Search PubMed⌕ Search

Biomedical subjects

S Kojima

Publications and source records attributed to S Kojima.

At least 361 records · Page 20Linked to original sources
PubMed1996

Chemical nature of the light emitter of the Aequorea green fluorescent protein.

The jellyfish Aequorea victoria possesses in the margin of its umbrella a green fluorescent protein (GFP, 27 kDa) that serves as the ultimate light emitter in the bioluminescence reaction of the animal. The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish fluorescence (lambda max = 508 nm) when irradiated with long ultraviolet light. The fluorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67-. GFP has been used extensively as a reporter protein for monitoring gene expression in eukaryotic and prokaryotic cells, but relatively little is known about the chemical mechanism by which fluorescence is produced. To obtain a better understanding of this problem, we studied a peptide fragment of GFP bearing the chromophore and a synthetic model compound of the chromophore. The results indicate that the GFP chromophore consists of an imidazolone ring structure and that the light emitter is the singlet excited state of the phenolate anion of the chromophore. Further, the light emission is highly dependent on the microenvironment around the chromophore and that inhibition of isomerization of the exo-methylene double bond of the chromophore accounts for its efficient light emission.

Amino Acid Sequence↗
PubMed1996

Cloning of a cDNA encoding the small subunit of cytochrome b558 (cybS) of mitochondrial fumarate reductase (complex II) from adult Ascaris suum.

Complex II in the mitochondria of the adult parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity in addition to succinate dehydrogenase activity and plays a key role in the anaerobic energy metabolism of the worm. In this study, the amino acid sequence of the small subunit of cytochrome b558 (cybS) in adult complex II was deduced from the cDNA isolated by immunoscreening an A. suum muscle cDNA library. Histidine residues, which are possible heme axial ligands in cytochrome b558, were found in the second transmembrane segment of the subunit. This is the first report of the primary structure of the small subunit in the two-subunit cytochrome b in mitochondrial complex II from a multicellular eukaryote.

Amino Acid Sequence↗
PubMed1996

Synthesis of alpha-helix-forming peptides by gene engineering methods and their characterization by circular dichroism spectra measurements.

Two kinds of peptides which were considered to form alpha-helices were designed and characterized. One was "alpha(3)-peptide' with 21 residues comprising three repeats of the seven-residue sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala. This peptide appeared to be amphipathic due to a hydrophobic surface of Leu residues and a hydrophilic surface of Lys and Glu residues, thus forming a bundle structure. The other was "alpha(3)-GPRRG-alpha(3) peptide' with 47 residues in which two alpha(3)-peptides were connected by the five-residue sequence Gly-Pro-Arg-Arg-Gly. The genes encoding these peptides were fused to the adenylate kinase gene via a methionine codon. The resulting fused protein was expressed as an inclusion body, and the peptides were purified after cleavage with BrCN. The stability of the peptides in various buffers was then examined by measuring their circular dichroism spectra. The alpha(3)-peptide showed concentration-dependent stabilization of the alpha-helix. Sedimentation equilibrium ultracentrifugation indicated that it formed a bundle structure composed of four polypeptide chains, and a dimer intermediate during oligomerization was also detected by analytical gel-filtration. The stability of the alpha(3)-peptide was decreased by shifting the pH to 2 or 12, due to electrostatic repulsion of charged residues. Thus, the alpha(3)-peptide was stabilized by increasing the ionic strength, particularly in acidic or alkaline buffer, through the masking of the repulsion by high salt concentration. In buffer of neutral pH and a high salt concentration, the alpha(3)-peptide at high concentration formed visible aggregates, due possibly to the exposed hydrophobic surfaces of the alpha-helical bundles. On the other hand, alpha(3)-GPRRG-alpha(3) peptide did not show concentration-dependent reversible dissociation and association. It was shown to exist as a trimer even at low concentration, indicating very tight association of the alpha(3)-GPRRG-alpha(3) peptide. In contrast to the alpha(3)-peptide, the alpha(3)-GPRRG-alpha(3) peptide was very stable at various pH values and salt concentrations. This seemed to be due to increased hydrophobic interactions resulting from the increase in the number of seven-residue repeats from three to six, even though each group of three repeats was separated by a five-residue sequence.

Amino Acid Sequence↗
PubMed1996

Inhibition of proliferation of chondrocytes by specific receptors in response to retinoids.

