Genetic mapping of mouse transient receptor potential (Trrp) genes responsible for capacitative calcium entry channels to chromosomes 3, 7, 9, and X.
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Animals↗
Biomedical subjects
T Okada
Publications and source records attributed to T Okada.
Electron-beam writing of large-area Fresnel zone plates.
High-resolution X-ray microscopy requires advanced fabrication technology for Fresnel zone plates (FZPs). As the resolution of an FZP depends on the width of the outermost zone, fine zone patterns for objective lenses have to be replicated. On the other hand, to achieve highly condensed X-ray beams by using FZPs for condenser lenses, large-field replication is required. A method of pattern replication of FZPs for X-ray microscopy is reported. Utilizing a 30 keV electron-beam writing tool and an FZP-generation computer program, FZP patterns for a condenser lens 1 mm in diameter with an outermost-zone width of 0.2 micro m and for an objective lens 0.5 mm in diameter with an outermost-zone width of 0.1 micro m were replicated.
Journal Article↗
Molecular cloning and functional characterization of a novel receptor-activated TRP Ca2+ channel from mouse brain.
Characterization of mammalian homologues of Drosophila TRP proteins, which induce light-activated Ca2+ conductance in photoreceptors, has been an important clue to understand molecular mechanisms underlying receptor-activated Ca2+ influx in vertebrate cells. We have here isolated cDNA that encodes a novel TRP homologue, TRP5, predominantly expressed in the brain. Recombinant expression of the TRP5 cDNA in human embryonic kidney cells dramatically potentiated extracellular Ca2+-dependent rises of intracellular Ca2+ concentration ([Ca2+]i) evoked by ATP. These [Ca2+]i transients were inhibited by SK&F96365, a blocker of receptor-activated Ca2+ entry, and by La3+. Expression of the TRP5 cDNA, however, did not significantly affect [Ca2+]i transients induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPases. ATP stimulation of TRP5-transfected cells pretreated with thapsigargin to deplete internal Ca2+ stores caused intact extracellular Ca2+-dependent [Ca2+]i transients, whereas ATP suppressed [Ca2+]i in thapsigargin-pretreated control cells. Furthermore, in ATP-stimulated, TRP5-expressing cells, there was no significant correlation between Ca2+ release from the internal Ca2+ store and influx of extracellular Ca2+. Whole-cell mode of patch-clamp recording from TRP5-expressing cells demonstrated that ATP application induced a large inward current in the presence of extracellular Ca2+. Omission of Ca2+ from intrapipette solution abolished the current in TRP5-expressing cells, whereas 10 nM intrapipette Ca2+ was sufficient to support TRP5 activity triggered by ATP receptor stimulation. Permeability ratios estimated from the zero-current potentials of this current were PCa:PNa:PCs = 14.3:1. 5:1. Our findings suggest that TRP5 directs the formation of a Ca2+-selective ion channel activated by receptor stimulation through a pathway that involves Ca2+ but not depletion of Ca2+ store in mammalian cells.
Amino Acid Sequence↗
Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex.
We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promoter and the SV40 intron/polyadenylation site. Transgenes were prepared for cloning into this vector by introduction of compatible restriction sites by PCR. A vector expressing rat granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed using DNA-protein complex as well as by traditional recombination techniques. The efficacy of our adenoviral cloning system utilizing DNA-protein complex was two logs higher than that seen using homologous recombination. All viruses generated by directional ligation of the insert into the vector DNA-protein complexes contained the desired transgene in the correct orientation. This technique greatly simplifies and accelerates the generation of recombinant adenoviral vectors.
Adenoviridae↗
Interactions of the amino acid residue at position 31 of the c-Ha-Ras protein with Raf-1 and RalGDS.
