Search PubMed⌕ Search

Biomedical subjects

T Sakaki

Publications and source records attributed to T Sakaki.

At least 73 records · Page 4Linked to original sources
PubMed2000

Practical application of mammalian cytochrome P450.

Heterologous expression systems play an important role in the analysis of structure-function relationships of mammalian P450s. In addition, these expression systems allow practical application of mammalian P450s. Genetically engineered fused enzymes between mammalian P450 and yeast NADPH-P450 reductase have possible applications in bioconversion processes. Combined use of techniques reported thus far could produce steroid hormones in the recombinant yeast cells harboring four P450 species, CYP11A1, CYP17A1, CYP21B1 and CYP11B1. In an Escherichia coli expression system, the technology of the construction of the mitochondrial P450 electron transport chain has been established. The recombinant E. coli cells expressing CYP27B1, adrenodoxin and NADPH-adrenodoxin reductase would be applicable to a bioconversion process to produce 1alpha,25-dihydroxyvitamin D3. We also demonstrated the usefulness of heterologous expression systems for human liver microsomal P450s for the prediction of drug metabolism in the human body. Microsomal fractions prepared from recombinant yeast, insect and mammalian cells are commercially available and play an important role in preclinical drug development. Application of mammalian P450 to bioremediation with genetic engineering has also been developed. Thus, mammalian P450s appear to have great potential for a wide range of practical applications.

Journal Article↗
PubMed2000

Use of spiral computerized tomography angiography in patients with subarachnoid hemorrhage in whom subtraction angiography did not reveal cerebral aneurysms.

OBJECT: Patients with subarachnoid hemorrhage (SAH) in whom angiography does not demonstrate diagnostic findings sometimes suffer recurrent disease and actually harbor undetected cerebral aneurysms. The management strategy for such cases remains controversial, but technological advances in spiral computerized tomography (CT) angiography are changing the picture. The purpose of this prospective study was to examine how spiral CT angiography can contribute to the detection of cerebral aneurysms that cannot be visualized on angiography. METHODS: In 134 consecutive patients with SAH, a prospective search for the source of bleeding was performed using digital subtraction (DS) and spiral CT angiography. In 21 patients in whom initial DS angiography yielded no diagnostic findings, spiral CT angiography was performed within 3 days. Patients in whom CT angiography provided no diagnostic results underwent second and third DS angiography sessions after approximately 2 weeks and 6 months, respectively. Six patients with perimesencephalic SAH were included in the 21 cases. Six of the other 15 patients had small cerebral aneurysms detectable by spiral CT angiography, five involving the anterior communicating artery and one the middle cerebral artery. Two patients in whom initial angiograms did not demonstrate diagnostic findings proved to have a ruptured dissecting aneurysm of the vertebral artery; in one case this was revealed at autopsy and in the other during the second DS angiography session. A third DS angiography session revealed no diagnostic results in 13 patients. CONCLUSIONS: Spiral CT angiography was useful in the detection of cerebral aneurysms in patients with SAH in whom angiography revealed no diagnostic findings. Anterior communicating artery aneurysms are generally well hidden in these types of SAH cases. A repeated angiography session was warranted in patients with nonperimesencephalic SAH and in whom initial angiography revealed no diagnostic findings, although a third session was thought to be superfluous.

Adult↗
PubMed2000

Effect of age on cerebral venous circulation disturbances in the rat.

