Biomedical subjects
Publications and source records attributed to K Morikawa.
We made an attempt to express a complaint of sleep disturbance by a self-rating scale of radar chart mode. The questionnaire for sleep disturbance is made up of eight items. Each item was scored from grade 1 to 4. The score of each item was projected to the MY radar chart, designed by us. It is noted that this method is useful in following the effect of hypnotics on sleep disturbance.
We investigated the mechanism of the trypanocidal activity of gallic acid (GA). GA-induced trypanocidal activity was significantly reduced by pretreatment with superoxide dismutase (SOD) and/or catalase. The ESR technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent revealed that a DMPO-OH adduct was detected in culture medium containing GA. The intensity of ESR signals of the DMPO-OH adduct was increased in a time dependent manner. SOD also inhibited the formation of GA-induced DMPO-OH adducts. Furthermore, GA enhanced DNA single-strand breaks induced by Fenton reagent. These results suggest the possibility that GA acts as pro-oxidant for trypanocidal activity.
We studied the central nervous system (CNS) effects of 3-[4-(8-fluoro-5, 11-dihydrobenz[b] oxepino[4, 3-b]pyridin-11-ylidene)piperidino]propionic acid dihydrate (HSR-609), a novel amphoteric antiallergic agent having antihistaminic activity. Its effects on gross behavior, spontaneous electroencephalograms (EEG) and some pharmacological parameters of unanesthetized, unrestrained dogs with chronic indwelling brain electrodes after oral administration were compared with typical antiallergic agents and 8-fluoro-5, 11-dihydro-11-(1-methyl-4-piperidylidene)benz[b] oxepino[4,3-b]pyridine (PY-608), a non-amphoteric basic compound having a similar chemical structure to HSR-609. HSR-609 (1, 10 and 100 mg/kg) and terfenadine (100 mg/kg) had no effect on the behavior, EEG patterns, sleep-wakefulness cycles or EEG power spectrum. Cyproheptadine (10 mg/kg), ketotifen (30 mg/kg) and PY-608 (10 mg/kg) increased slow waves with high amplitude in all EEG leads and caused dissociation between the slowing of EEG and waking behavior. Both azelastine (30 mg/kg) and oxatomide (100 mg/kg) caused generalized seizure discharges accompanied by agitation with the former and sedation with the latter. These findings suggest that observations of behavior and EEG in conscious dogs can be useful for clarifying the pharmacological characteristics of various antiallergic agents on the CNS. We were able to show that HSR-609 has no effect on the behavior and EEG of dogs because of its amphoteric chemical structure.
The fatty acid beta-oxidation multienzyme complex from Pseudomonas fragi, HDT, exhibits predominantly the three enzymic activities of 2-enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The HDT complex is encoded by the faoAB operon, consisting of the faoA and faoB genes that encode two individual constituents, the alpha-subunit and the beta-subunit. We have constructed Escherichia coli overexpression systems for the faoAB gene product (coexpression of the alpha- and beta-subunits), the alpha-subunit alone and the beta-subunit alone, and have purified the three respective products. Gel-filtration analysis revealed that the faoAB gene product forms a heterotetrameric structure, alpha2beta2, identical with the native HDT oligomeric state from P. fragi, whereas the alpha-subunit and beta-subunit individually form dimers. Electron microscopy demonstrated that each protein morphologically adopts the above oligomeric structures. The HDT complex, reconstituted in vitro from the isolated alpha- and beta-subunits, exhibits the three original enzymic activities and yields the same crystal as those from the native enzyme. CD measurements indicated that the alpha- and beta-dimers hardly alter their global conformations upon the formation of the HDT complex. Interestingly, the beta-dimer alone does not exhibit 3-oxoacyl-CoA thiolase activity, whereas the alpha-dimer alone exhibits both the 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities. These results suggest that the contact between the alpha- and beta-subunits is essential for the thiolase activity. We have identified several structurally important proteolytic sites within each subunit, which are protected in the intact heterotetrameric molecule. These findings allow the possible location of the interface between the two subunits, which should be crucial for the exhibition of thiolase activity.