All-trans retinoic acid inhibited the proliferation of chondrocytes derived from rat xiphoid cartilage when added to the culture medium at 10(-10)-10(-8) M. Proliferation of mouse clonal osteoblastic cells was also inhibited to a significant extent by all-trans retinoic acid. However, no such inhibitory effects on rat smooth muscle cells and human fibroblasts were observed. Flow cytometric analyses of chondrocytes labeled with propidium iodide revealed that all-trans retinoic acid arrested chondrocytes at the G1 phase of the cell cycle. Since 9-cis retinoic acid, which is synthesized enzymatically from all-trans retinoic acid, also inhibited the proliferation of chondrocytes, we investigated the subtypes of retinoic acid receptors in chondrocytes. Northern blot analysis revealed high levels of mRNA for retinoid X receptor alpha (RXR alpha), moderate levels of mRNA for retinoic acid receptor gamma (RAR gamma), and low levels of mRNA for RAR alpha and RXR beta. The mRNA for RAR beta and RXR gamma were not detected. These results suggest that retinoids are associated with the inhibition of proliferation of chondrocytes via both families of nuclear receptors, namely, RARs and RXRs.

Animals↗
PubMed1996

Restoration of endothelium-dependent relaxation in both hypercholesterolemia and diabetes by chronic taurine.

We examined the effects of taurine on levels of low-density lipoprotein (LDL) cholesterol and glucose, and an endothelium-dependent relaxation in response to acetylcholine in cholesterol-fed or streptozotocin-induced diabetic mice. The acetylcholine-induced concentration-dependent relaxation was significantly attenuated in aortic rings from cholesterol-fed and streptozotocin-induced diabetic mice. The attenuated vasodilation in both cholesterol-fed and streptozotocin-induced diabetic mice was normalized by the chronic administration of taurine. The endothelium-independent relaxation of aortic rings induced by sodium nitroprusside was not significantly different between control, cholesterol-fed and streptozotocin-induced diabetic mice. The increased serum levels of LDL cholesterol in cholesterol-fed and diabetic mice were returned to normal by the chronic administration of taurine. The chronic administration of taurine had no effects on serum glucose levels. These results suggest that the impaired endothelium-dependent vasodilation seen in both cholesterol-fed and streptozotocin-diabetic mice can be normalized by the chronic administration of taurine and this effect may be, at least in part, due to lowering of serum LDL levels.

Acetylcholine↗
PubMed1996

Retinoic acid-stimulated liver stellate cells suppress the production of albumin from parenchymal cells via TGF-beta.

We studied a cell-cell interaction via transforming growth factor-beta (TGF-beta ) between liver stellate cells (SCs) and parenchymal cells (PCs) using co-cultures of rat primary SCs and PCs. Both TGF-beta added exogenously to the culture medium, and TGF-beta produced endogenously from SCs after stimulation with retinoic acid (RA), suppressed the production and secretion of albumin from PCs. This effect occurred at the translational level, but not at the transcriptional level; TGF-beta, as well as SC culture medium conditioned by RA, did not affect the albumin mRNA levels, but decreased the biosynthesis of [35-S]methionine-labeled albumin without altering its post-translational degradation rate. These results suggest that TGF-beta generated from SCs facilitates the development of liver cirrhosis not only by inducing the production of fibrotic components from SCs, but also by impairing the function of the surrounding PCs.

Albumins↗
PubMed1996

Measurement of endogenous plasma granulocyte colony-stimulating factor in patients with acquired aplastic anemia by a sensitive chemiluminescent immunoassay.

Endogenous plasma granulocyte colony-stimulating factor (G-CSF) concentrations were serially measured in 68 patients with acquired aplastic anemia (AA). A very sensitive chemiluminescent immunoassay (CLEIA) was used to measure the plasma G-CSF concentration. The minimum detection limit of this assay (0.5 pg/mL) was sufficient for the determination of G-CSF concentrations in patients and normal subjects. The plasma G-CSF concentrations were significantly higher in 51 AA patients without signs of infection as compared with healthy control subjects. In AA patients with signs of infection, the G-CSF concentrations appeared to increase during the acute phase. There was a significant negative correlation between plasma G-CSF concentrations and absolute neutrophil counts (ANC) in AA patients without signs of infection. Although a decrease in the plasma G-CSF concentration was observed in all patients who achieved self-sustaining hematopoiesis following bone marrow transplantation (BMT) or immunosuppressive (IS) therapy, it was lower in patients undergoing BMT as compared with those receiving IS therapy for any given degree of neutropenia. Plasma G-CSF concentrations were higher in patients responding to IS therapy than in nonresponders. This study demonstrated the negative feedback regulation of G-CSF that plasma G-CSF concentrations may be useful in predicting the clinical response to IS therapy.

