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Biomedical subjects

S Koide

Publications and source records attributed to S Koide.

At least 19 recordsLinked to original sources
PubMed2000

Solution conformation and amyloid-like fibril formation of a polar peptide derived from a beta-hairpin in the OspA single-layer beta-sheet.

A 23-residue peptide termed BH(9-10) was designed based on a beta-hairpin segment of the single-layer beta-sheet region of Borrelia OspA protein. The peptide contains a large number of charged amino acid residues, and it does not follow the amphipathic pattern that is commonly found in natural beta-sheets. In aqueous solution, the peptide was highly soluble and flexible, with a propensity to form a non-native beta-turn. Trifluoroethanol (TFE) stabilized a native-like beta-turn in BH(9-10). TFE also decreased the level of solubility of the peptide, resulting in peptide precipitation. The precipitation process accompanied a conformational conversion to a beta-sheet structure, as judged with circular dichroism spectroscopy. The precipitate was found to be fibrils similar to those associated with human amyloid diseases. The fibrillization kinetics depended on peptide and TFE concentrations, and had a nucleation step followed by an assembly step. The fibrillization was reversible, and the dissociation reaction involved two phases. TFE appears to induce the fibrils by stabilizing a beta-sheet conformation of the peptide that optimally satisfies hydrogen bonding and electrostatic complementarity. This TFE-induced fibrillization is quite unusual, because most amyloidogenic peptides form fibrils in aqueous solution and TFE disrupts these fibrils. Nevertheless, the BH(9-10) fibrils have similar structure to other fibrils, supporting the emerging idea that polypeptides possess an intrinsic ability to form amyloid-like fibrils. The high level of solubility of BH(9-10), the ability to precisely control fibril formation and dissociation, and the high-resolution structure of the same sequence in the beta-hairpin conformation in the OspA protein provide a tractable experimental system for studying the fibril formation mechanism.

Amyloid↗
PubMed2000

Design of single-layer beta-sheets without a hydrophobic core.

The hydrophobic effect is the main thermodynamic driving force in the folding of water-soluble proteins. Exclusion of nonpolar moieties from aqueous solvent results in the formation of a hydrophobic core in a protein, which has been generally considered essential for specifying and stabilizing the folded structures of proteins. Outer surface protein A (OspA) from Borrelia burgdorferi contains a three-stranded beta-sheet segment which connects two globular domains. Although this single-layer beta-sheet segment is exposed to solvent on both faces and thus does not contain a hydrophobic core, the segment has a high conformational stability. Here we report the engineering of OspA variants that contain larger single-layer beta-sheets (comprising five and seven beta-strands) by duplicating a beta-hairpin unit within the beta-sheet. Nuclear magnetic resonance and small-angle X-ray scattering analyses reveal that these extended single-layer beta-sheets are formed as designed, and amide hydrogen-deuterium exchange and chemical denaturation show that they are stable. Thus, interactions within the beta-hairpin unit and those between adjacent units, which do not involve the formation of a hydrophobic core, are sufficient to specify and stabilize the single-layer beta-sheet structure. Our results provide an expanded view of protein folding, misfolding and design.

Antigens, Surface↗
PubMed2000

Molecular cloning and expression of a novel klotho-related protein.

Klotho protein is a novel beta-glucosidase-like protein produced predominantly in the kidney. The klotho mouse, which genetically lacks klotho gene expression, manifests various systemic phenotypes resembling aging. In the present study we succeeded in isolating a novel human protein structurally related to klotho protein. The protein possesses one beta-glucosidase-like domain and is 42% identical with klotho protein at the amino acid level. Unlike klotho protein, it possesses neither a signal sequence nor a transmembrane domain, suggesting that it is a cytosolic protein, and thus was termed cytosolic beta-glucosidase-like protein-1 (cBGL1). By Northern blot analysis cBGL1 mRNA was expressed most abundantly in the liver, followed by the small intestine, colon, spleen, and kidney. When klotho and cBGL1 gene expression was examined in renal cell carcinoma tissues, both klotho and cBGL1 mRNA levels in tumors were lower than those in nontumor regions, suggesting that renal epithelial cells may lose klotho and cBGL1 gene expression during the course of malignant transformation. In conclusion, we describe the primary structure and gene expression of a novel protein related to klotho protein.

