Development of SAAP3D force field and the application to replica-exchange Monte Carlo simulation for chignolin and C-peptide.

Single amino acid potential (SAAP) would be a prominent factor to determine peptide conformations. To prove this hypothesis, we previously developed SAAP force field for molecular simulation of polypeptides. In this study, the force field was renovated to SAAP3D force field by applying more accurate three-dimensional main-chain parameters, instead of the original two-dimensional ones, for the amino acids having a long side-chain. To demonstrate effectiveness of the SAAP3D force field, replica-exchange Monte Carlo (REMC) simulation was performed for two benchmark short peptides, chignolin (H-GYDPETGTWG-OH) and C-peptide (CHO-AETAAAKFLRAHA-NH2). For chignolin, REMC/SAAP3D simulation correctly produced native ß-turn structures, whose minimal all-atom root-mean-square deviation value measured from the native NMR structure (except for H) was 1.2 Å, at 300 K in implicit water, along with misfolded ß-hairpin structures with unpacked aromatic side chains of Tyr2 and Trp9. Similar results were obtained for chignolin analog [G1Y,G10Y], which folded more tightly to the native ß-turn structure than chignolin did. For C-peptide, on the other hand, the α-helix content was larger than the ß content on average, suggesting a significant helix-forming propensity. When the imidazole side chain of His12 was protonated (i.e., [His12Hip]), the α content became larger. These observations as well as the representative structures obtained by clustering analysis were in reasonable agreement not only with the structures of C-peptide that were determined in this study by NMR in 30% CD3CD in H2O at 298 K but also with the experimental and theoretical behaviors having been reported for protonated C-peptide. Thus, accuracy of the SAAP force field was improved by applying three-dimensional main-chain parameters, supporting prominent importance of SAAP for peptide conformations.

Nonconvulsive Status Epilepticus Associated with Cerebral Hyperperfusion Syndrome after Carotid Endarterectomy: A Case Report.

We report a case of a 73-year-old man who developed nonconvulsive status epilepticus as a complication of cerebral hyperperfusion syndrome after carotid endarterectomy for carotid artery stenosis. On postoperative day 1, the patient experienced headaches and vomiting. Resting N-isopropyl-p-[123I] iodoamphetamine single-photon emission computed tomography showed increased cerebral blood flow to the entire right hemisphere, and the patient was diagnosed with cerebral hyperperfusion syndrome. He was treated with antihypertensive and antiseizure medications, sedated using propofol, intubated, and placed under mechanical ventilation. On postoperative day 3, computed tomography perfusion imaging showed a reduction in hyperperfusion, and propofol sedation was terminated on postoperative day 4. However, the patient exhibited prolonged impaired awareness and roving eye movements, and long-term video electroencephalographic monitoring revealed electrographic seizures. The patient was diagnosed with nonconvulsive status epilepticus. Propofol sedation was resumed, and the antiseizure medication dose was increased. Subsequently, the state of hyperperfusion in the right hemisphere diminished, and electroencephalographic findings improved, allowing sedation to be terminated on postoperative day 7. The findings from this case suggest that when clinical subtle symptoms, such as impaired awareness and roving eye movements, are observed during treatment of cerebral hyperperfusion syndrome, video electroencephalography should be performed to detect electrographic seizures.

A Case of Acquired Factor XIII Deficiency with Systemic Lupus Erythematosus Diagnosed after Repeated Intracerebral Hemorrhages.

We present a case of autoimmune-acquired factor XIII deficiency associated with systemic lupus erythematosus, which was diagnosed as a cause of repeated intracerebral hemorrhage. An intracerebral hemorrhage occurred in a 24-year-old female patient. Craniotomy was performed to remove the hematoma, but rebleeding occurred at the same site on days 2 and 11, respectively. Detailed blood tests revealed that factor XIII activity decreased. Although autoimmune-acquired factor XIII deficiency is a very rare disease, it can sometimes be fatal when intracerebral hemorrhage occurs. If there is repeated intracerebral hemorrhage, factor XIII activity should be confirmed.

Chemical and genetic study of Ligularia duciformis and related species in Sichuan and Yunnan Provinces of China.