OBJECT: Mild cerebral venous circulation disturbances (CVCDs) in aged patients are frequently known to cause unexpectedly severe postoperative complications in neurosurgical practice. The object of the present study was to determine whether there are age-related differences involved in vulnerability to CVCDs. METHODS: Thirty-eight male Wistar rats were used. A single cortical vein with a 100-microm diameter was occluded photochemically by using rose bengal dye and fiberoptic illumination in young (Group Y, 19 animals aged 10-14 weeks) and aged (Group A, seven animals aged 80-100 weeks) rats. Five young and seven aged animals served as sham-operated controls. Regional cerebral blood flow (rCBF) was determined from local CBF, which was measured at 25 (5 x 5) identical locations, with the occluded vein located central to the scanning field, by using a laser Doppler scanning technique every 15 minutes for 90 minutes after venous occlusion. The cerebral venous flow pattern was examined using fluorescence angiography until 90 minutes after occlusion. Histological specimens were examined 24 hours after occlusion. In Group Y, rCBF did not change significantly after venous occlusion. However, in Group A, rCBF decreased rapidly beginning 15 minutes after occlusion. Significant intergroup differences were observed 30, 60, and 90 minutes after occlusion. Venous flow arrest, which resulted in venous infarct, was observed on angiography 90 minutes after occlusion in two (10.5%) of 19 young and six (85.7%) of seven aged rats. The venous thrombus in Group A rats was significantly larger than that in Group Y rats 90 minutes after occlusion. Venous infarction was seen in all aged rats (100%) and in six young rats (31.6%); the infarct size, expressed as a percentage of the size of the ipsilateral hemisphere, was significantly larger in aged rats than in young rats. CONCLUSIONS: This study demonstrated an age-related increase in the rate and size of venous infarct following vein occlusion, suggesting that the greater vulnerability to CVCDs in the aged brain might be attributed to early and extensive hypoperfusion of circumscribed brain areas drained by the occluded vein. The larger thrombus formation in aged animals indicates that a shift in the thrombogenetic/thrombolytic equilibrium is responsible for the observed effect.

Aging↗
PubMed2000

Effect of direct arterial bypass on the prevention of future stroke in patients with the hemorrhagic variety of moyamoya disease.

OBJECT: The authors evaluated the effects of superficial temporal artery-middle cerebral artery (STA-MCA) bypass in the prevention of future stroke, including rebleeding or an ischemic event, in patients suffering from hemorrhagic moyamoya disease by comparing this method with indirect bypass and conservative treatment. METHODS: Twenty-two patients who had hemorrhagic moyamoya disease but no aneurysm comprised the study group. These patients' clinical charts were examined with respect to their treatment and clinical course after an initial hemorrhagic episode. The mean age of the patients was 43 years and the follow-up period ranged from 0.8 to 15.1 years, with a mean of 8 years. Eleven patients (50%) were conservatively treated. Among the 11 patients who were surgically treated, STA-MCA bypass was performed in six patients (27%) and encephaloduroarteriosynangiosis (EDAS) in the other five patients (23%). Nine patients (41%) presented with an ischemic or rebleeding event during the follow-up period. The incidence of future stroke events in patients who had undergone an STA-MCA bypass was significantly lower (p<0.05) than that in patients who had been treated conservatively or with EDAS. Kaplan-Meier plots comparing stroke-free times in patients treated with direct bypass and those in patients who conservatively or with indirect bypass showed a significant difference (p<0.05) in favor of direct bypass. CONCLUSIONS: The effect of STA-MCA bypass on the prevention of recurrent hemorrhage or an ischemic event in patients with hemorrhagic moyamoya disease has been statistically confirmed in this study.

Adult↗
PubMed2000

[A surgical case of growing cavernous angioma at the pontomedullary junction].

We described a surgical case of growing cavernous angioma located at the pontomedullary junction. This 52-year-old woman presented with symptoms caused by a small hemorrhage in the right cerebellopontine angle. Magnetic resonance images (MRI) suggested cavernous angioma as the underlying pathology. 9 months after the first episode, the second hemorrhage occurred with a deteriorated neurological state that disappeared under conservative treatment except for right facial paresis and hearing disturbance. During careful observation for 1 year, the size of the lesion gradually increased on MRI and additional neurological deficits including left hemiparesis and right abducent nerve palsy were diagnosed. The first operation was carried out through the right lateral suboccipital approach, but only partial removal of the cavernous angioma was accomplished due to the overlying seventh and lower cranial nerves. After more than 4 months, a third hemorrhagic episode was presented with a sudden onset of right cerebellar signs and facial numbness. The cavernous angioma grew in size to reach the ventrolateral corner of the 4th ventricle with dense hemosiderin deposition around the core lesion on MRI. An enhancement inside the lesion was also demonstrated after gadolinium-diethylenetriaminepenta-acetic acid administration. The second operation through the midline suboccipital approach was selected for the complete resection of the residual cavernous angioma. The lesion was too hard to resect without internal decompression. The pontine part of the lesion was almost totally resected, but manipulation for the medullary part to create a discrete layer between the lesion and surrounding neural tissues was unsuccessful and generated severe bradycardia, so this part of the cavernous angioma had to be left. The problems for the management of cavernous angioma in the brain stem should be discussed, especially focussing on the surgical indication in reference to our experience and previous literatures.