The Ras and Rap1A proteins can bind to the Raf and RalGDS families. Ras and Rap1A have Glu and Lys, respectively, at position 31. In the present study, we analyzed the effects of mutating the Glu at position 31 of the c-Ha-Ras protein to Asp, Ala, Arg, and Lys on the interactions with Raf-1 and RalGDS. The Ras-binding domain (RBD) of Raf-1 binds the E31R and E31K Ras mutants less tightly than the wild-type, E31A, and E31D Ras proteins; the introduction of the positively charged Lys or Arg residue at position 31 specifically impairs the binding of Ras with the Raf-1 RBD. On the other hand, the ability of the oncogenic RasG12V protein to activate Raf-1 in HEK293 cells was only partially reduced by the E31R mutation but was drastically impaired by the E31K mutation. Correspondingly, RasG12V(E31K) as well as Rap1A, but not RasG12V(E31R), exhibited abnormally tight binding with the cysteine-rich domain of Raf-1. On the other hand, the E31A, E31R, and E31K mutations, but not the E31D mutation, enhanced the RalGDS RBD-binding activity of Ras, indicating that the negative charge at position 31 of Ras is particularly unfavorable to the interaction with the RalGDS RBD. RasG12V(E31K), RasG12V(E31A), and Rap1A stimulate the RalGDS action more efficiently than the wild-type Ras in the liposome reconstitution assay. All of these results clearly show that the sharp contrast between the characteristics of Ras and Rap1A, with respect to the interactions with Raf-1 and RalGDS, depends on their residues at position 31.
3T3 Cells↗
Molecular cloning of a cDNA encoding a pollen extracellular protein as a potential source of a pollen allergen in Brassica rapa.
A polyclonal antiserum was raised against the extracellular pollen proteins of Brassica rapa and used for screening the expression cDNA libraries made from immature anthers. We obtained five groups of cDNA clones, including cDNAs similar to PCP1, thioredoxin, and lipid transfer protein (LTP). Recombinant protein of the cDNA clone showing sequence similarity to LTP was demonstrated to bind IgE of a patient allergic to Brassica pollen. The cDNA clone reported here, therefore, represents a novel pollen allergen of Brassica rapa.
Allergens↗
MK-801 blocks monoamine transporters expressed in HEK cells.
(+)-MK-801 is known to be a specific non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors. However, besides having an anticonvulsant effect, this compound possesses a central sympathomimetic effect and an anxiolytic-like action, raising the possibility that (+)-MK-801 might affect monoamine uptake systems. To elucidate this possibility, we investigated the effects of (+)-MK-801 on monoamine transporters expressed in HEK cells. (+)-MK-801 significantly inhibited the uptake of all three monoamine transporters in a dose-dependent manner and the inhibitions were competitive with respect to monoamines. The Ki values of (+)-MK-801 on the norepinephrine, dopamine and serotonin transporters were 3.2 microM, 40 microM and 43 microM, respectively. In addition, (-)-MK-801, a less potent antagonist of NMDA receptors, also inhibited monoamine transporters with a similar potency as that of (+)-MK-801. These results clearly indicate that MK-801, a non-competitive antagonist of NMDA receptors, competitively inhibits monoamine transporters without stereoselectivity.
ATP-Binding Cassette Transporters↗
Differential distribution of TRP Ca2+ channel isoforms in mouse brain.
Mammalian homologues of the Drosophila TRP proteins, which are essential for light-activated, phosphatidyl-inositide (PI)-dependent Ca2+ conductance in Drosophila photoreceptors, were molecularly identified, to investigate receptor-activated Ca2+ influx in the mammalian nervous system. Two cloned mouse TRP homologues, TRP3 and TRP4, structurally related to the voltage-dependent Na+ channel, were expressed predominantly in the brain, where a sharp contrast in the distribution of the RNA transcripts for TRP isoforms was demonstrated by in situ hybridization analysis. TRP3 mRNA was concentrated in cerebellar Purkinje cells and sparsely localized in the cerebellar granule layer, pontine nuclei, and thalamus, whereas TRP4 mRNA was abundantly expressed in hippocampal CA1 pyramidal neurons, dentate gyrus granule cells, and cerebral cortical neurons, and in the septal nuclei and the mitral layer of olfactory bulb. The distinct spatial patterns of TRP isoforms implicate that neurons are highly heterogeneous in receptor-activated Ca2+ influx responsible for the second phase of PI-mediated rise in intracellular Ca2+.