Bcl-xL is a member of the Bcl-2 protein family, which regulates apoptosis. Preparation of recombinant rat Bcl-xL yielded two forms, one deamidated at -Asn-Gly- sequences to produce isoaspartates and the other not deamidated. The crystal structures of the two forms show that they both adopt an essentially identical backbone structure which resembles the fold of human Bcl-xL: three layers of two alpha-helices each, capped at one end by two short helices. Both forms have a long disordered region, which contains the potential deamidation sites. The molecular structure exhibits a low level of interhelical interactions, the presence of three cavities, and a notable hydrophobic cleft surrounded by walls rich in basic residues. These unique structural features may be favorable for its accommodation into membranes or for possible rearrangement to modulate homo-/heterodimerization. Homology modeling of Bcl-2 and Bax, based on the Bcl-xL structure, suggests that Bax has the strongest potential for membrane insertion. Furthermore, we found a possible interface for interaction with non-Bcl-2 family member proteins, such as CED-4 homologues.
SUG1 is an integral component of the 26 S proteasome. Belonging to a novel putative ATPase family, it shares four conserved motifs characteristic of ATP-dependent DNA/RNA helicases. Recombinant rat SUG1 (rSUG1) produced in Escherichia coli was highly purified and characterized in terms of its biochemical properties. The rSUG1 exhibited a Mg2+-dependent ATPase activity. The Km for ATP and Vmax of rSUG1 were 35 microM and 7 pmol of ATP/min/microg of protein, respectively. Both ATPase activity to release [32P]monophosphate and [32P]ATP-labeling activity were coordinately affected by cold ATP severely, GTP and UTP moderately, and CTP little. Interestingly, the rSUG1 ATPase activity was stimulated by poly(U) and poly(C), but not by poly(A), poly(G), or by any forms of DNAs tested. A UV cross-linking assay also indicated poly(U)- and poly(C)-stimulated labeling of rSUG1 with [alpha-32P]ATP. Moreover, the ATPase activity was facilitated by cellular poly(A)+ RNA, but not by poly(A)- RNA. RNA transcribed in vitro from cDNA encoding a b-Zip protein could stimulate the ATPase activity. This is the first report to demonstrate a specific RNA requirement for ATPase with respect to the proteasomal ATPases. Our present work suggests that SUG1 can specifically interact with protein-coding RNA (mRNA) and play some roles in mRNA metabolism.
Transcription initiation sites and the promoter sequence of the ubiquitously expressed mouse type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) gene were determined. In contrast to the nervous system-enriched IP3R1, the IP3R2 gene had multiple (seven major) transcription initiation sites located 334 to 269 bp upstream from the first ATG codon. Transient luciferase assay revealed promoter activity of the IP3R2 sequence upstream from the transcription initiation sites. The IP3R2 promoter was GC-rich and had no conventional TATA box, but had a GC box in the proximal promoter. Multiple transcription start sites were flanked by CpG islands, and various cis elements were located in the promoter. These structural features are considered to be responsible for a profile of IP3R2 gene expression.
BACKGROUND: Several strains of yeasts and fungi produce proteinous substances, termed killer toxins, which kill sensitive strains. The SMK toxin, secreted by the halotolerant yeast Pichia farinosa KK1 strain, uniquely exhibits its maximum killer activity under conditions of acidic pH and high salt concentration. The toxin is composed of two distinct subunits, alpha and beta, which tightly interact with each other under acidic conditions. However, they are easily dissociated under neutral conditions and lose the killer activity. The three-dimensional structure of the SMK toxin will provide a better understanding of the mechanism of toxicity of this protein and the cause of its unique pH-dependent stability. RESULTS: Two crystal structures of the SMK toxin have been determined at 1.8 A resolution in different ionic strength conditions. The two subunits, alpha and beta, are jointly folded into an ellipsoidal, single domain structure belonging to the alpha/beta-sandwich family. The folding topology of the SMK toxin is essentially the same as that of the fungal killer toxin, KP4. This shared topology contains two left-handed split betaalphabeta motifs, which are rare in the other proteins. Many acidic residues are clustered at the bottom of the SMK toxin molecule. Some of the carboxyl sidechains interact with each other through hydrogen bonds. The ionic strength difference induces no evident structural change of the SMK toxin except that, in the high ionic strength crystal, a number of sulfate ions are electrostatically bound near the basic residues which are also locally distributed at the bottom of the toxin molecule. CONCLUSIONS: The two killer toxins, SMK and KP4, share a unique folding topology which contains a rare structural motif. This observation may suggest that these toxins are evolutionally and/or functionally related. The pH-dependent stability of the SMK toxin is a result of the intensive interactions between the carboxyl groups. This finding is important for protein engineering, for instance, towards stabilization of the toxin molecule in a broader pH range. The present crystallographic study revealed that the structure of the SMK toxin itself is hardly affected by the ionic strength, implying that a high salt concentration affects the sensitivity of the cell against the toxin.