Brain Stem Neoplasms↗
PubMed1999

BDNF atelocollagen mini-pellet accelerates facial nerve regeneration.

We investigated the effect of BDNF mini-pellet on the GAP-43 mRNA expression and functional status of facial nerve in a rat model of facial nerve transection and immediate repair. The facial function started to recover at 17 days in the placebo group and 14 days in the BDNF group. BDNF group had shorter period of increased GAP-43 mRNA expression than the placebo group. Topically applied BDNF may accelerate the facial nerve regeneration.

Animals↗
PubMed1999

Dynamic mobility of genetically expressed fusion protein between cytochrome P4501A1 and NADPH-cytochrome P450 reductase in yeast microsomes.

A fusion protein of rat liver CYP1A1 with NADPH-cytochrome P450 reductase was expressed genetically in yeast microsomal membranes. This flavo-cytochrome is active in 6-hydroxylation of zoxazolamine. Rotational diffusion of the fusion protein was examined by observing the flash-induced absorption anisotropy r(t) of the P450.CO complex. Theoretical analysis of r(t) was performed based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 2 ms to a time-independent value r(3). Forty percent of the fusion protein rotated with a rotational relaxation time phi of 1.35 ms. Treatment with high salt increased the mobile population of the fusion protein to 62% with phi = 0.96 ms. The mobile population of the fusion protein is close to that of CYP1A1 coexpressed with the P450 reductase and greater than that of CYP1A1 alone [Iwase et al. (1991) Biochemistry 30, 8347-8351]. The large mobile population of the fusion protein provides evidence that CYP1A1 is mobilized by forming associations with P450 reductase in microsomal membranes.

Animals↗
PubMed1999

Electrostatic interaction between cytochrome P450 and NADPH-P450 reductase: comparison of mixed and fused systems consisting of rat cytochrome P450 1A1 and yeast NADPH-P450 reductase.

The electrostatic interaction between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase was analyzed by using recombinant yeast microsomes containing both native enzymes or their fused enzyme. The Vmax of the 7-ethoxycoumarin O-deethylation in the recombinant microsomes containing both rat cytochrome P4501A1 and yeast NADPH-P450 reductase (the mixed system) was maximal when the ionic strength of the reaction mixture was 0.1-0.15. However, on the fused enzyme between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase (the fused system), the activity was uniformly reduced with increasing ionic strength. The pH profiles of Vmax were also different between the mixed and the fused systems. Based on these results, we propose a hypothesis that cytochrome P450 and NADPH-P450 reductase have more than one binding mode. The maximal activity of the mixed system at ionic strength of 0.1-0.15 is explained by change of the binding mode. On the other hand, the fused enzyme appears to have only one binding mode due to the limited topology of cytochrome P450 and NADPH-P450 reductase domains.

Animals↗
PubMed1999

Effects of bypass on CO2 cerebrovascular reactivity in ischaemic cerebrovascular diseases--based on the intra-operative LCBF and CO2 cerebrovascular reactivity studies.