Amino Acid Sequence↗
Effects of maternal uninephrectomy on the development of fetal rat kidney: numerical and volumetric changes of the glomerulus and formation of the anionic site in the glomerular basement membrane.
Developmental processes of the fetal rat kidney from uninephrectomized mothers were studied. The uninephrectomy was performed on day 5 of gestation. Glomerular number and volume in the fetal kidneys on days 18, 20, and 22 of gestation were morphometrically determined. To investigate the anionic site formation in the glomerular basement membrane, distribution of cationized ferritin (CF) in the fetal glomerulus was examined electron microscopically after CF injection. Blood urea nitrogen (BUN) concentration in the pregnant rats was also determined on various days after uninephrectomy. On fetal days 20 and 22, the glomerular volume was significantly larger in the fetuses from uninephrectomized mothers than in those from sham-operated ones. On fetal day 20, the CF particles were clustered in the laminae rarae interna and externa of the glomerular basement membrane in the fetuses from uninephrectomized mothers, while the clusters were arrayed in three to four layers in the glomerular basement membrane in the fetuses from sham-operated ones. On fetal day 22, the CF particles noted in the lamina rara externa in the fetuses from uninephrectomized mothers were slightly larger in number than such particles in the age-matched control fetuses. The BUN concentration of the uninephrectomized pregnant rats was significantly higher than that of the sham-operated pregnant ones on each postoperative day. These results suggest that the development of the fetal renal glomerulus is accelerated by the elevated BUN level following maternal uninephrectomy when the fetal kidney is functional in effective filtration in the rat.
Animals↗
Circadian rhythm of autonomic activity in patients with obstructive sleep apnea syndrome.
BACKGROUND AND HYPOTHESIS: Although the immediate effects of sleep apnea on hemodynamics and the neurological system have been studied, little is known about the circadian rhythm of heart rate variability in patients with obstructive sleep apnea syndrome (OSAS). The purpose of the present study was to investigate the effects of sleep apnea on the autonomic activity during daytime, which may play some role in the pathogenesis of cardiovascular complications in OSAS. METHODS: We studied 18 middle-aged male patients with OSAS and 10 age-matched control subjects. Patients with OSAS were classified according to the severity of OSAS: patients with an apnea index (AI) < 20 were considered to have mild OSAS (Group 1, n = 8) and patients with an AI > or = 20 were considered to have severe OSAS (Group 2, n = 10). Heart rate variability was calculated from the 24-h ambulatory electrocardiograms by the Fourier transformation. Power spectra were quantified at 0.04-0.15 Hz [low frequency power (LF)ln(ms2)] and 0.15-0.40 Hz [high frequency power (HF)ln(ms2)]. The HF component and the ratio of LF to HF were used as indices of the parasympathetic and sympathetic activity, respectively. RESULTS: The circadian rhythms of the LF, HF, and LF/HF ratio differed significantly in Group 2 compared with Group 1 and control subjects (p < 0.05). Hypertension (> 160/95 mm Hg) was found in 7 (70.0%) of 10 patients in Group 2, and in 1 (12.5%) of 8 patients in Group 1. Echocardiographic evidence of left ventricular hypertrophy (LVH) (an interventricular septal thickness or a left ventricular posterior wall thickness > or = 12 mm) was found in 3 (30.0%) of 10 patients in Group 2, and in 1 (12.5%) of 8 patients in Group 1. The mean HF from 4 A.M. to 12 noon was significantly lower in Group 2 than in Group 1 and the control group, and it correlated significantly with the lowest nocturnal SaO2 (r = 0.58, p < 0.05). The mean LF/HF ratio during the same period was significantly higher in Group 2 than in Group 1 and the control group, and it correlated significantly with total time of the nocturnal oxygen saturation < 90% (r = 0.64, p < 0.005) and the lowest nocturnal SaO2 (r = 0.56, p < 0.05). Ventricular tachycardia was found in the early morning in one patient, ST-T depression in two patients, and sinus arrest in two patients in Group 2. CONCLUSION: These findings suggest that sleep-disordered breathing associated with severe oxygen desaturation might influence heart rate variability not only during sleep but also during daytime. OSAS per se might contribute to altered circadian rhythm in autonomic activity leading to the development of cardiovascular diseases.
