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Biomedical subjects

T Miyazawa

Publications and source records attributed to T Miyazawa.

At least 325 records · Page 18Linked to original sources
PubMed1992

A 1H-15N NMR study of human c-Ha-ras protein: biosynthetic incorporation of 15N-labeled amino acids.

A 1H-15N NMR study was performed on the GDP-bound form of a truncated human c-Ha-ras oncogene product (171 amino acid residues). Resonance cross peaks of the backbone amide 1H-15N nuclei of a uniformly 15N-labeled protein were observed with heteronuclear single-quantum coherence spectroscopy (HSQC). In order to resolve overlapping cross peaks, selective 15N-labeling of one or two types of amino acid residues (Ala, Arg, Asx, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Tyr and/or Val) was carried out using appropriate E. coli mutant strains. By this procedure, all the backbone 1H-15N cross peaks were classified into amino acid types.

Amino Acids↗
PubMed1992

Chemiluminescence detection of 8a-hydroperoxy-tocopherone in photooxidized alpha-tocopherol.

Photosensitized oxidation products of alpha-tocopherol were analyzed by reversed phase high-performance liquid chromatography (CL-HPLC) in combination with chemiluminescence detection, to measure hydroperoxide derivatives. An intense peak was observed on the chemiluminescence chromatogram, corresponding to 8a-hydroperoxy-alpha-tocopherone (alpha-tocopherol hydroperoxide), the identity of which was confirmed by 1H nuclear magnetic resonance and mass spectrometry. Observed peak intensities correlated with the peroxide value of oxidized alpha-tocopherol. As little as 50 pmol of 8a-hydroperoxy-alpha-tocopherone was detectable. The CL-HPLC method, which is specific for the detection of hydroperoxides, should prove useful in studies on tocopherol oxidation in foods and biological systems.

Chromatography, High Pressure Liquid↗
PubMed1992

Sequences within the feline immunodeficiency virus long terminal repeat that regulate gene expression and respond to activation by feline herpesvirus type 1.

We constructed a series of deletion mutants of feline immunodeficiency virus (FIV) long terminal repeat (LTR) to identify the regions that regulate gene expression and are responsive to trans-activation of FIV LTR by feline herpes-virus type 1 (FHV-1). We demonstrated that sequences between -124 and -79, and between -21 and -32 (relative to the cap site) are essential for gene expression of FIV in Felis catus whole fetus 4 (fcwf-4) cells. Further, we demonstrated that the sequence between -63 and -23 responds to trans-activation of FIV LTR by FHV-1 in fcwf-4 cells.

Animals↗
PubMed1992

Cortical synaptogenesis in congenitally hydrocephalic HTX-rats using monoclonal anti-synaptic vesicle protein antibody.

We attempted to develop a method for investigating impairment of synaptogenesis quantitatively involving measurement of the fluorescence intensity emitted by immunohistochemically stained rat brain using a monoclonal antibody (Mab 171B5) against a synaptic vesicle protein (SVP-38). We applied this method to congenitally hydrocephalic and non-hydrocephalic brains of HTX-rats, and compared the postnatal changes in the fluorescence intensity in the molecular layer of the cerebral cortex. In non-hydrocephalic HTX-rats, the fluorescence intensity remained nearly unchanged from the 1st to 7th postnatal day and then increased at an almost linear rate until the 21st postnatal day, when it reached 4.5 times the value on the 7th postnatal day. The increase thereafter was gradual until the 28th postnatal day. In hydrocephalic HTX-rats, the fluorescence intensity increased nearly in parallel with that in non-hydrocephalic HTX-rats up to the 21st postnatal day. On the 28th postnatal day, the fluorescence intensity showed a marked reduction (P less than 0.01). This finding indicates that despite the presence of continuous pressure due to progressive ventricular dilatation after birth, synaptogenesis in the cerebral cortex may be relatively resilient.

Aging↗
PubMed1992

Methodological requirements for accurate measurements of brain and body temperature during global forebrain ischemia of rat.

