Biomedical subjects

S Kojima

Publications and source records attributed to S Kojima.

At least 55 records · Page 3Linked to original sources

PubMed2001

The NK cell MHC class I receptor Ly49A detects mutations on H-2Dd inside and outside of the peptide binding groove.

The NK cell inhibitory receptor Ly49A recognizes the mouse MHC class I molecule H-2D(d) and participates in the recognition of missing self. Previous studies indicated that the determinant recognized by Ly49A exists in alpha1/alpha2 domain of H-2D(d). Here we have substituted polymorphic as well as conserved residues of H-2D(d) alpha1/alpha2 domain (when compared with H-2K(d), which does not interact with Ly49A). We then tested the ability of the H-2D(d) mutants to interact with Ly49A by soluble Ly49A tetramer binding and NK cell cytotoxicity inhibition assays. Individual introduction of mutations converting the H-2D(d) residue into the corresponding H-2K(d) residue (N30D, D77S, or A99F) in H-2D(d) partially abrogated the interaction between Ly49A and H-2D(d). Introduction of the three mutations into H-2D(d) completely abolished Ly49A recognition. Individual introduction of D29N or R35A mutation into the residues of H-2D(d) that are conserved among murine MHC class I severely impaired the interaction. The crystal structure of H-2D(d) reveals that D77 and A99 are located in the peptide binding groove and that N30, D29, and R35 are in the interface of the three structural domains of MHC class I: alpha1/alpha2, alpha3, and beta(2)-microglobulin. These data suggest that Ly49A can monitor mutations in MHC class I inside and outside of the peptide binding groove and imply that inhibitory MHC class I-specific receptors are sensitive to mutations in MHC class I as well as global loss of MHC class I. Our results also provide insight into the molecular basis of Ly49A to distinguish MHC class I polymorphism.

Amino Acid Sequence↗

PubMed2001

An "elongated" translation elongation factor Tu for truncated tRNAs in nematode mitochondria.

We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematode Caenorhabditis elegans. Because the corresponding protein was detected immunologically in a nematode mitochondrial (mt) extract, it could be regarded as a nematode mt EF-Tu. The protein possesses an extension of about 57 amino acids (we call this domain 3') at the C terminus, which is not found in any other known EF-Tu. Because most nematode mt tRNAs lack a T stem, domain 3' may be related to this feature. The nematode EF-Tu bound to nematode T stem-lacking tRNA, but bacterial EF-Tu was unable to do so. A series of domain exchange experiments strongly suggested that domains 3 and 3' are essential for binding to T stem-lacking tRNAs. This finding may constitute a novel example of the co-evolution of a structurally simplified RNA and the cognate RNA-binding protein, the latter having apparently acquired an additional domain to compensate for the lack of a binding site(s) on the RNA.

Amino Acid Sequence↗

PubMed2001

Rhodium complex-catalyzed reaction of isonitriles with carbonyl compounds: catalytic synthesis of pyrroles.

[figure: see text] Low-valent rhodium complexes are efficient catalysts for the activation of alpha-C-H bond of isonitriles. Addition of isonitriles to carbonyl compounds proceeds under mild and neutral conditions to give the corresponding alpha,beta-unsaturated formamides. Catalytic synthesis of pyrroles can be performed by cyclocondensation of isonitriles with 1,3-dicarbonyl compounds.

Journal Article↗

PubMed2001

Effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transfer activity in rat brain slices assessed by gas-tissue autoradiography using.