A killer toxin from a halotolerant yeast, Pichia farinosa strain KK1, was crystallized at high- and low-salt concentrations. Crystals from the high-salt solution belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell dimensions of a = b = 81.10, c = 118.46 A. The low-salt solution provided crystals that belonged to the same space group, with nearly same cell dimensions. Preliminary diffraction studies showed that the intensity distributions are significantly different between the two crystals. Both types of crystals contained either two or three molecules per asymmetric unit. They diffracted X-rays beyond 2.0 A resolution and were stable to X-ray irradiation.
Recent crystallographic studies of DNA-repair enzymes have provided the structural basis for the recognition of damaged DNA. The results imply that flipping out of the base is a common and crucial event in DNA repair. Two classes of repair enzymes that recognize distinct types of damage may exist. DNA-repair enzymes that share similar folds and DNA binding motifs have been proposed to belong to a superfamily.
SMK toxin is a killer toxin produced by a halotolerant yeast, Pichia farinosa. It is a heterodimer consisting of alpha (63 aa) and beta (77 aa) subunits, between which no disulfide bond exists. The two subunits interact tightly with each other below pH 5. However, the subunits dissociate under neutral conditions, resulting in the aggregation of the alpha subunit and the concomitant loss of killer activity. CD spectral measurements showed that the secondary structure of the SMK toxin changes drastically in the pH range 5.1-5.5 and that after the dissociation of the subunits, the soluble beta subunit alone cannot take any secondary structure. It was also shown that the concentration of NaCl does not affect the secondary structure of the SMK toxin.
OBJECTIVES: Comparison of fracture healing with two different implants: a conventional Dynamic Compression Plate (DCP) and a new internal Point Contact Fixator (PC-Fix). DESIGN: Randomized, prospective study in experimental animals. Observation times: 12, 24, 48 and 96 weeks, with six sheep per group. SETTING: Following surgery, animals were kept with unrestricted weight-bearing in individual stalls for 12 weeks, thereafter in groups. ANIMALS: 56 adult Swiss mountain sheep. INTERVENTION: A standardized oblique fracture of the sheep tibia was reduced and compressed by a lag screw and "neutralized" with one of the implants. MAIN OUTCOME MEASUREMENTS: Standard radiographs were used for callus size measurements. After sacrifice the implant was removed and both the treated bone and the contralateral bone were tested for static strength in bending with the plate side under tension. Broken bones were processed for histological evaluation. RESULTS: In the DCP group all six bones failed through the original fracture at 12 weeks. At 24 and 48 weeks two out of six, at 96 weeks one out of six bones failed through the original fracture, others through one of the screw holes. In the PC-Fix group there were no failures through the original fracture with a single exception at 96 weeks. The strength values in the PC-Fix groups of 12 and 96 weeks were significantly higher then in the corresponding DCP groups. CONCLUSIONS: Healing of simple diaphysial fractures treated by PC-Fix was superior to that achieved by conventional plating. The histological evaluation suggested that the observed differences can be accounted for by the absence of implant-related cortical necrosis and by the circumferentially uninterrupted (if smaller) callus in the PC-Fix group.
We studied the pharmacological characteristics of HSR-609 (3-[4-(8-fluoro-5,11-dihydrobenz[b]oxepino[4,3-b]pyridin-11- ylidene)- piperidino]propionic acid dihydrate), a novel amphoteric antiallergic agent, on the central nervous system (CNS). Its selectivity for the histamine H1-receptor and its ability to penetrate into the CNS were compared with those of typical antiallergic agents and the nonamphoteric basic compound PY-608 (8-fluoro-5,11-dihydro-11-(1-methyl-4-piperidylidene)benz[b]oxe pino- [4,3-b]pyridine), which has a chemical structure similar to that of HSR-609. In the in vitro study, HSR-609 had a high affinity for H1-receptors in the guinea pig cerebral cortex in comparison to affinities for muscarinic and serotonin 5-HT2-receptors in the rat cerebral cortex, while the selectivity of PY-608 for the H1-receptor was low. The inhibitory effects of these antiallergic agents on histamine-induced increase of vascular permeability in mice (ED50) were compared with the displacement of [3H]mepyramine binding to H1-receptors in mouse brain ex vivo (ID50). The ID50/ED50 ratio of HSR-609 was much larger than those of cyproheptadine, ketotifen and PY-608 and larger than those of terfenadine and cetirizine. HSR-609 was found to display selective displacement of the [3H]mepyramine binding to H1-receptors for lung vs cerebral cortex as found with terfenadine in guinea pigs ex vivo. These findings suggest that HSR-609 has high selectivity for the H1-receptor and poor ability to penetrate into the CNS in mice and guinea pigs due to its amphoteric chemical structure.