The methodological requirements for accurate measurements of brain and body temperature during brain ischemia have been validated in Wistar rats submitted to 30 min of four-vessel occlusion. During ischemia, brains were exposed to three different temperature profiles: spontaneous cooling from 36 to 31 degrees C (n = 10), constant hypothermia at 30 degrees C (n = 19), and constant normothermia at 36 degrees C (n = 21). Direct and indirect brain temperature recordings were carried out by placing fine thermocouples (200 microns diameter) into the striate nucleus, the temporal muscle, and the epidural space. Body temperature was measured with a flexible thermocouple inserted at various depths into the rectum. Accurate measurements of body temperature required insertion of the rectal probe to a depth of at least 6 cm; lesser insertion resulted in an underestimation of up to 6 degrees C. Accurate estimates of brain temperature were obtained in all three experimental conditions by recording of the epidural temperature. The temperature in the temporal muscle, by contrast, differed from the brain temperature by up to 2 degrees C, depending upon the experimental condition and the duration of ischemia. We therefore suggest that indirect measurements of brain temperature during ischemia are carried out in the epidural space in order to avoid misinterpretations of temperature-sensitive pathological changes.

Animals↗
PubMed1992

Immunocytochemical study of an early microglial activation in ischemia.

Transient arrest of the cerebral blood circulation results in neuronal cell death in selectively vulnerable regions of the rat brain. To elucidate further the involvement of glial cells in this pathology, we have studied the temporal and spatial distribution pattern of activated microglial cells in several regions of the ischemic rat brain. Transient global ischemia was produced in rats by 30 min of a four-vessel occlusion. Survival times were 1, 3, and 7 days after the ischemic injury. The microglial reaction was studied immunocytochemically using several monoclonal antibodies, e.g., against CR3 complement receptor and major histocompatibility complex (MHC) antigens. Two recently produced monoclonal antibodies against rat microglial cells, designated MUC 101 and 102, were also used to identify microglial cells. Following ischemia, the microglial reaction was correlated with the development of neuronal damage. The earliest presence of activated microglial cells was observed in the dorsolateral striatum, the CA1 area, and the dentate hilus of the dorsal hippocampus. However, the microglial reaction was not confined to areas showing selective neuronal damage, but also occurred in regions that are rather resistant to ischemia, such as the CA3 area. Particularly in the frontoparietal cortex, the appearance of MHC class II-positive microglial cells provided an early indication of the subsequent distribution pattern of neuronal damage. The microglial reaction would thus seem to be an early, sensitive, and reliable marker for the occurrence of neuronal damage in ischemia.

Animals↗
PubMed1992

Production of feline immunodeficiency virus in feline and non-feline non-lymphoid cell lines by transfection of an infectious molecular clone.

An infectious molecular clone of the TM1 strain of feline immunodeficiency virus (FIV) was transfected into each of one feline (CRFK), two simian (COS and Vero) and two human (SW480 and HeLa) non-lymphoid cell lines, and virus production was assayed on feline T lymphoblastoid MYA-1 cells by monitoring reverse transcriptase activity. Infectious virus was produced in CRFK, Vero and HeLa cells, but not in COS and SW480 cells. When the basal promoter activity of the FIV long terminal repeat (LTR) was examined in these cell lines by using a chloramphenicol acetyltransferase assay, the activity correlated with the virus production in each cell line. Furthermore, when the activity of the FIV LTR was compared with those of three primate lentivirus LTRs, the highest activity in all the cell lines examined was produced by the LTR of simian immunodeficiency virus from African green monkey (SIVAGM), suggesting that it has a wide expression range. In COS and SW480 cells, the activity of the FIV LTR was much lower than that of the SIVAGM LTR. These results indicate that, whereas the primary block to FIV infection of certain cells may occur at the cell surface, the FIV LTR may also participate in controlling virus replication, as an intracellular mechanism.

Animals↗
PubMed1992

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the mixed anhydride and related methods.

In segment couplings by the mixed anhydride method using isobutyloxycarbonyl chloride, the use of copper(II) chloride as an additive suppressed racemization completely in the same manner as in the carbodiimide method reported previously. This was confirmed by employing a number of couplings between Z-dipeptides and amino acid esters. The racemization-suppressing effect of other compounds were also evaluated by employing one of these model couplings to be at best only limitedly effective. Copper(II) chloride was effective also in the related method using EEDQ. Thus, in the couplings where a low level of racemization was observed without an additive, the addition of copper(II) chloride eliminated racemization even at ambient temperature where EEDQ is usually used. The effectiveness of copper(II) chloride was confirmed also in the BOP-Cl method. In the presence of HOBt racemization was reduced to a low but still detectable level, while it was suppressed completely by the addition of copper(II) chloride.