Aged↗
PubMed2000

[Treatment by corticosteroid and plasma exchange in 5 cases of renal cholesterol embolic disease].

Cholesterol arterial embolization is a systemic disease resulting from cholesterol crystal embolization to multiple organs, including the kidney, skin, brain, eye, gastrointestinal tract and extremities. In general, it is associated with high morbidity and mortality, but no optimal treatment has yet been developed. In this paper, we report five patients with cholesterol atheroembolic renal failure. In three of the five patients, combined therapy with corticosteroids and plasma exchange was performed. The three patients survived. On the other hand, the two remaining patients died of multifactorial causes. In this report, the literature on steroid therapy for cholesterol atheroembolic renal disease is reviewed and the efficacy of combined therapy by use of corticosteroids and plasma exchange is evaluated.

Acute Kidney Injury↗
PubMed2000

[A case of Chinese herbs nephropathy in which the progression of renal dysfunction was slowed by steroid therapy].

The patients was a 43-year-old woman whose chief complaints were nausea and heaviness of the heads. There was a history of toxemia of pregnancy. The patient had previously taken Tenshin Tokishigyaku-ka-goshuyu-shokyo-to for two years because of cold sensitivity. Fever, thirst, and loss of appetite developed from approximately 18 months after she started treatment with the Chinese herbal preparation, and she presented at our outpatient clinic 2.5 years later. On initial examination, deterioration of renal function was evident and the serum creatinine level was 3.4 mg/dl. A renal biopsy specimen showed marked interstitial fibrosis without inflammatory cell infiltration, leading to the diagnosis of Chinese herbs nephropathy (CHN). Steroid therapy was started on the 36th hospital day after a sharp rise in the serum creatinine level to 5.1 mg/dl. This resulted in the rapid improvement of renal function and reduction of the serum creatinine to 2.6 mg/dl by 8 weeks after the initiation of treatment. In a study on the use of steroids for patients with progressive moderate renal dysfunction caused by Chinese herbs, Vanherweghem et al. reported that the progression of renal failure was appreciably slowed in patients given steroids when compared with the control group. We were also able to slow the progression of renal dysfunction in our patient by steroid therapy, although the prognosis of CHN is generally considered to be very poor.

Adult↗
PubMed1999

Multistep denaturation of Borrelia burgdorferi OspA, a protein containing a single-layer beta-sheet.

Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi, is a dumbbell-shaped protein in which two globular domains are connected by a three-stranded beta-sheet segment that is solvent-exposed on both faces. Previous studies showed that the whole protein, including the single-layer beta-sheet, is highly rigid. To elucidate the folding mechanism and the role of the central beta-sheet in the formation of the rigid molecule, we investigated the equilibrium thermal denaturation reaction of OspA. We applied differential scanning calorimetry, heteronuclear NMR spectroscopy, and solution small-angle X-ray scattering (SAXS) to characterize the reaction in detail. All three techniques revealed that OspA denatures in two separable cooperative transitions. NMR measurements on OspA specifically 15N-labeled at Lys residues identified the locations of the two folding units and revealed that the C-terminal segment is less stable than the remaining N-terminal segment. The boundary between the two folding units is located within the central beta-sheet. The interconversion among the three folding states (fully folded, C-terminus unfolded, and fully denatured) is slow relative to chemical shift differences (<24 Hz), indicating that there are significant kinetic barriers in the denaturation reactions. SAXS measurements determined the radius of gyration of the native protein to be 25.0 +/- 0.3 A, which increases to 34.4 +/- 1.0 A in the first transition, and then to 56.1 +/- 1.6 A in the second transition. Thus, the intermediate state, in which the C-terminal folding unit is already denatured, is still compact. These results provide a basis for elucidating the folding mechanism of OspA.