Adult↗
Twenty-four-hour ambulatory oxygen desaturation and electrocardiographic recording in obstructive sleep apnea syndrome.
BACKGROUND: Although nocturnal pulseoximetry is routinely performed in obstructive sleep apnea syndrome (OSAS), pulseoximetry over a 24-h period has not been studied. HYPOTHESIS: The purpose of the study was to determine whether simultaneous 24-h oxygen desaturation and electrocardiographic (ECG) recording might be used to screen for daytime sleep sequelae in patients with OSAS. METHODS: Simultaneous recording of arterial oxygen saturation (SpO2) and ECG was conducted over a 24-h period in 18 male patients with OSAS (mean age 51.3 years) who were diagnosed by standard polysomnography (PSG), and in 15 age-matched healthy subjects (mean age 52.7 years) as controls to evaluate circadian variation of these parameters. The measures of heart rate variability (HRV) were calculated from 24-h ambulatory ECGs. Seventeen patients with OSAS showed excessive daytime sleepiness (EDS). We calculated the duration in which SpO2 decreased to < 90% (duration of SpO2 < 90%). The number of apnea/hypopneas per hour (AHI) during sleep was investigated with Apnomonitors (Chest MI, Co., Tokyo) on the same day as the SpO2 recordings. RESULTS: Controls showed no episodes of oxygen desaturation. In patients with OSAS, driving (33.3% of patients with OSAS) was the most common activity in which SpO2 decreased to < 90%, followed by daytime napping (27.8%) and resting after meals (22.2%). The duration of SpO2 < 90% over a 24-h period correlated significantly with the duration levels recorded during sleep (r = 0.99, p < 0.05) and in the afternoon (r = 0.62, p < 0.05), and with the AHI (r = 0.55, p < 0.05), but not with the duration of SpO2 < 90% in the morning. The number of ventricular premature beats correlated significantly with the duration of SpO2 < 90% for a 24-h period, but not with measures of HRV. Ventricular tachycardia was found in two (11.1%) and ST-T depression in three patients (16.6%) with underlying cardiac diseases. CONCLUSION: Our results suggest that daytime sleep attacks accompanied by oxygen desaturation in patients with moderate to severe OSAS may contribute to the occurrence of traffic or cardiovascular accidents. We conclude that 24-h ambulatory recordings of SpO2 and ECG are useful for screening for daytime sleep sequelae associated with the potential risk of this pathology in OSAS during social activities.
Adult↗
Strategy for lipid suppression in lactate imaging using STIR-DQCT: a study of hypoxic-ischemic brain injury.
In vivo lactate detection using gradient enhanced double quantum coherence transfer (DQCT) was significantly improved by addition of short-time-inversion-recovery (STIR). Phantom studies demonstrated lipid suppression down to the background noise level with 33% loss of lactate signal. In vivo studies using a rabbit model of hypoxic and unilateral-ischemic brain injury showed reduction down to 29 +/- 11% in lipids with inversion times between 140 and 170 ms. Lactate signals on the ischemic side were 51 +/- 53% higher than the nonischemic side at the peak of hypoxia. STIR-DQCT can be a useful robust method of obtaining metabolic maps of lactate in vivo.