We studied the central nervous system (CNS) effects of HSR-609 (3-[4-(8-fluoro-5,11-dihydrobenz[b]oxepino[4,3-b]pyridin-11- ylidene) piperidino]propionic acid dihydrate), a novel amphoteric antiallergic agent having antihistaminic activity. Its effects on the behavior of mice and the electroencephalograms (EEG) of unanesthetized and unrestrained rabbits after oral administration were compared with those of typical antiallergic agents and the non-amphoteric basic compound PY-608 (8-fluoro-5,11-dihydro-11-(1-methyl-4-piperidylidene)benz [b]oxepino[4,3-b]pyridine), which has chemical structure similar to that of HSR-609. HSR-609 (3-300 mg/kg) had no effect on general behavior, spontaneous locomotor activity, hexobarbital-induced sleeping time and reserpine-induced hypothermia in mice. HSR-609 (10-100 mg/kg) and terfenadine (100 mg/kg) had no effect on spontaneous EEG, sleep-wakefulness cycles and EEG power spectra in rabbits. On the other hand, cyproheptadine (3-30 mg/kg), ketotifen (30-100 mg/kg) and PY-608 (0.3-100 mg/kg) caused increases and/or decreases of spontaneous locomotor activity, prolongation of hexobarbital-induced sleeping time and antagonistic effects on reserpine-induced hypothermia in mice. These agents (30 mg/kg) increased slow wave sleep and enhanced EEG power spectra at low frequency bands such as delta and theta in rabbits. These findings suggest that HSR-609 has no inhibitory effect on the CNS due to its amphoteric chemical structure.
When observers are presented directionally ambiguous motion, they exhibit a bias for experiencing movement in the direction in which shapes appear to face. We examined the influence of rigidity of a shape on the forward-facing bias with stimuli whose directionality is biologically specified. In general, the lack of shape correspondence during a nonrigid transformation should weaken the motion percept and decrease forward-facing bias. In contrast, representational momentum cues associated with a biologically likely nonrigid transformation should enhance the motion percept and increase forward-facing bias. Analysis for both rigid and nonrigid conditions indicated statistically significant forward-facing biases, but strength of bias did not differ significantly. The lack of difference between the two conditions suggests that the transformation was not one which allowed the influence of either consistency of correspondence or representational momentum to dominate and confirms that a comparably sized forward-facing bias can occur with both rigidly and nonrigidly transformed shapes.
A new method has been developed that is suitable for physiological studies of urethral motility in the conscious state with a strain-gauge force transducer (force transducer). In anesthetized dogs, the contractile or relaxing responses of the urethral smooth muscle in the direction of the circular layers measured with the force transducer, elicited by either phenylephrine or isoproterenol, were significantly correlated to the increase or decrease in the intra-urethral pressure (r = 0.998 and 0.780, respectively). In conscious dogs, the fluctuation in the urethral tone was observed to be less than in the intra-urethral pressure, and a dose-dependent response to phenylephrine (1-30 micrograms./kg. i.v.) and isoproterenol (0.1-3 micrograms./kg. i.v.) was clearly recognized. Moreover, this conscious model provided a fairly reproducible pattern with the urethral contraction or relaxation related to the filling or voiding phase of the cystometrogram, which was abolished by anesthetization. These results indicate that the present force transducer method makes it possible to evaluate the further physiological characteristics of urethral motility in conscious dogs.
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Two enzymes, dUTP pyrophosphatase and uracil-DNA glycosylase, prevent the misincorporation of uracil into the genome in distinct manners. The atomic structures of these proteins complexed with substrate analogs reveal the structural basis for uracil recognition and suggest a novel mechanism of DNA repair.