The authors evaluated the effects of superficial temporal to middle cerebral artery (STA-MCA) bypass on CO2 cerebrovascular reactivity (CVR) in ischaemic cerebrovascular diseases (CVDs). Local cerebral blood flow (LCBF) and CO2 CVR in 19 patients with ischaemic CVD subjected to standard STA-MCA bypasses were examined during surgery. Single photon emission computed tomography (SPECT) with acetazolamide (ACZ) activation was also performed before and at 1 month after surgery. The results are as follows. 1) Before bypass, the average CO2 CVR value was -1.50 +/- 2.30%/mmHg (mean +/- SD). SPECT showed disturbed response to ACZ in all cases. Fifteen cases showed the steal phenomenon. After bypass, the mean CO2 CVR value significantly (p < 0.05) increased, and four cases resolved their steal phenomenon. 2) Before bypass, the mean LCBF was significantly (p < 0.05) lower than the control level. After bypass, the mean LCBF significantly (p < 0.05) increased. 3) In the postoperative SPECT findings, 13 cases showed a disturbed response to ACZ. The CO2 CVR value in these 13 cases was -1.21 +/- 1.19%/mmHg, which was significantly (p < 0.05) low compared to the values for the 6 cases showing normal postoperative ACZ responses. In ischaemic CVDs before bypass, the CO2 CVR values were extremely low. After bypass, however, CO2 CVR and LCBF values significantly improved. SPECT findings, including ACZ challenge, correlated well to the LCBF and CO2 CVR values. STA-MCA by pass exerted a favourable effect on the CO2 CVR and LCBF values immediately after bypass in the cases showing a reduced pre-operative response to CO2.

Acetazolamide↗
PubMed1999

Cyclopropenone-containing cysteine proteinase inhibitors. Synthesis and enzyme inhibitory activities.

By focusing on the amphiphilic properties of cyclopropenone (e.g. a good electrophile and a precursor for a stable 2pi-aromatic hydroxycyclopropenium cation), a new class of cysteine proteinase inhibitors containing a cyclopropenone moiety was designed. For the purpose of the present research, we needed to devise a new method to introduce a peptide-related moiety as a substituent on the cyclopropenone residue. We investigated the reaction of metalated cyclopropenone acetal derivatives (2, R2 = metal) with N-protected alpha-aminoaldehydes 4 to obtain the adduct 5, and succeeded in the preparation of highly potentiated cysteine proteinase inhibitors 8 after several steps transformations. They showed strong inhibitory activities only to cysteine proteinases such as calpain, papain, cathepsin B, and cathepsin L and not to serine (e.g. thrombin and cathepsin G) and aspartic proteinases (e.g. cathepsin D). Kinetic studies indicated that they are competitive inhibitors, and by the examinations of their inhibitory mechanism it became clear that they are reversible inhibitors.

Calpain↗
PubMed1999

Enzymatic properties of mouse 25-hydroxyvitamin D3 1 alpha-hydroxylase expressed in Escherichia coli.

Renal 25-hydroxyvitamin D3 1 alpha-hydroxylase cDNA cloned from the kidneys of mice lacking the vitamin D receptor was expressed in Escherichia coli JM109. As expected, the bacterially-expressed enzyme catalyzes the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 with a Michaelis constant, K(m), value of 2.7 microM. Unexpectedly, the enzyme also hydroxylates the 1 alpha-position of 24,25-dihydroxyvitamin D3 with a K(m) of 1.3 microM, and a fourfold higher Vmax/K(m) compared with the 25-hydroxyvitamin D3 hydroxylase activity, suggesting that 24,25-dihydroxyvitamin D3 is a better substrate than 25-hydroxyvitamin D3 for 1 alpha-hydroxylase. In addition, the enzyme showed 1 alpha-hydroxylase activity toward 24-oxo-25-hydroxyvitamin D3. However, it showed only slight activity towards 23,25-dihydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and no detectable activity towards vitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3. These results suggest that the 25-hydroxyl group of vitamin D3 is essential for the 1 alpha-hydroxylase activity and the 24-hydroxyl group enhances the activity, but the 23-hydroxyl group greatly reduced the activity. Another remarkable finding is that living recombinant E. coli cells can convert the substrates into the 1 alpha-hydroxylated products, suggesting the presence of a redox partner of 1 alpha-hydroxylase in E. coli cells.