Adolescent↗
PubMed1996

New subtilisin-trypsin inhibitors produced by Streptomyces: primary structures and their relationship to other proteinase inhibitors from Streptomyces.

Three new proteinaceous inhibitors of trypsin and subtilisin of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family were isolated and purified from culture media of Streptomyces strains; SIL5 from S. fradiae, SIL7 from S. ambofaciens and SIL12 from S. hygroscopicus. Their complete amino-acid sequences were determined by sequence analysis of the intact SIL proteins and peptides obtained by enzymatic digestion of S-pyridylethylated proteins. SIL7 showed high sequence similarity to other Arg-possessing SSI-family inhibitors at the P1 site. SIL12 is unique in having a two-residue insertion in the flexible loop region. Based on the amino-acid sequences of these inhibitors and other SSI-family inhibitors whose sequences have already been determined, the phylogenetic relationship of SSI-family inhibitors and Streptomyces strains was considered. Among about 110 amino-acid residues possessed by SSI-family inhibitors, 28 are completely conserved. The contribution of these conserved residues to the function and stability of the inhibitor molecules is discussed on the basis of the results obtained from mutational analysis of SSI and its crystal structure.

Amino Acid Sequence↗
PubMed1996

Effects of endogenously activated transforming growth factor-beta on growth and differentiation of retinoic acid-treated HL-60 cells.

Retinoic acid (RA)-treated HL-60 cells were used as a model to study differentiation of granulocytic leukemias. RA induces these cells to mature into granulocytes and to decrease growth. Mediators of these RA effects have not been identified definitively, but transforming growth factor-beta (TGF-beta) has been implicated in regulating proliferation and differentiation of myelogenous leukemic cells. The role of TGF-beta in RA-dependent differentiation and cessation of growth was examined by adding neutralizing anti-TGF-beta IgG to RA-treated HL-60 cells, followed by assessing cell growth and markers of granulocytic differentiation over 5 days. After addition of neutralizing anti-TGF-beta IgG, growth of RA-treated HL-60 cells was maintained at control levels, but granulocytic differentiation continued. These experiments demonstrated that the antiproliferative activity of RA was TGF-beta dependent but that differentiation was not. Because most cell types secrete TGF-beta in a biologically inactive complex, a TGF-beta-dependent effect requires cells to activate the latent form of TGF-beta. Active and total TGF-beta levels were quantitated in media harvested from control and RA-treated cells using a luciferase-based bioassay for TGF-beta activity. Similar levels of total TGF-beta were observed between control and RA-treated cells. RA-treated cells produced active TGF-beta (18-24 pg/ml) after 1, 2, and 3 days of treatment, whereas negligible levels were produced by control cultures. Activation of endogenous latent TGF-beta by RA-treated cells occurred through a plasmin-independent mechanism.

Cell Differentiation↗
PubMed1996

Taxonomic characterization of closely related Streptomyces spp. based on the amino acid sequence analysis of protease inhibitor proteins.

Amino acid sequences of protease inhibitors (Streptomyces subtilisin inhibitor-like proteins) widely distributed in Streptomyces were compared to clarify the taxonomic status of three strains of Streptomyces spp., S. coelicolor A3(2), S. lividans 66 and S. coelicolor Müller, which are closely related by conventional taxonomical procedures. The sequence comparison indicated that S. coelicolor A3(2) is distinct from the type strain S. coelicolor Müller, but belongs to the same taxon as S. lividans 66.

Amino Acid Sequence↗
PubMed1996

Two hydrophobic subunits are essential for the heme b ligation and functional assembly of complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli.

Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits encoded by the sdhCDAB operon. Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein) to the cytoplasmic membrane and are believed to be the components of cytochrome b556 in E. coli complex II. In the present study, to elucidate the role of two hydrophobic subunits in the heme b ligation and functional assembly of complex II, plasmids carrying portions of the sdh gene were constructed and introduced into E. coli MK3, which lacks succinate dehydrogenase and fumarate reductase activities. The expression of polypeptides with molecular masses of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, indicating that sdhC encodes the large subunit (cybL) and sdhD the small subunit (cybS) of cytochrome b556. An increase in cytochrome b content was found in the membrane when sdhD was introduced, while the cytochrome b content did not change when sdhC was introduced. However, the cytochrome b expressed by the plasmid carrying sdhD differed from cytochrome b556 in its CO reactivity and red shift of the alpha absorption peak to 557.5 nm at 77 K. Neither hydrophobic subunit was able to bind the catalytic portion to the membrane, and only succinate dehydrogenase activity, not succinate-ubiquinone oxidoreductase activity, was found in the cytoplasmic fractions of the cells. In contrast, significantly higher amounts of cytochrome b556 were expressed in the membrane when sdhC and sdhD genes were both present, and the catalytic portion was found to be localized in the membrane with succinate-ubiquitnone oxidoreductase and succinate oxidase activities. These results strongly suggest that both hydrophobic subunits are required for heme insertion into cytochrome b556 and are essential for the functional assembly of E. coli complex II in the membrane. Accumulation of the catalytic portion in the cytoplasm was found when sdhCDAB was introduced into a heme synthesis mutant, suggesting the importance of heme in the assembly of E. coli complex II.