Antigens, Surface↗
PubMed1999

Fentanyl increases dopamine release in rat nucleus accumbens: involvement of mesolimbic mu- and delta-2-opioid receptors.

The effects of the mu-receptor agonist fentanyl on extracellular levels of dopamine in rat nucleus accumbens were studied in awake animals by in vivo brain microdialysis. Fentanyl dose-dependently increased the levels of dopamine when given intravenously (microg/kg) or via a microdialysis probe placed into the ventral tegmental area or the nucleus accumbens (nmol). The effect of fentanyl given into the nucleus accumbens was blocked by systemic administration of the non-selective opioid receptor antagonist naloxone and by accumbens administration of D-Phe-Cys-Tyr-D-Trp-Om-Thr-Phe-Thr-NH2 (nmol), a mu-opioid receptor antagonist, and naltrindole (nmol), a non-selective delta-opioid receptor antagonist, in a dose-dependent manner. The delta2-opioid receptor antagonist, naltriben (nmol), also blocked the effects of fentanyl, whereas the delta1-opioid receptor antagonist, (E)-7-benzylidenenaltrexone (nmol), was ineffective. When marginally effective doses of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2 and naltriben were given simultaneously, the effect of fentanyl was nearly fully blocked; the pretreatment itself had no effect. Administration of the mu-opioid receptor agonist [D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin (nmol), the delta1-opioid receptor agonist [D-Pen2,5]-enkephalin (nmol) or the delta2-opioid receptor agonist [D-Ala2,Glu4]-deltorphin (nmol) into the nucleus accumbens enhanced the amount of accumbal dopamine. This study provides evidence that not only activation of delta1- and delta2-opioid receptors, but also activation of mu-opioid receptors in the nucleus accumbens increases the release of accumbal dopamine in freely moving rats. We suggest that the effect of intra-accumbens administration of fentanyl upon accumbal release of dopamine is either due to the simultaneous activation of mu-opioid receptors and delta2-opioid receptors or due to activation of mu-opioid receptors that interact with delta2-opioid receptors in a complex manner.

Analgesics, Opioid↗
PubMed1999

Human subtilisin-like proprotein convertase, PACE4 (SPC4) gene expression is highly regulated through E-box elements in HepG2 and GH4C1 cells.

PACE4 (SPC4) is a member of the mammalian subtilisin-like proprotein convertase (SPC) family, which participates in maturation of precursor proteins. PACE4 is expressed at high levels in the anterior pituitary, central nervous system, the developing olfactory bulb, heart, and liver. Recently, we determined the gene structure of human PACE4. [Tsuji et al. (1997) J. Biochem. 122, 438-452]. The 5'-flanking region of PACE4 gene contains 12 E-boxes (E1 to E12) within 1 kb upstream of the transcription initiation site. To examine the function of these E-box elements in the regulation of PACE4 expression, deletion and mutation constructs of the 5'-flanking region were ligated to the luciferase gene and analyzed for promoter activity in HepG2 and GH4C1 cells, which express PACE4 at high level. Some differences were observed in the activity of each promoter construct between HepG2 and GH4C1 cells, although the overall profiles of activity for the promoter fragment series were similar regardless of cell type. We showed that the basal promoter activity of the PACE4 gene is first determined by sequences lying between -315 and -1 bp and further regulated by positive and negative elements in the upstream region. Site-directed mutagenesis of E-boxes in these regulatory elements showed that the E10 E-box act as positive regulator, whereas an E-box cluster (E4-E9) acts as a negative regulator in both cells. E2 E-box acts as a positive regulator only in HepG2 cells. Other E-boxes (E1, E3, and E12) had no effect on the promoter activity. These results indicate that E-box elements play a critical role in controlling PACE4 expression in HepG2 and GH4C1 cells and that PACE4 expression is regulated by a mechanism distinct from that of other SPC family proteases.

