Biomedical subjects
Publications and source records attributed to K Shimizu.
Assuming that substrate-assisted catalysis is the mechanism of GTP hydrolysis for ras p21 and other GTP-binding proteins, we used the PM3 semiempirical molecular orbital method to predict from the calculated reaction profiles of GTP hydrolysis reactions the changes in GTPase activities caused by mutations. We succeeded in making qualitative predictions for mutants.
The RHO1 gene encodes a homolog of mammalian RhoA small G protein in the yeast Saccharomyces cerevisiae. We have shown that Bni1p is one of the downstream targets of Rho1p and regulates reorganization of the actin cytoskeleton through the interaction with profilin, an actin monomer-binding protein. A Bni1p-binding protein was affinity purified from the yeast cytosol fraction and was identified to be Tef1p/Tef2p, translation elongation factor 1alpha (EF1alpha). EF1alpha is an essential component of the protein synthetic machinery and also possesses the actin filament (F-actin)-binding and -bundling activities. EF1alpha bound to the 186 amino acids region of Bni1p, located between the FH1 domain, the proline-rich profilin-binding domain, and the FH2 domain, of which function is not known. The binding of Bni1p to EF1alpha inhibited its F-actin-binding and -bundling activities. The BNI1 gene deleted in the EF1alpha-binding region did not suppress the bni1 bnr1 mutation in which the actin organization was impaired. These results suggest that the Rho1p-Bni1p system regulates reorganization of the actin cytoskeleton through the interaction with both EF1alpha and profilin.
We have developed a computational method that detects 'identities' in tRNA genes by using principal component analysis to classify the sequences of bases in tRNA genes into groups of similar sequences and then comparing the distribution of sequences of bases, in order to extract characteristic bases that are conserved within a group but differ between groups. These classification and comparison procedures are applied recursively to classify the sequences into hierarchical groups, so that multiple levels of characteristic sites can be detected. By using this computational method, we were able to detect many characteristic sites in the T and D domains of tRNAs, as well as the characteristic sites that had already been detected experimentally. This suggests that bases not only in the contact regions but also in the elbow regions, which determine the structure and dynamics of the whole tRNA molecule, are important to the tRNA-aminoacyl tRNA synthetase recognition.
We have recently isolated SMAP (Smg GDS-associated protein; Smg GDS: small G protein GDP dissociation stimulator) as a novel Smg GDS-associated protein, which has Armadillo repeats and is phosphorylated by Src tyrosine kinase. SMAP is a human counterpart of mouse KAP3 (kinesin superfamily-associated protein) that is associated with mouse KIF3A/B (a kinesin superfamily protein), which functions as a microtubule-based ATPase motor for organelle transport. We isolated here a SMAP-interacting protein from a human brain cDNA library, identified it to be a human homolog of Xenopus XCAP-E (Xenopus chromosome-associated polypeptide), a subunit of condensins that regulate the assembly and structural maintenance of mitotic chromosomes, and named it HCAP (Human chromosome-associated polypeptide). Tissue and subcellular distribution analyses indicated that HCAP was ubiquitously expressed and highly concentrated in the nuclear fraction, where SMAP and KIF3B were also present. SMAP was extracted as a ternary complex with HCAP and KIF3B from the nuclear fraction in the presence of Mg-ATP. The results suggest that SMAP/KAP3 serves as a linker between HCAP and KIF3A/B in the nucleus, and that SMAP/KAP3 plays a role in the interaction of chromosomes with an ATPase motor protein.