24,25-Dihydroxyvitamin D 3↗
PubMed1999

Metabolic studies using recombinant escherichia coli cells producing rat mitochondrial CYP24 CYP24 can convert 1alpha,25-dihydroxyvitamin D3 to calcitroic acid.

Previously we expressed rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) cDNA in Escherichia coli JM109 and showed that CYP24 catalyses three-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 [Akiyoshi-Shibata, M., Sakaki, T., Ohyama, Y., Noshiro, M., Okuda, K. & Yabusaki, Y. (1994) Eur. J. Biochem. 224, 335-343]. In this study, we demonstrate further oxidation by CYP24 including four- and six-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3, respectively. When the substrate 25-hydroxyvitamin D3 was added to a culture of recombinant E. coli, four metabolites, 24, 25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3, 24-oxo-23, 25-dihydroxyvitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3 were observed. These results indicate that CYP24 catalyses at least four-step monooxygenation toward 25-hydroxyvitamin D3. Furthermore, in-vivo and in-vitro metabolic studies on 1alpha,25-dihydroxyvitamin D3 clearly indicated that CYP24 catalyses six-step monooxygenation to convert 1alpha,25-dihydroxyvitamin D3 into calcitroic acid which is known as a final metabolite of 1alpha,25-dihydroxyvitamin D3 for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3.

Animals↗
PubMed1999

Enzymatic properties of human 25-hydroxyvitamin D3 1alpha-hydroxylase coexpression with adrenodoxin and NADPH-adrenodoxin reductase in Escherichia coli.

We have cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase cDNAs from normal subjects and patients with pseudovitamin D-deficient rickets (PDDR), and expressed the cDNAs in Escherichia coli JM109 cells. Kinetic analysis of normal 1alpha-hydroxylase in the reconstituted system revealed that Km values for 25(OH)D3 and (24R), 25(OH)2D3 were 2.7 and 1.1 microM, respectively. The lower Km value and higher Vmax/Km value for (24R),25(OH)2D3 indicated that it is a better substrate than 25(OH)D3 for 1alpha-hydroxylase. These results are quite similar to those of mouse 1alpha-hydroxylase. To establish a highly sensitive in vivo system, 1alpha-hydroxylase, adrenodoxin and NADPH-adrenodoxin reductase were coexpressed in E. coli cells. The recombinant E. coli cells showed remarkably high 1alpha-hydroxylase activity, suggesting that the electrons were efficiently transferred from NADPH-adrenodoxin reductase through adrenodoxin to 1alpha-hydroxylase in E. coli cells. Using this system, the activities of four mutants of 1alpha-hydroxylase, R107H, G125E, R335P and P382S, derived from patients with PDDR were examined. Although no significant reduction in expression of these mutants was observed, none showed detectable activity. These results strongly suggest that the mutations found in the patients with PDDR completely abolished 1alpha-hydroxylase activity by replacement of one amino acid residue.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1999

Overexpression of midkine in lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine in rats and its increase with progression.

The expression of midkine (MK) in lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was examined. The animals were administered 2000 p.p.m. of BHP in their drinking water for 12 weeks, then maintained without further treatment until being killed 20-28 weeks after the beginning of the experiment. MK mRNA expression of adenocarcinomas and squamous cell carcinomas assessed by means of the reverse transcriptase-polymerase chain reaction and northern blot analysis was significantly higher than in rat embryonic tissues (positive controls) and contrasted strongly with the lack in normal lungs. MK protein was detected immunohistochemically in 58.3% of alveolar hyperplasias, 92.3% of adenomas and 100% of adenocarcinomas and squamous cell carcinomas. The extent of staining significantly increased along with malignant progression in adenomatous (pre-)neoplastic lesions and tended to become more pronounced with malignant progression in squamous lesions. The results suggest that MK may play some essential roles in the development and progression of lung tumors induced by BHP in rats.