Carbon Dioxide↗
PubMed1992

Racemization studies in peptide synthesis through the separation of protected epimeric peptides by reversed-phase high performance liquid chromatography.

Separation of protected epimeric peptides, Z-Gly-Xaa-Xbb-OMe (where Xaa and Xbb = chiral amino acid residues), by reversed-phase HPLC was utilized for studying racemization in peptide synthesis. Thus, the following factors which might affect the extent of racemization during the coupling by the carbodiimide method were investigated: the combination of amino acid residues to be coupled, coexisting tertiary amine salts, and the relative configuration of the amino acid residues. The following points were revealed: the combination of bulky residues at the coupling site results in extensive racemization in a polar solvent such as DMF, the amine hydrochlorides cause less racemization than the p-toluene-sulfonates in DMF, and the influence of relative configuration differs depending on the solvent and the individuality of the amino components. Furthermore, the racemization-suppressing effect of some additives in the carbodiimide method was reevaluated by employing the same procedure.

Amino Acids↗
PubMed1992

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the carbodiimide method.

Copper(II) chloride was found to be an extremely efficient racemization-suppressing additive in the DCC method as compared with the hitherto known ones, by employing the model coupling Z-Gly-L-Val-OH + H-L-Val-OMe in DMF. Although some other copper salts also had a profound effect, copper(II) chloride was the best from the viewpoint of both racemization suppression and coupling efficiency. The effectiveness of copper(II) chloride was further confirmed by employing the EDC-mediated couplings of Z-Gly-containing dipeptides with amino acid esters or dipeptide esters, and those of Z-L-Ala (or L-Val)-L-Val-OH with amino acid esters or dipeptide esters. In almost all the cases studied, no detectable amount (less than 0.1%) of epimer was observed by the HPLC analysis in the presence of copper(II) chloride. This was also the case even with an extremely stringent coupling system Z-L-Pro-L-Val-OH + H-L-Pro-OMe. With reference to the mechanism of racemization suppression, it was found that copper(II) chloride has a strong ability to suppress the racemization of the 5(4H)-oxazolone, which may be formed from an activated carboxyl component during the coupling.

Amino Acid Sequence↗
PubMed1992

Simultaneous use of 1-hydroxybenzotriazole and copper(II) chloride as additives for racemization-free and efficient peptide synthesis by the carbodiimide method.

In the carbodiimide mediated coupling of Z-Gly-L-Val-OH with H-L-Val-OMe in DMF, the simultaneous use of HOBt and copper(II) chloride as additives was found to give the desired peptide in a high yield without racemization. In the presence of HOBt, reducing the amount of copper(II) chloride produced a higher yield. Besides improving the coupling efficiency as compared with the case using copper(II) chloride alone as an additive, the present procedure offered another advantage for racemization suppression. Thus, even for the couplings where a low level of racemization was observed in the presence of copper(II) chloride, the simultaneous addition of HOBt and copper(II) chloride resulted in the elimination of racemization. The effectiveness of this new procedure using the two carbodiimide additives in the synthesis of biologically active peptides was assessed by the preparation of a protected Leu-enkephalin. In the 4 + 1 segment condensation using HOBt and copper(II) chloride simultaneously as additives, no racemization was detected and the yield was high enough. The elimination of racemization and improvement of coupling efficiency produced by the present procedure can be attributable to a reduced tendency for the activated forms of the carboxyl component to form a 5(4H)-oxazolone by the action of HOBt, and to the prevention of racemization by copper(II) chloride of the small amount of the oxazolone formed which is not eliminated by the action of HOBt alone.

Amino Acid Sequence↗
PubMed1992

Identification of a feline immunodeficiency virus gene which is essential for cell-free virus infectivity.

Feline immunodeficiency virus (FIV) contains at least three small open reading frames (ORFs) in the genome, in addition to the three structural genes. Two of these ORFs (putative vif and ORF-A) have unknown functions. Northern (RNA) blot analysis of mRNAs from an FIV-infected cell line showed that the putative-vif-specific mRNA was expressed as a 5.2-kb species. To examine the function of the putative vif gene, we constructed mutants carrying a deletion in either the vif-like gene or the rev gene from an infectious molecular clone of FIV. Although the vif mutant produced virion-associated reverse transcriptase at a normal level upon transfection, cell-free virus prepared from the transfected cells could not infect feline CD4+ cells. The infectivity of the vif mutant, however, was demonstrated in a coculture of the transfected cells and feline CD4+ cells. We conclude that FIV contains the vif gene, which is structurally and functionally similar to that of the primate lentiviruses.