We have investigated the effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transport function in brain slices using gas-tissue autoradiography technique with [15O]O2. Brain slices were preincubated in Krebs-Ringer phosphate medium bubbled with 100% O2 for 30 min at 37 degrees C. (1) Control culture was incubated in the same medium bubbled with 100% O2 for 5-40 min at 37 degrees C, then for another 30 min under the same conditions. (2) In vitro ischemia was induced by placing the culture in the medium deprived of glucose and bubbled with 100% N2 for 5-40 min, then returning it to control conditions and culturing for another 30 min. (3) In vitro hypoxia was induced by placing the culture in the medium with glucose and bubbled with 100% N2 for 5-40 min, then returning it to the control conditions for 30 min. After the three different treatments, the [15O]O2 fixation by brain slices reflect to mitochondrial electron transport function was determined using gas-tissue autoradiography technique with [15O]O2. The fixation of [15O]O2 by striatum, cerebral cortex and hippocampus was reduced dependent upon the period of in vitro ischemic treatment. In contrast, the [15O]O2 fixation by those brain regions was only slightly reduced by hypoxia treatment. The reduction in [15O]O2 fixation induced by ischemic treatment was prevented by an antioxidant: glutathione, glutathione monoethyl ester or acetylsalicylic acid. The preventive effect of antioxidants on the mitochondrial damage induced by ischemia was more remarkable in the striatum than in the cerebral cortex and hippocampus. In the comparison of [15O]O2 fixation between ischemia-treated young and senescent brain slices, reduction of 15O fixation by every brain region examined was more prominent in senescence than in the young. These results suggest that gas-tissue autoradiography using [15O]O2 is useful to assess mitochondrial electron transport dysfunction induced by ischemia treatment in brain slices and that the oxidative stress participates in the mechanism of ischemia-induced dysfunction in mitochondria.

Age Factors↗

PubMed2001

Functional roles of ionic and hydrophobic surface loops in smooth muscle myosin: their interactions with actin.

This investigation ascertains whether, in (smooth muscle) myosin, certain residues engage in functional interactions with their actin conjugates in an actomyosin complex. Such interactions have been postulated from putting together crystallographic models of the two proteins [Rayment, I., Rypniewski, W. R., Schmidt-Bäse, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G., and Holden, H. M. (1993) Science 261, 50-58]. Here, in several instances, we ask whether mutation of a particular residue significantly impairs a function, and find that the answers are largely rationalized by the original postulation. Additionally, a novel element emerges from our investigation. To assess function, we test the wild type and mutant systems as they perform in the steady state of ATP degradation. In doing so, we assume, as usual, that degradation proceeds from an early stage in which the complex forms (and is described by parameter K(app)) to a later stage during which the product leaves the complex (and is described by parameter V(max)). Interestingly, certain defects induced by the mutations are associated with changes in K(app), and other defects are associated with changes in V(max), suggesting that our procedure at least roughly distinguishes between events according to the time in the degradation at which they occur. In this framework, we suggest that (1) in the actin-myosin association phase, cationic residues Lys-576 and Lys-578 interact with anionic residues of the so-called second actin, and (2) in the product leaving phase, hydrophobic residues Trp-546, Phe-547, and Pro-548, as well as the Thr-532/Asn-533/Pro-534/Pro-535 sequence, sever connections with the so-called first actin. The role of Glu-473 is also examined.

Actins↗

PubMed2001

Explanation of the crystallization rate of amorphous nifedipine and phenobarbital from their molecular mobility as measured by (13)C nuclear magnetic resonance relaxation time and the relaxation time obtained from the heating rate dependence of the glass transition temperature.

To gain further insight into the effect of molecular mobility on the crystallization rate of amorphous drugs, the mean relaxation times of amorphous nifedipine and phenobarbital were calculated based on the Adam-Gibbs-Vogel (AGV) equation, using the parameters D, T(0), and T(f), derived from the heating rate dependence of the glass transition temperature (T(g)) of the amorphous drugs and heat capacity of the drugs in the amorphous and crystalline states. These relaxation times were compared with the crystallization rate of amorphous nifedipine and phenobarbital reported previously. The spin-lattice relaxation time (T(1)) and the spin-lattice relaxation time in the rotating frame (T(1rho)) of phenobarbital and nifedipine carbons were also determined. The temperature dependence of the crystallization rate of nifedipine and phenobarbital on the T(g) was coincident with that of the mean relaxation time calculated according to the AGV equation within experimental error, indicating that the crystallization of nifedipine and phenobarbital is largely correlated with molecular mobility at the temperatures studied. A (13)C nuclear magnetic resonance relaxation study indicated that the molecular motion of nifedipine and phenobarbital in the mid-kHz frequency range became significant at temperatures higher than T(g)-20 and T(g), respectively.