Adenocarcinoma↗
PubMed1999

The effects of prostaglandin E1 on intraoperative temperature changes and the incidence of postoperative shivering during deliberate mild hypothermia for neurosurgical procedures.

UNLABELLED: We investigated the effects of i.v. prostaglandin E1 (PGE1) on intraoperative changes of core temperature and the incidence of postoperative shivering in neurosurgical patients undergoing deliberate mild hypothermia. Eighty-three patients were randomly assigned to one of three groups: patients in the control group did not receive PGE1, whereas patients in the PG20 group and PG50 group received PGE1 at a dose of 0.02 and 0.05 microg x kg(-1) x min(-1), respectively. The administration of PGE1 was started just after the induction of anesthesia and continued until the end of anesthesia. Anesthesia was maintained with nitrous oxide in oxygen, sevoflurane, and fentanyl. After the induction of anesthesia, patients were cooled using a water blanket and a convective device blanket. Tympanic membrane temperature was maintained at 34.5 degrees C. During surgical wound closure, patients were rewarmed. Intraoperative changes in tympanic membrane and skin temperatures and the incidence of postoperative shivering were compared among groups. Demographic and intraoperative variables were similar among groups. There were no significant differences in tympanic temperatures among groups at each point during the operation. Skin temperature 30 min after rewarming and just after tracheal extubation was significantly lower in the PG20 group than in the PG50 group. Postoperative shivering was more frequent in the PG20 group (43%) than in the control (13%) and PG50 (17%) groups. These results suggest that the intraoperative administration of PGE1 does not affect changes in core temperature during deliberate mild hypothermia and that PGE1 at a dose of 0.02 microg x kg(-1) x min(-1) may increase the occurrence of postoperative shivering. IMPLICATIONS: Deliberate mild hypothermia has been proposed as a means of providing cerebral protection during neurosurgical procedures. Vasodilating drugs may be used during deliberate mild hypothermia to maintain peripheral circulation and to enhance the cooling and rewarming rate. In the present study, however, we found no benefit from i.v. prostaglandin E1 administration during deliberate mild hypothermia in neurosurgical patients.

Alprostadil↗
PubMed1999

Accuracy and availability of the computed assisted neurosurgery navigation system during epilepsy surgery.

The magnetic-force-based Computed Assisted Neurosurgery System was used for epilepsy surgery to localize targets accurately in the operative field. The correlation between X components of target coordinates in the axial plane and the coronal plane for the same target was strong in all cases. Concerning Y components, there were statistically significant differences in 2 cases. There was a case that showed statistically significant differences only in the Z dimension. The interdisk distance by data sets of coordinates obtained from neuronavigation was calculated to quantify localization error, and the measuring error ranged from -5 to 13.3 mm (1.3 +/- 3.2 mm). The magnitude of the application errors in this study tended to be large in the frontal and vertex regions.

Adolescent↗
PubMed1999

Usefulness of the dipole tracing method with a scalp-skull-brain head model: relationship between the epileptic focus and equivalent current dipole locations.

PURPOSE: We examined whether the dipole tracing (DT) method with a realistic three-shell head model and inhomogeneous electric conductivity is useful to estimate the epileptic focus from interictal spikes. METHOD: This study included 17 temporal lobe epilepsy (TLE) cases, classified into three types as type A (unilateral), type B (intermediate) and type C (bilateral) and 5 extratemporal epilepsy (XTLE) cases. The epileptic areas were determined by noninvasive and/or invasive examinations. Selected interictal spikes were analyzed and the calculated equivalent current dipoles (ECDs) of dipolarity greater than 0.98 were superimposed over the realistic head model in each patient. We evaluated the ECD concentration within and around the epileptic area. RESULTS: In TLE cases, types A showed better ECD concentration (87%) within the epileptic area than other types (type B: 68%; type C: 74%). XTLE exhibited variable ECD distribution within and around the epileptic area. CONCLUSION: The DT method with a realistic head model and inhomogeneous electric conductivity can be useful to estimate the epileptic area from interictal spikes, especially in unilateral TLE cases.

Action Potentials↗