Base Sequence↗
PubMed1999

Lambert-Eaton myasthenic syndrome associated with idiopathic thrombocytopenic purpura and diffuse panbronchiolitis: long-term remission after a course of intravenous immunoglobulin combined with low-dose prednisolone.

We report a case of Lambert-Eaton myasthenic syndrome (LEMS) associated with idiopathic thrombocytopenic purpura (ITP) and diffuse panbronchiolitis (DPB). An extensive search for malignancy yielded negative results. Interestingly, ITP and DPB developed simultaneously when the patient suffered from myasthenic symptoms. This is the first report in the Japanese or English literature of an association of LEMS, ITP, and DPB. The use of cholinesterase blocker alone did not improve the myasthenic symptoms, and the symptoms and signs relapsed with the tapering of prednisolone (PSL) dosage. However, after administration of immunoglobulin (IVIG) (0.4 g/kg/day x 5 days), low-dose PSL (20 mg/day) alleviated the LEMS and ITP, and the diseases have remained in remission for 8 months without additional IVIG. We suspect that there is a synergistic relationship between IVIG and PSL.

Anti-Inflammatory Agents↗
PubMed1999

Serum thrombopoietin levels in patients with chronic hepatitis and liver cirrhosis.

OBJECTIVE: Thrombocytopenia is a common manifestation of cirrhosis. The aim of this study was to examine the relationship between serum thrombopoietin concentrations, circulating platelet levels, and the stage of hepatic fibrosis in patients with chronic viral hepatitis. METHODS: The study included 48 patients with chronic viral hepatitis (14 with stage 1 fibrosis; five with stage 2 fibrosis; three with stage 3 fibrosis; 26 with cirrhosis) and 30 healthy volunteers. Serum thrombopoietin levels were measured using an enzyme-linked immunosorbent assay. Spleen size, platelet counts, and prothrombin time were measured. RESULTS: Thrombopoietin levels of patients with fibrosis stage 1 (2.50 +/- 1.60 fmol/ml) or stage 2 (1.89 +/- 0.65) were significantly higher than those in patients with cirrhosis (1.21 +/- 0.55) or healthy volunteers (1.26 +/- 0.74). Mean platelet counts of patients with cirrhosis (8.0 +/- 4.6 x 10(4)/microl) were significantly lower than those with fibrosis stage 1 (18.6 +/- 3.9) or stage 2 (16.0 +/- 5.8), or healthy volunteers (24.5 +/- 7.3). Patients with cirrhosis had larger spleens (30.9 +/- 18.4 cm2) than those with fibrosis stage 1 (18.2 +/- 6.4). Platelet counts showed a significant inverse relationship to spleen size (p = -0.51, p < 0.0005) and a significant positive relationship with thrombopoietin levels (p = 0.34, p < 0.02). Thrombopoietin levels were significantly correlated to prothrombin time (p = 0.45, p < 0.005). CONCLUSIONS: Serum thrombopoietin levels are elevated in patients with an early stage of chronic viral hepatitis. As the disease progresses from mild fibrosis to cirrhosis, decreased production of thrombopoietin may contribute to the further development of thrombocytopenia in cirrhosis.

Adult↗
PubMed1998

The fibronectin type III domain as a scaffold for novel binding proteins.

The fibronectin type III domain (FN3) is a small autonomous folding unit which occurs in many animal proteins involving in ligand binding. The beta-sandwich structure of FN3 closely resembles that of immunoglobulin domains. We have prepared a phage display library of FN3 in which residues in two surface loops were randomized. We have selected mutant FN3s which bind to a test ligand, ubiquitin, with significant affinities, while the wild-type FN3 shows no measurable affinity. A dominant clone was expressed as a soluble protein and its properties were investigated in detail. Heteronuclear NMR characterization revealed that the selected mutant protein retains the global fold of FN3. It also has a modest conformational stability despite mutations at 12 out of 94 residues. These results clearly show the potential of FN3 as a scaffold for engineering novel binding proteins.