The chemical constituents of the root extracts and the evolutionarily neutral DNA base sequences were studied for 28 samples of Ligularia duciformis, L. kongkalingensis, and L. nelumbifolia collected in Sichuan and Yunnan Provinces of China. The samples could be classified into four chemotypes (1-4). Sesquiterpenoids having eremophilane and oplopane skeletons were isolated from two (Chemotype 1) and three (Chemotype 2) samples, respectively. Two new oplopane derivatives were isolated and their structures were determined. In 18 samples, phenylpropenoids were the major components (Chemotype 3). In five samples, neither phenylpropenoids nor sesquiterpenoids were found (Chemotype 4). Despite this large chemical variety, no correlation was found between the chemotype and the morphological criteria of species identification. The analysis of the evolutionarily neutral DNA regions also indicated that the samples were not separated into distinct clades and that introgression was extensive.

Effect of contact with titanium alloys on the proliferation of mouse osteoblastic cells in culture.

This study was aimed at studying the effect of contact with titanium alloy plates of different surface textures on the proliferative capability of mouse osteoblastic MC3T3-E1 cells. First, the proliferation characteristics of MC3T3-E1 cells were investigated. MC3T3-E1 cells showed a high capacity for proliferation and survived for a long period even under nutritionally starved conditions. During logarithmic cell growth, the consumption of Ser, Gln, Val, Ile and Leu increased time-dependently. Contact with an hydoxyapatite (HA)-coated titanium alloy plate resulted in the increase in the recovery of cells from the plate by trypsin, and an increase in the consumption of these amino acids, suggesting enhanced cell proliferation. On the contrary, contact with the sandblasted and anodized titanium alloy plates resulted in the reduction of the recovery of the cells from the plate, but a slight increase in the amino acid consumption, suggesting the tight adhesion of the cells to the plates. This study demonstrates that the present method, based on the amino acid consumption of the cells, is useful for monitoring the cell proliferative capability, without detachment of the cells from the plate. This method may be applicable to the study of the interaction between cells and metal plates.

Simple and reliable method to simultaneously determine urinary 1- and 2-naphthol using in situ derivatization and gas chromatography-mass spectrometry for biological monitoring of naphthalene exposure in occupational health practice.

OBJECTIVES: The aim of this study was to develop and validate a simple and reliable gas chromatography-mass spectrometry (GC-MS) method to simultaneously determine urinary 1-naphthol (1-NAP) and 2-naphthol (2-NAP) for biological monitoring of occupational exposure to naphthalene. METHODS: NAPs were derivatized in situ with acetic anhydride after enzymatic hydrolysis, extracted with n-hexane, and analyzed using GC-MS. Validation of the proposed method was conducted in accordance with US Food and Drug Administration guidance. A final validation was performed by analyzing a ClinChek® -Control for phenolic compounds. RESULTS: The linearity of calibration curves was indicated by a high correlation coefficient (>0.999) in the concentration range 1-100 µg/L for each NAP. The limits of detection and quantification for each NAP were 0.30 and 1.00 µg/L, respectively. The recovery was 90.8%-98.1%. The intraday and interday accuracies, expressed as the deviation from the nominal value, were 92.2%-99.9% and 93.4%-99.9%, respectively. The intraday and interday precision, expressed as the relative standard deviation, was 0.3%-3.9% and 0.4%-4.1%, respectively. The ClinChek® values obtained using our method were sufficiently accurate. CONCLUSIONS: The proposed method is simple, reliable, and appropriate for routine analyses, and is useful for biological monitoring of naphthalene exposure in occupational health practice.

Adhesion and Proliferation of Osteoblastic Cells on Hydroxyapatite-dispersed Ti-based Composite Plate.

BACKGROUND/AIM: Biocompatibility of a novel and more stable hydroxyapatite (HA)-dispersed titanium (Ti)-based composite was investigated, using the mouse osteoblast precursor cell line MC3T3-E1. MATERIALS AND METHODS: Surface of powders and plates was observed by scanning electron microscopy. Distribution of calcium and phosphorus on the surface of the composite was evaluated by an electron beam microanalyzer. Crystal structure was analyzed by X-ray diffractometer. Cell viability was determined by WST-1 assay. RESULTS: HA was stable against the compressive force, shearing stress and sintering heat at 800°C, but it slightly decomposed at 1100°C. With the increase of HA in the composites, the adhesion/proliferation of MC3T3-E1 cells was reduced. The growth inhibition by HA does not seem to be due to materials released from the plate, but rather to the contact to the surface of the plate. Sintering of the HA plate at 1100°C increased the number of attached viable cells. On the other hand, culturing on the synthesized calcium phosphate (apatite containing carbonic acid) increased the number of attached cells to a greater extent. CONCLUSION: HA inhibits the growth of osteoblastic cells, but sintering at 1100°C changes the surface properties of the composite so as to stimulate cell growth.