Local cerebral blood flow (LCBF) was measured in cats by magnetic resonance imaging (MRI), using 17O-labeled water (H[2]17O) as a tracer. Cat brain images were obtained using a 256 x 240 matrix fast spin-echo sequence with a total acquisition time of 124 seconds per image. Intravenous injection of H(2)17O and sampling of arterial blood were simultaneously performed during the MRI scan. Injection of H(2)17O over 40 seconds caused a transient decrease in brain signal intensity, from which the changes in H(2)17O concentration of brain tissue were calculated. The concentration of H(2)17O in arterial blood was measured directly by 17O nuclear magnetic resonance (NMR) spectroscopy, and used as an input function for the calculation of LCBF. LCBF was successfully determined using an in vivo autoradiographic strategy in a total of three cats. In one, LCBF measurements were also performed under hypercapnic conditions, and LCBF maps of the cat brain during normocapnia and hypercapnia were constructed. These LCBF maps reflected well the changes in LCBF induced by hypercapnia, which indicated the validity of the used method.
The ultrasonically-induced in vitro cell damaging effect of fluorine-containing anthracycline derivative (FAD104) was investigated. Sarcoma 180 cells suspended in air-saturated PBS were exposed to ultrasound for up to 60 s in the presence and absence of FAD104. The rate of inducing cell damage with ultrasound was doubled with 80 microM FAD104, while no cell damage was observed with FAD104 alone. This enhancement was significantly inhibited by histidine, which may suggest a sonochemical mechanism.
Defects in mismatch repair function can lead to the microsatellite instability (MI+; replication error) phenotype in certain human cancers. We previously reported that MI+ tumor-specific repeat number alteration at 13 consecutive trinucleotide (CAG) repeats within a coding exon of the E2F4 gene is a possible target of the defective repair pathway. Additional investigations revealed that E2F4 mutations are common (11 of 17 cases, 65%, mostly deletions) in a subset of human colorectal cancers with extensive MI+ phenotype, with respect to the proportion of loci affected and that most of these E2F4-mutated tumors (9 of 11, 82%) were accompanied by frameshift mutations in a polyadenine stretch within the seventh exon of the hMSH3 gene, a known mismatch repair gene that is responsible for repair of mismatch loops of two to four nucleotides. However, neither of these mutations was detected in 15 tumors with a lower incidence of MI+ loci. Similar repeat number alterations were less frequent in CAG repeats from other genes in all of the MI+ tumors we examined. These results indicate the presence of a novel cascade of mutational events that may be involved in acquisition of the malignant phenotype of human colorectal cancers with genetic instability.
A 64-year-old woman presented with cardiomegaly, Sweet's syndrome, and refractory anemia (RA), and died of sudden cardiac arrest. The autopsy revealed a perivascular and myocardial infiltration by neutrophils, which could be responsible for the cardiomegaly and probably had caused disturbances in the conduction system leading to sudden cardiac arrest. Myocardial infiltration by functionally defective neutrophils can develop in a patient with myelodysplastic syndrome (MDS) without peripheral neutrophilia or leukemic blood picture and needs a special diagnostic and therapeutic consideration.
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The mean centerline red blood cell (RBC) velocity of the rat pial artery was measured using an image-intensified high-speed (1000 frames/s) video camera system and RBCs labeled with fluorescein isothiocyanate (FITC). Some investigations measuring RBC velocity have been made in most organs, but the RBC velocity of the pial artery has not yet been measured with this system using FITC labeled RBC. After recording the emission of the FITC labeled RBC through a closed cranial window using this system, the authors analyzed the videotape. The movement of each individual RBC for several milliseconds over a distance of 50 microm could be pursued. The mean centerline RBC velocity in normal rats varied between 1.0 and 9.0 mm/s (most of the measurements we taken in vessels ranging between 20 and 80 microm in diameter). As the diameter of the pial artery becomes smaller, the blood flow rate (pi x (diameter/2)2 x (mean centerline velocity/1.6)) tends to become smaller. During CO2 inhalation, the pial artery diameter, mean centerline RBC velocity, and blood flow rate increased with statistical significance. Mean centerline RBC velocities in the cerebral microcirculation could not be measured directly with accuracy using the older methods (30 frames/s). However, this method is useful for investigation of the cerebral microcirculation and is considered to be applicable for studying the behavior of leukocytes or platelets, which will be examined in a subsequent study.