Base Sequence↗
PubMed1996

Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state.

Streptomyces subtilisin inhibitor (SSI) contains three methionine residues in a subunit: two (at positions 73 and 70) in the crucial enzyme-recognition sites P1 and P4, respectively, and one (Met 103) in the hydrophobic core. The motions of the side chains of these three Met residues and the changes in mobility on binding with subtilisin were studied by deuterium NMR spectroscopy in solution and in crystalline and powder solids. For this purpose, the wild-type SSI was deuterium-labeled at the methyl groups of all three Met residues, and three artificial mutant proteins were labeled at only one specific Met methyl group each. In solution, for methionines 73 and 70, the effective correlation times were only 0.8-1.0 x 10(-10)s indicating that the two side chains on the surface fluctuate almost freely. On formation of a complex with subtilisin, however, these high mobilities were quenched, giving a correlation time of 1.1 x 10(-8)s for the side chains of methionines 70 and 73. The correlation time of Met 103, located in the hydrophobic core, was at least 1.0 x 10(-8)s in free SSI, showing that its side chain motion is highly restricted. The nature of the internal motions of the three Met side chains was examined in more detail by deuterium NMR spectroscopy of powder and crystalline samples. The spectral patterns of the powder samples depended critically on hydration: immediately after lyophilization, the side-chain motions of the three Met residues were nearly quenched. With gradual hydration to 0.20 gram of water per gram protein-water, the orientational fluctuation of the methyl axes of methionines 70 and 73 was selectively enhanced in both amplitude and frequency (to about 1 MHz) and, at nearly saturating hydration (0.60 gram of water per gram protein-water), became extremely high in amplitude and frequency (> 10 MHz). In contrast, the polycrystalline wild-type SSI spectrum showed fine structures, reflecting characteristic motions of the Met side chains. The polycrystalline spectrum could be reproduced reasonably well by the same motion models and parameters used to simulate the powder spectrum at the final level of hydration, suggesting that the side-chain motions are similar in the fully hydrated powder and in crystals. Spin-lattice relaxation measurements gave evidence that, even in crystals, the methyl axes of all three Met residues undergo rapid motions with correlation times between 10(-8) and 10(-10)s, comparable to the correlation times in solution. Finally, in the hydrated stoichiometric complex of SSI with subtilisin BPN' in the solid state, large-amplitude motions are absent, but the side chains of methionines 70 and/or 73 are likely to have small-amplitude motions.

Bacterial Proteins↗
PubMed1996

Impaired synthesis of retinol-binding protein and transthyretin in rat liver with bile duct obstruction.

To gain further insight into the protein metabolism in bile duct-obstruction, we examined the synthesis of retinol-binding protein (RBP) and transthyretin (TTR) in rats with common bile duct-ligation. In these rats, liver and plasma levels of RBP and TTR decreased markedly, whereas liver retinoid contents remained unchanged. Although there appeared no decrease in the total amount of RBP or TTR mRNA expressed in the liver, the subcellular distribution of these mRNAs changed from the membrane-bound polysome fraction to the membrane-unbound polysome fraction. This abnormal distribution recovered rapidly after biliary drainage, resulting in the subsequent recovery of the plasma RBP and TTR levels. These observations suggest that cholestasis inhibits the synthesis and secretion of RBP and TTR by disrupting the binding of their mRNAs to membrane-bound polysomes. Plasma levels of RBP and TTR might be sensitive indicators of the recovery of protein synthesis after biliary drainage in patients with obstructive biliary disorders.

Animals↗
PubMed1996

Extraction of Schistosoma haematobium antigens from infected human urine and generation of potential diagnostic monoclonal antibodies to urinary antigens.