Method for comparing the structures of protein ligand-binding sites and application for predicting protein-drug interactions.

Many drugs, even ones that are designed to act selectively on a target protein, bind unintended proteins. These unintended bindings can explain side effects or indicate additional mechanisms for a drug's medicinal properties. Structural similarity between binding sites is one of the reasons for binding to multiple targets. We developed a method for the structural alignment of atoms in the solvent-accessible surface of proteins that uses similarities in the local atomic environment, and carried out all-against-all structural comparisons for 48,347 potential ligand-binding regions from a nonredundant protein structure subset (nrPDB, provided by NCBI). The relationships between the similarity of ligand-binding regions and the similarity of the global structures of the proteins containing the binding regions were examined. We found 10,403 known ligand-binding region pairs whose structures were similar despite having different global folds. Of these, we detected 281 region pairs that had similar ligands with similar binding modes. These proteins are good examples of convergent evolution. In addition, we found a significant correlation between Z-score of structural similarity and true positive rate of "active" entries in the PubChem BioAssay database. Moreover, we confirmed the interaction between ibuprofen and a new target, porcine pancreatic elastase, by NMR experiment. Finally, we used this method to predict new drug-target protein interactions. We obtained 540 predictions for 105 drugs (e.g., captopril, lovastatin, flurbiprofen, metyrapone, and salicylic acid), and calculated the binding affinities using AutoDock simulation. The results of these structural comparisons are available at http://www.tsurumi.yokohama-cu.ac.jp/fold/database.html.

Notoamides F-K, prenylated indole alkaloids isolated from a marine-derived Aspergillus sp.

Six new prenylated indole alkaloids, named notoamides F-K (8-13), were isolated from a marine-derived Aspergillus sp. Their structures, including absolute configurations, were elucidated by spectroscopic methods. Notoamide I (11) showed weak cytotoxicity against HeLa cells with an IC(50) value of 21 microg/mL.

Effect of saliva, epigallocatechin gallate and hypoxia on Cu-induced oxidation and cytotoxicity.

We have previously reported that contact with copper (Cu) induced immediate cell death via an oxidation-involved mechanism in human promyelocytic leukemic HL-60 cells, whereas contact with other metals (Au, Ag, Pd) produced no discernible effect. In the present study, we investigated the conditions under which Cu-induced oxidative stress can be reduced. Contact with a Cu plate in the absence of cells enhanced the rate of consumption of cystine to the greatest extent, followed by that of methionine and histidine. Under hypoxic conditions, the consumption of all these amino acids was significantly reduced. On the other hand, the addition of saliva slightly, but not significantly, reduced the amino acid oxidation. The addition of epigallocatechin gallate (EGCG) slightly, but significantly reduced the consumption of cystine and histidine. The inhibitory effect of EGCG on the methionine consumption was more prominent, especially at higher concentrations. The Cu-induced cell death was significantly inhibited when freshly-prepared human gingival fibroblasts were incubated under hypoxic conditions. The present study demonstrates for the first time that the Cu-induced oxidation and cell death were effectively alleviated under hypoxic conditions.

Disruption of amino acid metabolism in human myelogenous leukemic cell lines destined to die after contact with metal plates.

Changes in amino acid metabolism during cell death of human myelogenous leukemic cell lines (HL-1, ML-1, KG-1) induced by contact with gold (Au), silver (Ag) or palladium (Pd) were investigated. All three leukemic cell lines consumed glutamine and serine at the highest rate (amounting to 50%-58% and 12%-16% of the total amino acid consumption, respectively). HL-60 cell growth was slightly stimulated by contact with any metal plate. Contact with Ag or Pd, but not Au plates occasionally induced cytotoxicity against ML-1 and KG-1 cells. In such cases, glutamine consumption was inhibited by 88%-90% and consumption of other amino acids completely ceased. This was accompanied by the enhanced production of arginine, glycine and glutamic acid. These data suggest the tight association of the disruption of amino acid metabolism with the cell death induced in human myelogenous leukemic cell lines by contact with metal plates.

Rapid changes in amino acid and polyamine metabolism during copper-induced cell death of human gingival fibroblast.