Calcium Channel Blockers↗

PubMed2001

Alteration of inhibitory properties of Pleurotus ostreatus proteinase A inhibitor 1 by mutation of its C-terminal region.

Pleurotus ostreatus proteinase A inhibitor 1 (POIA1), which is composed of 76 residues without disulfide bridges, is a unique inhibitor in that it exhibits sequence similarity to the propeptides of subtilisins. In order to elucidate the inhibitory mechanism of POIA1, we constructed an expression system for a synthetic POIA1 gene. The wild-type POIA1 was found to inhibit subtilisin BPN' with an inhibitor constant (K(i)) of 3.2 x 10(-9) M, but exhibited a time-dependent decrease of inhibitory activity as a consequence of degradation by the protease, showing that the wild-type POIA1 was a temporary inhibitor when subtilisin BPN' was used as a target protease. Since POIA1 shows sequence similarity to the propeptide of subtilisin, which is known to inhibit the protease via its C-terminal region, the C-terminal six residues of POIA1 were replaced with those of the propeptide of subtilisin BPN'. The mutated POIA1 inhibited subtilisin BPN' with a K(i) value of 2.8 x 10(-11) M and did not exhibit time-dependent decrease of inhibitory activity, showing about 100-fold increases in binding affinity for, and resistance to, the protease. These results clearly indicate that the C-terminal region of POIA1 plays an important role in determining the inhibitory activity toward the protease, and that the increase in binding ability to the protease is closely related to resistance to proteolytic degradation. Therefore, the inhibitory properties of POIA1 can be altered by mutation of its C-terminal region.

Amino Acid Sequence↗

PubMed2001

Large amounts of apicoplast nucleoid DNA and its segregation in Toxoplasma gondii.

Apicoplasts (apicomplexan plastids) are nonphotosynthetic, secondary endosymbiotic plastids that are found in most apicomplexans. Although these organelles are essential for parasite survival, their functions, activities, and structures are not well understood. We examined the apicoplast nucleoid of Toxoplasma gondii from a morphological aspect by high-resolution epifluorescence microscopy and electron microscopy. We found unexpectedly large amounts of DNA in the nucleoid and the presence of several division-related structures. Initially, we identified the organellar nucleoids by staining with the DNA-specific dye 4',6-diamidino-2-phenylindole. A single nucleoid was observed per apicoplast, and the fluorescent spot representing the nucleoid was bright and spherical in contrast to the weak and filamentous spot representing the mitochondrial nucleoid. We also measured the DNA content of each apicoplast nucleoid by a video-intensified microscope photon-counting system and determined that the genomic copy number was at least 25, a figure over four times greater than that reported previously. Moreover, several groups of apicoplasts had significantly higher genomic copy numbers. The DNA molecules were accurately divided into two daughter apicoplasts just before nuclear division. In addition, we examined nucleoid segregation and the division apparatus using electron microscopy. However, we failed to observe nucleoid structures, suggesting that the apicoplasts are predominantly composed of nucleoid material. In addition, we observed "cap" structures at the termini of dividing apicoplasts, a possible plastid-dividing ring, and a microbody-like granule around the constriction. These structures may be involved in apicoplast division.

Animals↗

PubMed2001

Abortive assembly of succinate-ubiquinone reductase (complex II) in a ferrochelatase-deficient mutant of Escherichia coli.