Animals↗
Encoding target-trunk relative position: cervical versus vestibular contribution.
The contribution of cervical and vestibular cues in signaling the changes in target-trunk relative positions during self-motion was investigated. Normal subjects (Ss) were shown a LED flashed in the peripheral visual field in a dark room. Ss were then passively rotated about the vertical axis in one of three different conditions: (1) head chair-fixed (vestibular condition); (2) head earth-fixed (relaxed neck condition); and (3) head earth-fixed, but with the Ss actively attempting to turn it (activated neck condition). The Ss were then required to indicate, with their unseen index finger, the position of the previously flashed target. It was found that pointing at the memorized target was similarly accurate in the relaxed neck condition and in the activated neck condition. In the vestibular condition, pointing accuracy dropped significantly. These results suggest that neck proprioceptive signals are more effective than vestibular ones in signaling relative changes in the position of stationary objects with respect to the body during head-trunk motion. The finding that cervically mediated estimates were unchanged during active contraction of the neck muscles may suggests that efference copy signals may help interpret the change in the afferent signals caused by voluntary neck muscle activation.
Adult↗
A case of Rathke's cleft cyst with apoplexy.
We report a case of Rathke's cleft cyst associated with cholesterin granuloma in an 8-year-old girl with apoplexy. She was admitted to our hospital in April 1996 because of repeated headache and deep ophthalmic pain, without any visual disturbance. Computed tomography (CT) of the pituitary demonstrated an intrasellar isodense mass extending to the suprasellar cistern. Magnetic resonance imaging (MRI) showed a high-intensity mass on both T1- and T2-weighted images. The preoperative diagnosis of this lesion was Rathke's cleft cyst associated with a craniopharyngioma and/or hemorrhage. Transsphenoidal microsurgery was performed, and a bloody coffee-like serous and mucinous-yellowish substance was evacuated. Curettage of the wall removed the yellowish hard mass and soft membranous tissue. Histological examination of this tumor revealed a Rathke's cleft cyst with cholesterin granuloma.
Cerebrovascular Disorders↗
Combination chemotherapy (cisplatin, vinblastin) and low-dose irradiation in the treatment of pineal parenchymal cell tumors.
Pineal parenchymal cell tumors (PPCTs) with or without metastasis into the lumbar region by way of the cerebrospinal fluid were treated successfully with combination chemotherapy using cisplatin, vinblastin, and bleomycin (PVB) or cisplatin and vinblastin (PV) and low-dose irradiation (25 approximately 30 Gy). Our series included a case of pineoblastoma, two cases of mixed pinocytoma/pineoblastoma, and a case of pineocytoma, compared to which the data held by the All Japan Brain Tumor Registry (AJBTR) included information on 47 cases pineocytoma and 20 of pineoblastoma. All our patients have survived, with scores of 90% or over on Karnofsky's performance scale, for 2-12 years of follow-up so far; however, the 5-year survival rates of the patients recorded by AJBTR were 83% for pineocytoma treated with radiation and 43% without radiation; and 42% for pineoblastoma treated with radiation and 50% without radiation. Incomplete or varied chemotherapeutic regimens used in different medical centers to treat PPCTs precluded an evaluation such as was made by AJBTR. Our results suggested that combination chemotherapy with low-dose back-up radiotherapy may be the treatment of choice for primary or recurrent disease with or without dissemination in PPCTs.
Adult↗
Effective endless-loop bougienage through the oral cavity and esophagus to the gastrostomy in corrosive esophageal strictures in children.