It would seem that a large discrepancy exists between the "chronological" age and "apparent" age of elderly patients, and we often observe that the latter reflects the results of surgical procedures very well. In the present study, we reviewed 258 patients aged 70 years or older who underwent elective abdominal operations under general anesthesia, to evaluate an outcome predictor representing their "physiological" age. A total of 24 preoperative variables were compared between patients who left the hospital in a satisfactory condition, being survivors, and those who died in hospital despite the operative procedure performed, being nonsurvivors. In the group of patients aged between 70 and 79 years, there was no significant difference between the survivor and nonsurvivor groups for any of the variables examined; however, in the group of patients aged over 80 years old, the oldest of whom was 93 years, there were significant differences in the total lymphocyte count (TLC) and the performance status (PS), as well as in age, between the survivor and nonsurvivor groups. Utilizing the three variables of age, PS, and TLC, a computer-generated discriminant function analysis yielded an equation which discriminated survival with 97% accuracy, and mortality with 83% accuracy. These findings indicate that the PS and TLC scores added to the chronological age should be considered when deciding whether a surgical procedure is appropriate for an elderly patient.
OBJECTIVE AND DESIGN: Development of nasal blockage and sneezing during repeated inhalation challenges with Japanese cedar pollens was evaluated in guinea pigs. SUBJECTS: Male Hartley guinea pigs. TREATMENT: Guinea pigs were sensitized by intranasal instillation of cedar pollen extracts + Al(OH)3 2 times a day for 7 days. The animal was then forced to inhale the pollens for challenge, which was restrictively trapped in the upper airways, once a week. METHODS: Change of specific airway resistance (sRaw), sneezing frequency, and titers of anaphylactic antibodies in the serum were measured after each of the 30 challenges. RESULTS: At the first challenge, no obvious increase in sRaw was observed. However, the second and third challenges to the animals caused modest biphasic elevations of sRaw, with peaks at the first and the fourth to sixth hour. At the fourth to tenth challenges, marked elevations of sRaw were observed. However, with repetition of the inhalation challenge, the early and the late responses became almost indistinguishable because of partial overlapping as the responses expanded. All guinea pigs sneezed immediately after each pollen inhalation challenge. Apparent increases of both circulating gamma1 and IgE antibodies were seen after the seventh challenge. CONCLUSIONS: These results indicate that the experimental allergic rhinitis established in the present study can be a valuable model for analyzing the pathogenesis of the disease and developing new therapeutic drugs.
An 8-year-old girl showed symptoms of encephalitis during acute Epstein-Barr virus (EBV) infection. The diagnosis of EB virus infection was made by changes in the titres of EB virus-specific antibody. Cranial MRI demonstrated abnormal low and high signal intensities in the striatal body (putamen and caudate nucleus) on T1-weighted and T2-weighted images, respectively, during the acute phase. These abnormal findings had almost completely resolved 1 month later. EBV infection should be considered when lesions are localised to the basal ganglia.
We report studies of two unrelated Japanese patients with 17alpha-hydroxylase deficiency caused by mutations of the 17alpha-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17alpha-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient's mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17alpha-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation.
We report a type IIa early gastric cancer associated with xanthoma cell proliferation in a 61-year-old man. The patient was admitted to our hospital because of a gastric polyp detected at a medical checkup. An irregular protruding lesion with xanthoma cell proliferation was detected endoscopically. Histological examination showed a well differentiated tubular adenocarcinoma in the mucosa associated with xanthoma cell proliferation. The distribution of the xanthoma cells in the stroma corresponded closely with that of the cancer cells. Neither atypism nor mitotic figures were recognized in the xanthoma cells. In an immunohistochemical study, almost all the xanthoma cells were stained positive for alpha 1-antitrypsin, while relatively few exhibited positive S-100 protein staining. Specific monocyte chemotactic and activating factor immunoreactivity was present only in the xanthoma cells, and not in the cancer cells. On the basis of these findings, it was speculated that the gastric cancer cells may have caused the xanthoma cell proliferation via an autocrine mechanism i.e., by a chemical mediator acting in a paracrine or juxtacrine manner.