Heme molecules play important roles in electron transfer by redox proteins such as cytochromes. In addition, a structural role for heme in protein folding and the assembly of enzymes has been suggested. Previous results obtained using Escherichia coli hemA mutants, which are unable to synthesize 5-aminolevulinic acid, a precursor of porphyrins and hemes, have demonstrated a requirement for heme biosynthesis in the assembly of a functional succinate-ubiquinone reductase (SQR or complex II), which is a component of the aerobic respiratory chain. In the present study, in order to investigate the role of the heme in the assembly of E. coli SQR, we used a hemH (encodes ferrochelatase) mutant that lacks the ability to insert iron into the porphyrin ring. The hemH mutant failed to insert functional SQR into the cytoplasmic membrane, and the catalytic portion of SQR [the flavoprotein subunit (Fp) and the iron-sulfur protein subunit (Ip)] was localized in the cytoplasm of the cell. It is of interest to note that protoporphyrin IX accumulated in the mutant cells and inactivated the cytoplasmic succinate dehydrogenase (SDH) activity associated with the catalytic Fp-Ip complex. In contrast, SQR was assembled into the membrane of a heme-permeable hemH double mutant when hemin was present in the culture. Only a low level of SQR activity was found in the membrane when hemin was replaced by non-iron metalloporphyrins: Mn-, Co-, Ni-, Zn- and Cu-protoporphyrin IX, or protoporphyrin IX These results indicate that heme iron is indispensable for the functional assembly of SQR in the cytoplasmic membrane of E. coli, and provide a new insight into the biological role of heme in the molecular assembly of the multi-subunit enzyme complex.

Cell Membrane↗

PubMed2001

Morphological characteristics of the bony birth canal in patients with developmental dysplasia of the hip (DDH): investigation by three-dimensional CT.

We investigated the three-dimensional morphological characteristics of the pelvis in adult female patients with developmental dysplasia of the hip (DDH), using computerized tomography (CT) images. Forty-two subjects with normal hips and 40 DDH patients were recruited for the study. In the DDH group, the average transverse diameter of the pelvic inlet was significantly less and the average transverse diameter of the pelvic outlet was significantly greater than the measurements in the normal group. Further, the bony birth canal in DDH patients exhibited a higher incidence of anthropoid-type geometry, as defined by a longer sagittal diameter relative to the transverse diameter of the pelvic inlet. These findings indicate a characteristic pelvic geometry and suggest different development of the pelvis in the transverse direction in DDH patients. In addition, the obstetric conjugate length/transverse diameter of the pelvic inlet ratio was correlated to the degree of severity of acetabular dysplasia. This finding suggests that DDH is a manifestation of a developmental characteristic of the pelvis.

Adult↗

PubMed2001

Fluoride concentrations at and near the neonatal line in human deciduous tooth enamel obtained from a naturally fluoridated and a non-fluoridated area.

This study sought to obtain a precise profile of fluoride concentrations at and near the neonatal line in deciduous incisors and canines from the naturally fluoridated area (1.0--1.3 parts/10(6) F in drinking water) of West Hartlepool and the non-fluoridated area (less than 0.1 parts/10(6) F in drinking water) of Leeds in England. An abrasive microsampling method was used to determine the distribution of fluoride and phosphorus concentrations. The profile of fluoride concentrations in 100-microm layers before and after the neonatal line, that is, in the prenatal and postnatal enamel, were significantly higher in teeth from the fluoridated than non-fluoridated areas. It was concluded that the fact that the fluoride concentrations were about the same prenatally and postnatally in deciduous enamel obtained from the fluoridated and non-fluoridated areas indicates that fluoride enters the prenatal deciduous enamel and that it is transferred through the placenta.

Calcium↗

PubMed2001

Neural substrates for recognition of familiar voices: a PET study.

Identification of familiar people is essential in our social life. We can identify familiar people by hearing their voices as well as by viewing their faces. By measuring regional cerebral blood flow (rCBF) by positron emission tomography (PET), we identified neural substrates for the recognition of familiar voices. The brain activity during discrimination of voices of the subjects' associates and friends from those of unfamiliar people was compared with that during an analogous discrimination of their own voice from unfamiliar voices as well as during vowel discrimination. The left frontal pole, right temporal pole, right entorhinal cortex, and left precuneus were activated to a greater extent during discrimination of familiar voice than during control discriminations, suggesting that these brain regions are involved in the recognition of familiar voices. Furthermore, the adjusted values of rCBF in the left frontal pole and right temporal pole correlated with the number of subjects' correct identification of familiar voices. The present results suggest that these two regions are coactively associated with matching the currently heard voice to familiar voices in one's memory.