Proteins in Schistosoma haematobium infected human urine were concentrated by precipitation with saturated ammonium sulphate 50% (v/v) and various fractions obtained at different stages of precipitation tested for presence of schistosome antigens (ShAgs) by dot-ELISA. The protein fraction (UP2S) obtained following two-times precipitation was found to contain high concentrations of ShAg. Fraction UP2S was dialysed against phosphate-buffered saline (pH 7.4) and further purified by Sephadex G-200 column chromatography. Two protein peaks were eluted of which the first peak UP2S(pkI) was found to contain high concentrations of ShAgs as determined by microplate-ELISA. The second peak UP2S(pkII) consisted of human urine proteins. Further analysis of UP2S(pkI) revealed that ShAgs were mainly in the form of immune complexes with human IgG, IgM, IgA, IgE and complement C3. The ShAgs in both UP2S and UP2S(pkI) were found to be active as they induced immune responses in mice which produced antibodies reactive with S. haematobium worm as well as soluble egg antigens (SEA). Pure ShAgs were obtained from UP2S following dissociation of immune complexes with a carbonate buffer (pH 11.42) and further purification on Sephadex G-200. Immunizations with UP2S led to the generation of MoAbs which could bind both SEA and UP2S.

Animals↗
PubMed1996

Reference intervals for serum granulocyte colony-stimulating factor levels in children.

OBJECTIVE: To determine age-adjusted reference intervals for children for serum granulocyte colony-stimulating factor (G-CSF) levels. DESIGN: We determined the serum G-CSF levels of 168 disease-free children who were term neonates to 15 years of age with a highly sensitive chemiluminescent enzyme immunoassay. RESULTS: The lowest values (5 ng/L) exceeded the detectable limit (1 ng/L) of this assay technique. The G-CSF levels were highest on the day of birth (mean +/- SD 147 +/- 146 ng/L); thereafter values decreased to 46 +/- 33 ng/L in the early neonatal period and to 23 +/- 10 ng/L in the late neonatal period. The G-CSF levels both on the day of birth and in the early neonatal period were significantly higher than in all older age groups. No significant differences were found among any of the age groups after 4 weeks of age. The G-CSF values were correlated with both blood leukocyte counts (r = 0.496, p < 0.0001) and neutrophil counts (r = 0.547, p < 0.0001). In children older than 4 weeks of age (n = 128), the 95% reference interval for G-CSF values was 5 to 42 ng/L. CONCLUSIONS: Our study in disease-free children revealed that change in serum G-CSF levels are age dependent and are correlated with neutrophil counts. Determination of reference intervals for the neonatal period requires further study.

Adolescent↗
PubMed1996

Histological grading of carcinoma in situ of the bladder: its clinical significance in patients who underwent intravesical mitomycin C and doxorubicin sequential therapy.

PURPOSE: The clinical behavior of carcinoma in situ of the bladder seems rather complicated. Although some have advocated the histological grading of carcinoma in situ, to our knowledge no sufficient clinical information has been reported. Therefore, we evaluated the clinical significance of histological grading of carcinoma in situ of the bladder. MATERIALS AND METHODS: From January 1984 to December 1991, 58 patients with carcinoma in situ of the bladder were treated initially with intravesical mitomycin C and doxorubicin sequential therapy. Of the patients 20 had grade 2 and 38 had grade 3 anaplasia according to the modified World Health Organization grading system. Those who failed the initial therapy received another course of mitomycin C and doxorubicin sequential therapy or intravesical bacillus Calmette-Guerin. RESULTS: Following initial therapy, 13 patients (65%) with grade 2 and 28 (74%) with grade 3 disease achieved a complete response. Subsequent intravesical therapy resulted in complete response in 17 patients (85%) with grade 2 and 31 (82%) with grade 3 cancer. The local recurrence rate was higher in the grade 2 than in the grade 3 cases after a median followup of 48 months (range 10 to 84). The recurrent tumor configuration was significantly different between the 2 groups. Papillary cancer recurred only in grade 2 cases, while only nodular cancer recurred in grade 3 cases. The progression-free and survival curves were slightly higher in grade 2 than in grade 3 cases, although the difference was not significant. CONCLUSIONS: There may be some difference in response to initial intravesical chemotherapy and the local recurrence rate between grades 2 and 3 carcinoma in situ, both of which are detrimental to grade 2 lesions. Moreover, it appears likely that grade 2 carcinoma in situ is a precursor of papillary high grade cancer and grade 3 carcinoma in situ is a precursor of nodular cancer. However, patient prognosis in the 2 groups was not significantly different.

Administration, Intravesical↗