The role of the patellar tendon (PT) in the contractile properties of quadriceps muscle was investigated. After PT resection (partial or complete), PT reconstruction, or a sham operation, rats were forced to run on a rodent treadmill. The histochemical and mechanical characteristics of the vastus medialis (VM) and vastus lateralis (VL) muscles were examined 2, 4, and 8 weeks after treatment. There were no significant changes in either fiber type population or the contractile properties of glycerinated single muscle fibers from VM and VL 2 and 4 weeks after partial PT resection. Complete PT resection caused a decrease in the population of type I fibers in VM, while the running training after PT reconstruction increased the population of type I fibers in VM. For both the PT resection and PT reconstruction groups, the Hill coefficient, an indicator of the Ca2+ sensitivity of myofilaments, was calculated from the pCa-tension relationship of glycerinated single muscle fibers. In the PT reconstruction group, the Hill values for fibers isolated from VM were significantly decreased by running training, but the Hill values for VL were not. The effect of running training on the Ca2+ sensitivity of myofilaments in muscle fibers with PT reconstruction was different from that in muscle fibers without PT treatment. Both resection and partial resection of the PT in this animal model had heterogeneous effects on the contractile properties of quadriceps muscles; we presume that, clinically, we must give careful consideration to PT treatment.
PURPOSE: To evaluate the involvement of the anterior ocular segment in Harada disease. METHODS: In a prospective study, ultrasound biomicroscopy was performed in 18 consecutive eyes of nine patients with Harada disease in its acute phase before and after onset of systemic corticosteroid therapy. Examination was repeated every 3 days until the resolution of serous retinal detachment. RESULTS: Serous detachment of the ciliary body was present in 11 (61%) of 18 eyes before treatment. The circumference of the ciliary body was detached in 10 of the 11 eyes. The height of ciliary detachment ranged from 20% to 250% of the scleral thickness. Ciliary detachment was more prominent in eyes with more extensive retinal detachment. There was no relation between the frequency of ciliary detachment and the severity of iritis. On day 3 of treatment, ciliary detachment had disappeared in seven eyes. In the other four eyes, it diminished in height to less than 10% of scleral thickness. Retinal detachment disappeared on days 6 through 17 (mean, day 11) of treatment. Two eyes of one patient showed very shallow anterior chambers. These eyes showed high ciliary detachment at 250% of scleral thickness. In another patient, both eyes developed -5.0 diopters of transient myopia. CONCLUSION: Ciliary detachment was a frequent finding in the acute stage of Harada disease. Its resolution was more prompt than that of serous retinal detachment after treatment. Ciliary detachment may be related to the development of a shallow anterior chamber.
PURPOSE: To demonstrate the drainage routes that compensate the venous congestion in central retinal vein occlusion. METHODS: Indocyanine green angiography was performed in 13 eyes of 13 patients with central retinal vein occlusion at the involutional stage using a scanning laser ophthalmoscope. The interval between onset of central retinal vein occlusion and indocyanine green angiography ranged from 1.0 to 9.3 years (mean, 3.6 years). Panoramic indocyanine green angiograms that covered the entire choroidal vasculature were produced by composing regional angiograms. RESULTS: Retinochoroidal collateral routes were found in 10 eyes that had papillary vascular loops. They were not observed in three eyes that did not have the vascular loops. Papillary vascular loops served as the collaterals to connect retinal veins and choroidal veins. The blood in the retinal veins was drained through the papillary vascular loops into the choroidal veins. These choroidal veins were selectively dilated and acted as drainage routes to the vortex veins. Nasal vortex veins served as extraocular exits of the drainage routes in all 10 eyes, and temporal vortex veins served as extraocular exits of the drainage routes in three of the 10 eyes. CONCLUSIONS: In central retinal vein occlusion with papillary loops, blood in the retinal veins ultimately drained into the vortex veins through the retinochoroidal collaterals.