptor-mediated genomic DNA fragmentation in rat thymus following administration of glucocorticoids with different biopotencies: an approach by a cell-free system.

To further understand a receptor-mediated apoptosis in rat thymus, we undertook experiments to examine the relationship between the induction of genomic DNA fragmentation and the depletion and the replenishment of cytosolic glucocorticoid receptor in rat thymus. Following administration of dexamethasone and prednisolone which had been known as synthetic glucocorticoids with different biological activities, we observed the fragmentation of thymus DNA in a dose-dependent manner. The time course and the extent of DNA fragmentation induced by these glucocorticoids were closely related to the degree of receptor depletion and the length of the depletion period. Relative biopotencies of dexamethasone and prednisolone estimated by their abilities to induce the DNA fragmentation were approximately 50:1. Administration of dexamethasone in combination with 2-deoxy-D-glucose, a potent inhibitor of the glycolytic pathway, significantly decreased the ability of dexamethasone to induce the DNA fragmentation, suggesting that a receptor-mediated DNA fragmentation by glucocorticoids is an ATP-dependent process. As an attempt to elucidate the molecular mechanism of DNA fragmentation in vivo, we reconstituted a cell-free system of thymus nuclei and cytosol fraction. Using the thymus cytosol fraction from dexamethasone-treated rats, we demonstrated for the first time an ATP-dependent fragmentation of nuclear DNA into nucleosomal units in vitro. Toward further understanding of the biochemical process of glucocorticoid-induced apoptosis in the thymocyte, the cell-free system reconstituted in the present study might provide a useful model system.

Animals↗
PubMed1996

High-efficiency full-length cDNA cloning by biotinylated CAP trapper.

We have devised a method for efficiently constructing high-content full-length cDNA libraries based on chemical introduction of a biotin group into the diol residue of the cap structure of eukaryotic mRNA, followed by RNase I treatment to select full-length cDNA. The selection occurs by trapping the biotin residue at the cap sites using streptavidin-coated magnetic beads, thus eliminating incompletely synthesized cDNAs. When this method was used to construct a mouse brain full-length cDNA library, our evaluation showed that more than 95% of the total clones were of full length, and recombinant clones could be produced with high efficiency (1.2 x 10(7)/10 micrograms starting mRNA). The analysis of 120 randomly picked clones indicates an unbiased representation of the starting mRNA population.

Animals↗
PubMed1996

Activation of the JNK pathway by distantly related protein kinases, MEKK and MUK.

JNK/SAPKs are identified as new members of the MAPK family; they phosphorylate c-Jun protein in response to several cellular stimuli including ultraviolet irradiation, TNF and osmotic shock. We have identified a protein kinase, MUK, as an activator of the JNK-pathway, whose kinase domain shows significant homology to MAPKKK-related proteins such as c-Raf and MEKK. The over-expression of MUK or MEK kinase (MEKK) in NIH3T3 or COS1 cells results in the activation of JNK1 and the accumulation of a hyper-phosphorylated form of c-Jun. While MEKK also activates the ERK pathway, MUK is a rather selective activator of the JNK pathway. On the other hand, c-Raf activates the JNK pathway only slightly despite its remarkable ability to activate the ERK pathway. Even though we originally identified MUK as a MAPKKK-related protein kinase, a greater similarity to mixed lineage kinase (MLK) is found not only in the catalytic domain but also in the 'leucine-zipper'-like motifs located at the C-terminal side of the catalytic domain. The structural divergence between MUK and MEKK reveals the multiplicity of signaling pathways that activate JNK/SAPKs.

3T3 Cells↗