Adult↗

PubMed2001

Different neural systems for recognizing plants, animals, and artifacts.

The purpose of this study was to investigate functional organization in the human brain involved in the representation of knowledge regarding plants. We measured the brain activity of eight male volunteers during the recognition of visual stimuli representing plants, animals and artifacts, using positron emission tomography. The participants were presented with and were required to name silently two different images each of 15 entities belonging to three ontological categories, and 30 series of four to six digits. Marked increases in regional cerebral blood flow were found in the hippocampus and the parahippocampal areas bilaterally and the right lateral occipital cortex during the silent naming of all three categories, compared with that during the silent reading of digits. The right lateral occipital cortex was specifically activated in association with the naming of plants, and the right fusiform cortex was specifically activated in association with the naming of animals. In addition, the right temporo-occipital cortex was activated only during animals and plants, not artifacts. Our results indicate that there were a few characteristic activations for the different categories, and that entities belonging to the different categories are not necessarily represented in different locations of the brain.

Cerebral Cortex↗

PubMed2001

Renal macrostructure and cortical circulation in hypertension assessed by dynamic computed tomography.

The aim of this study was to assess the macrostructure of the kidney and the grade of heterogeneity in renal cortical circulation in the early stages of essential hypertension. The subjects consisted of 84 patients (<50 years old) who underwent dynamic computed tomography (CT) because of various abdominal diseases and who had no serious hemodynamic abnormalities. The volumes of the whole kidney, cortex, and medulla were measured with the program installed in the CT instrument. In dynamic CT under appropriate conditions, the CT number of each pixel (image element) reflects the blood volume in the pixel. The means and standard deviations were calculated from the CT numbers within the renal cortex. The coefficient of variation (CV) of CT numbers was used as the index of the heterogeneity of renal cortical circulation. The volume ratio of cortex/medulla was significantly (P < .01) smaller in the hypertensive patients (0.80 +/- 0.03 (mean +/- SE)) than in the normotensive subjects (0.92 +/- 0.03). The CV was significantly (P < .01) greater in the hypertensives (n = 23, 0.124 +/- 0.008) than in the normotensives (n = 61, 0.106 +/- 0.003). There was a significant correlation (r = -0.391, P < .001) between the CV of CT numbers and the volume ratio of cortex/medulla, and the relationship between the two variables was independent of other variables. These results suggest that the heterogeneity of renal cortical circulation is increased in the early stages of essential hypertension and is related to changes in renal macrostructure.

Adult↗

PubMed2001

Age-related changes of glutathione content, glucose transport and metabolism, and mitochondrial electron transfer function in mouse brain.

To evaluate the oxidative stress-related parameters and to determine their order of appearance in the brain aging process, radionuclide experiments were carried out on male DBF1 mice at 3, 12, 24 and 30 months of age. The content of nonprotein sulfhydryl compounds, mainly glutathione, was estimated with technetium-99m meso-hexamethyl propyleneamine oxime ([99mTc]meso-HMPAO) tissue sampling. Glucose transport and metabolism was examined with [1-14C]2-deoxy-D-glucose (2-DG) tissue sampling. Mitochondrial electron transport function was estimated with [15O]O2 gas-tissue ARG. [99mTc]Meso-HMPAO uptake in brain expressed as standardized uptake value (SUV), (radioactivity in brain tissue/tissue weight)/(total administered radioactivity/body weight), reached maximum at 12 months of age and decreased at 24 and 30 months of age in every region examined. The pattern of 2-DG, expressed as SUV, showed a tendency to increase rather than decrease with aging. [15O]O2 fixation in brain slices remained constant until 24 months, while it decreased significantly at 30 months of age. The results suggested the possibility of using imaging techniques in vivo for longitudinal evaluation of the aging process and indicated reduction of nonprotein sulfhydryl compounds including GSH at the early stages of aging may also accelerate the dysfunction of mitochondrial electron transport and neurodegeneration.