Base Sequence↗
PubMed1992

Low-level chemiluminescence and life span of Drosophila melanogaster.

Spontaneous photon emission (chemiluminescence, CL) as a monitor of free radical evolution in Drosophila melanogaster which had been maintained at 25 or 30 degrees C for 5 days after emergence was measured. When maintained at 30 degrees C the fly CL intensity was stronger than at 25 degrees C. Under the condition of the higher temperature, the fly life span was shorter (mean life span = 29 days at 30 degrees C and 63 days at 25 degrees C), and oxygen consumption (3.7 microliters/mg.h at 25 degrees C, 4.9 microliters/mg.h at 30 degrees C) and the mobility (movement distance = 25 mm/min at 25 degrees C, 700 mm/min at 30 degrees C) increased, together with augmentation of phospholipid hydroperoxide in the fly total lipids. The CL spontaneously emitted from fly homogenate was decreased by the free radical scavengers both in experiments in vivo and in vitro. The hypothesis is proposed that as the oxygen metabolism grows active, the chemiluminescent reactions that involve oxygen-dependent free radical metabolism, including membrane phospholipid hydroperoxidation, contribute to the acceleration of senescence of fly bodies.

Aging↗
PubMed1992

15N and 13C labeling of Escherichia coli tRNAs toward the NMR analysis.

Escherichia coli tRNAs were labeled with stable isotope 15N in vivo. Three species of tRNA, tRNA(Glu), tRNA(Lys) and tRNA(Ile), were purified by an HPLC system and their NMR spectra were observed. In heteronuclear 1H-15N multiple or single quantum coherence (HMQC or HSQC) spectra, the crosspeaks corresponding to NH3 of U and NH1 of G can be distinguished clearly since their 15N chemical shifts are significantly different from each other. Thus, this combination of 15N-labeling and the proton detected heteronuclear experiments are useful for the signal assignment and the conformational analysis of tRNAs. Furthermore, C1'- selective 13C-labeling of nucleotides was examined in vivo in order to resolve the H1' signals of tRNAs. By using a newly constructed E. coli mutant strain, the isotopic enrichments of more than 90% at C1' and of less than 10% for other ribose carbons were achieved.

Carbon Isotopes↗
PubMed1992

[A case of acetaminophen-induced pneumonitis].

A 20-year-old woman took 1.2 g of acetaminophen for toothache. She subsequently developed a dry cough, pyrexia, and dyspnea. Chest X-ray revealed diffuse reticulo-nodular shadows in both lung fields. Broncho-alveolar lavage examination showed a marked increase in the total cell number and an increase in the percentage of eosinophils, neutrophils, and lymphocytes. Because drug-induced pneumonitis was suspected, all drugs were stopped and she was administered methylprednisolone. Consequently her symptoms resolved, and pulmonary function and chest X-ray findings improved remarkably. The lymphocyte stimulation test was positive for Norshin and its acetaminophen element. Based on these findings, the diagnosis of acetaminophen-induced pneumonitis was made. Acetaminophen intoxication is well-known, but to our knowledge this is the first reported case of acetaminophen-induced allergic pneumonitis in Japan.

Acetaminophen↗
PubMed1992

[Evoked potentials following cerebral ischemia in the rat: the effect of the stimulus frequency].

EEG and somatosensory evoked potentials were recorded in anesthetized rats before and up to 24 h after 30 min of global forebrain ischemia. Before ischemia, SEP (sweep time 1000 ms) included primary (N25P50) components and late potentials of low amplitude. During ischemia, SEP and EEG were isoelectric. During postischemic recirculation SEP evoked by stimulation at 1.0 Hz remained severely suppressed for 24 h, although long-latency potentials (> 100 ms) recovered. Using a very low stimulation frequency of 0.1 Hz, late components strongly increased in amplitude and the overall evoked activity in the averaged post-stimulus-EEG reached 90% of control. These results demonstrate that the SEP evoked at very low stimulation frequency may serve as an early indicator of functional brain recovery after prolonged cerebro-circulatory arrest.

Animals↗