Amino Acid Sequence↗
PubMed1998

Neurological function after deep hypothermic circulatory arrest in the rat.

BACKGROUND: Integrated neurological function, behavior, and somatic recovery were studied in 35 rats undergoing 5 to 80 minutes of hypothermic circulatory arrest (HCA). METHODS AND RESULTS: A closed extracorporeal circulation system (ECC) consisting of a miniature oxygenator and heat exchanger, primed with 6 mL of asanguinous solution, was connected to a closed-chest rat with cannulae in the right atrium for venous drainage (ID = 1.7 mm) and in the ascending aorta for arterial return (ID = 1.0 mm). The rat was surface- and core-cooled until rectal temperature reached 18 degrees C, when ECC was stopped and cardioplegic solution delivered. After 5, 10, 20, 40 (each n = 5), and 80 minutes (n = 15) of HCA, the rat was reperfused, weaned from ECC, and followed with behavioral scoring, passive avoidance tasks, and cardiopulmonary exercise testing until euthanized for morphological study. Every rat resumed weight gain in the first week after HCA and regained preoperative exercise capacity by the fourth week. Only rats undergoing 80 minutes of HCA showed behavioral abnormalities such as stereotypy and incomplete righting reflex, which eventually disappeared in the fourth week. Learning ability was preserved in all except for rats after 80 min of HCA, who failed to acquire new memory to avoid electric stimuli (n = 10) up to 3 months after HCA, when pyramidal cells were partly replaced by astroglia in the cerebral cortex and CA1 sector of hippocampus. Nonetheless, old memory established before HCA was preserved even after 80 minutes of HCA and allowed rats (n = 5) to avoid electric stimuli. CONCLUSIONS: Homogeneity of animals, miniature ECC system, and an established testing system allowed evaluation of rats after HCA, which disclosed learning disability (functional disorder) and pyramidal cell loss (organic defect) after 80 minutes of HCA despite recovery of somatic function, behavior, and growth.

Animals↗
PubMed1998

NMR identification of epitopes of Lyme disease antigen OspA to monoclonal antibodies.

Outer surface protein A (OspA) from the Lyme disease spirochete Borrelia burgdorferi has been a focus of vaccine development. We have identified epitopes of OspA to two monoclonal antibodies (mAbs) by comparing NMR chemical shifts of free OspA and those in Fab complexes. Deuteration of non-labile protons in OspA extended the size limit of this technique so that it was applicable to the 78 kDa complexes of OspA and the Fab fragment. The epitope identified by NMR to an mAb, 184.1, agrees well with that previously defined by the crystal structure of the same complex, indicating the ability of the NMR method to accurately map an epitope in a large protein complex. The technique mapped the epitope to mAb 336, a mAb of clinical interest, to a region centered at the C-terminal alpha-helix. The results provides a basis for rational design of OspA-based Lyme disease vaccines.

Amino Acid Sequence↗
PubMed1998

A solution SAXS study of Borrelia burgdorferi OspA, a protein containing a single-layer beta-sheet.

The crystal structure of a soluble form of Borrelia burgdorferi outer surface protein A (OspA) complexed with the Fab fragment of a monoclonal antibody has revealed an unusual structure that has a repetitive antiparallel beta topology with a nonglobular, single layer beta-sheet connecting the globular N- and C-terminal domains. Earlier NMR studies have shown that the local structure of OspA including the single layer beta-sheet is similar to the crystal structure. Here we report a small angle X-ray scattering (SAXS) study of the global conformation of OspA in solution. The radius of gyration (Rg) and the length distribution function (P(r)) of OspA measured by SAXS in solution are nearly identical to the calculated ones from the crystal structure, respectively. The NMR and SAXS experiments complement each other to show that OspA including the central single-layer beta-sheet is a stable structure in solution, and that the OspA crystal structure represents the predominant solution conformation of the protein.

Antigens, Surface↗