Aging↗

PubMed2001

Design and synthesis of a dimeric derivative of RK-682 with increased inhibitory activity against VHR, a dual-specificity ERK phosphatase: implications for the molecular mechanism of the inhibition.

BACKGROUND: VHR is a dual-specificity phosphatase, which dephosphorylates activated ERK1/2 and weakens the ERK signaling cascade in mammalian cells. A selective inhibitor is expected to be useful for revealing the physiological function of VHR. RESULTS: First, we investigated the molecular mechanism of VHR inhibition by a known natural product, RK-682. Kinetic analysis indicated that inhibition was competitive toward the substrate, and two molecules of RK-682 were required to inhibit one molecule of VHR. Based on the structure-activity relationships for VHR inhibition by RK-682 derivatives, we constructed a binding model using molecular dynamics calculation. Based on this model, we designed and synthesized a novel dimeric derivative. As expected, the dimeric derivative showed increased inhibition of VHR, supporting our proposed mechanism of VHR inhibition by RK-682. CONCLUSION: We have developed a novel inhibitor of VHR based on the results of kinetic analysis and docking simulation.

Dimerization↗

PubMed2001

Phylogenetic identification of Sparganum proliferum as a pseudophyllidean cestode by the sequence analyses on mitochondrial COI and nuclear sdhB genes.

Sparganum proliferum is a larval cestode for which the adult stage is unknown. It is characterized by the continuous branching and budding when parasitized to humans, and causes fatal human sparganosis. However, the biological features of S. proliferum, including its taxonomic status, still remain obscure. Our previous investigation suggested that S. proliferum might be phylogenetically distinct from Spirometra erinaceieuropaei, by the analysis on mitochondrial NADH dehydrogenase subunit 3 (ND3) gene. However, mitochondrial DNA sequence in Platyhelminth is known to have heteroplasmy within a species. Therefore, in the present study, we have investigated the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene and the partial nucleotide sequences of nuclear coded succinate dehydrogenase iron-sulfur protein subunit gene (sdhB). The results clearly demonstrated that S. proliferum is a distinct species from S. erinaceieuropaei, and that S. proliferum belongs to the order Pseudophyllidea.

Amino Acid Sequence↗

PubMed2001

Membrane localization and demonstration of isoforms of nucleoside triphosphate hydrolase from Toxoplasma gondii.

Toxoplasma gondii has a unique enzyme, a NTPase, which has a wide specificity toward NTP. In the present study, we produced a monoclonal antibody (IgG1, 6C6) against the enzyme which could recognize NTPase isozymes among several strains of T. gondii. Three avirulent strains of T. gondii, ME49, Beverley and Nakayama, were found to have 1 NTPase (63 kDa, pI 6.0), while a virulent strain RH and an avirulent strain Fukaya had 2 isozymes (63 kDa) with different pIs (pIs 6.0 and 6.5 for the former, and pIs 6.2 and 6.4 for the latter, respectively), suggesting that this monoclonal antibody recognizes a common epitope of NTPase among T. gondii strains. Furthermore, 6C6 could inhibit NTPase activity in the presence of dithiothreitol in a dose-dependent manner, and immuno-EM study of NTPase revealed that this molecule is located on the surface membrane of T. gondii tachyzoites. When Vero cells were co-cultured with tachyzoites pre-treated with 6C6, the number of infected cells significantly decreased, suggesting that 6C6 inhibits invasion of the parasites to host cells. These data suggest that the molecule recognized by 6C6 might be considered a potential candidate antigen for vaccines against T. gondii tachyzoites.

Acid Anhydride Hydrolases↗