The authors present a safe, conservative method of endless-loop bougienage (ELB) through the oral cavity and esophagus to a gastrostomy without general anesthesia in three children with corrosive esophageal burns treated since 1966. Esophagogastroscopy was performed to evaluate for esophagitis at an early phase after ingestion of the caustic substance. When esophageal stricture formation was recognized after subsequent conservative treatment, a feeding gastrostomy was made. A continuous string loop with plummets of progressively larger size was positioned to pass through the patient's oral cavity and esophagus to the gastrostomy. Strictures were found in the upper esophagus in two patients and in the middle and lower esophagus in one. The gastrostomy was performed 15 months, 20 days, and 2 months after the injury, respectively, and the periods of ELB were 3, 5, and 2(1/2) years, respectively. The patients were able to start eating at 26, 42, and 29 months after injury, respectively. They are now 30, 18, and 17 years old, and slight dysphagia remains in patients 1 and 2. No patient developed esophageal carcinoma at the site of the corrosive stricture. Our method of ELB through the patient's oral cavity and esophagus to the gastrostomy appears to be safe, reliable, and useful. We believe that most caustic esophageal strictures in children can be treated by this conservative measure.
Burns, Chemical↗
Long-term survival in a patient with congenital laryngeal atresia and multiple malformations.
Laryngeal atresia (LA) is an uncommon congenital anomaly, and only a few cases with long-term survival are reported in the literature. The authors describe a 2-year-old boy with LA and esophageal (EA), intestinal, and urethral atresia (UA). Immediately after birth, severe respiratory distress and mild abdominal distension became evident. Endotracheal intubation was unsuccessful and emergency tracheal puncture was performed, after which a tracheostomy was constructed. Direct laryngoscopy revealed LA at the vocal cord level. A cystostomy and gastrostomy were constructed immediately because of UA and EA. A cystocutaneostomy, ileocolic anastomosis, and resection of a tracheoesophageal fistula (TEF) were simultaneously performed 1 day after birth. The EA was proximal with a distal TEF. The gap between the ends of the upper and lower esophagus was 4 cm in length. It was thought impossible to perform a primary anastomosis, and therefore, a gastrostomy and resection of the TEF using multiple-stage surgery was undertaken. Intestinal resection and anastomosis were performed due to intestinal stenosis from necrotizing enterocolitis at the age of 3 months. Hypoxic encephalopathy developed due to accidental obstruction of the tracheostomy tube at the age of 10 months, and physical therapy was begun. He required a cutaneous nephrostomy due to a right hydromegaureter with vesicoureteric reflux and a left non-functioning kidney at the age of 23 months. He has been hospitalized for partial ventilatory assistance for 2 years at our institution. The course of this patient seems noteworthy in relation to the genesis of the multiple malformations.
Abnormalities, Multiple↗
Intracellular dynamics of alkaline phosphatase-containing granules in electropermeabilized human neutrophils.
Human neutrophils possess alkaline phosphatase-containing intracellular granules which are upregulated to the cell surface upon stimulation. The mechanism that governs the intracellular dynamics of these granules is, however, poorly understood. The aim of the present study was to investigate the possible participation of GTP-binding proteins in the reorganization and exocytosis of the alkaline phosphatase-containing granules using electropermeabilized cells. Biochemical assays using intact neutrophils showed that the alkaline phosphatase activity was upregulated and exocytosed into the extracellular space upon stimulation with AIF4 and N-formyl peptide. This upregulation was inhibited by treatment of cells with pertussis toxin and botulinum toxin. Alkaline phosphatase activity was also upregulated in electropermeabilized cells stimulated with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), but not with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). Cytochemically, alkaline phosphatase-containing granules were dispersed throughout the cytoplasm in unstimulated electropermeabilized neutrophils. Upon stimulation with GTPgammaS, but not with GDPbetaS, these granules fused to form elongated tubular structures which eventually became associated with the plasma membrane. Nocodazole disturbed the reorganization of the alkaline phosphatase-containing granules in cells stimulated with GTPgammaS. The results from this study indicate that GTP-binding proteins participate in the reorganization and exocytosis of alkaline phosphatase-containing granules associated with the microtubules in electropermeabilized human neutrophils.
Alkaline Phosphatase↗