There are very few studies on the interaction between dental alloys and oral tissues. The effect of direct contact with copper (Cu) on the cellular function of human gingival fibroblast (HGF) derived from the periodontal tissues was investigated. When HGF cells were inoculated onto a Cu plate, the viability of HGF cells immediately declined. This was accompanied by vacuolization and chromatin condensation near the nuclear membrane. The intracellular concentration of spermidine and spermine declined, whereas that of putrescine slightly increased. Amino acid analysis of the medium revealed that glutamine was consumed at the greatest rate, amounting to more than half of the total amino acid consumption. Contact with the Cu plate resulted in the complete elimination of glutamine utilization and a simultaneous increase in the production of most amino acids, possibly due to enhanced proteolysis. This was accompanied by a time-dependent increase in the consumption of cystine, possibly due to oxidative reactions, and the enhanced production of glycine and glutamic acid. These data suggest that the contact with the Cu plate induced non-apoptotic cell death in HGF cells, which was tightly coupled with a rapid dysfunction of amino acid and polyamine metabolism.

Chemical and genetic diversity of Ligularia latihastata and Ligularia villosa in Yunnan Province of China.

The chemical constituents of the root extracts and the nucleotide sequences of the atpB-rbcL intergenic region of Ligularia latihastata and L. villosa, collected in northwestern Yunnan Province, were studied. In the twelve collected samples of L. latihastata, two major benzofurans, 5,6-dimethoxy-2-(1-methylethenyl)-1-benzofuran (1) and euparin (2) were detected as major components. The minor compound (2R*,3S*)-5-acetyl-2,3-dihydro-6-hydroxy-2-(1-methylethenyl)-1-benzofuran-3-yl (2Z)-2-[(acetoxy)methyl]but-2-enoate (4) was found to be susceptible to artifact formation upon extraction with EtOH. The intra-specific diversity in chemical composition of the samples was small, but the diversity in the atpB-rbcL sequence was fairly large. Compounds 1 and 2 were also found in the three collected samples of L. villosa, indicating that the two species are chemically close to each other, in agreement with morphological taxonomy.

The identification of á´ -tryptophan as a bioactive substance for postembryonic ovarian development in the planarian Dugesia ryukyuensis.

Many metazoans start germ cell development during embryogenesis, while some metazoans possessing pluripotent stem cells undergo postembryonic germ cell development. The latter reproduce asexually but develop germ cells from pluripotent stem cells or dormant primordial germ cells when they reproduce sexually. Sexual induction of the planarian Dugesia ryukyuensis is an important model for postembryonic germ cell development. In this experimental system, hermaphroditic reproductive organs are differentiated in presumptive gonadal regions by the administration of a crude extract from sexual planarians to asexual ones. However, the substances involved in the first event during postembryonic germ cell development, i.e., ovarian development, remain unknown. Here, we aimed to identify a bioactive compound associated with postembryonic ovarian development. Bioassay-guided fractionation identified ʟ-tryptophan (Trp) on the basis of electrospray ionization-mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy. Originally masked by a large amount of ʟ-Trp, ᴅ-Trp was detected by reverse-phase high-performance liquid chromatography. The ovary-inducing activity of ᴅ-Trp was 500 times more potent than that of ʟ-Trp. This is the first report describing a role for an intrinsic ᴅ-amino acid in postembryonic germ cell development. Our findings provide a novel insight into the mechanisms of germ cell development regulated by low-molecular weight bioactive compounds.

Protection against copper-induced cytotoxicity by inclusion of gold.

BACKGROUND: We previously reported that contact with copper (Cu) induced immediate cell death via an oxidation-involved mechanism, and the Cu-induced oxidation and cell death were effectively alleviated under hypoxic conditions. In order to explore alternative strategies for the protection from the Cu-induced cytotoxicity, we investigated whether the inclusion of gold (Au) in the Cu plate, as alloy,has a protective effect. MATERIALS AND METHODS: Human gingival fibroblast (HGF) cells, established from periodontal tissues, were inoculated on Au/Cu alloy of different Au ratios. After incubation at 37°C for different times under normoxic conditions, cellular viability and amino acid consumption were determined. Changes in the elemental composition of the alloy and in the culture medium were chemically analyzed by X-ray photoelectron spectroscopy and by inductively coupled plasma-optical emission spectrometry. RESULTS: Contact with the Cu plate induced cytotoxicity and cystine oxidation in time-dependent manners. Inclusion of Au at more than 10% in the alloy, completely abrogated the cytotoxicity and reduced the oxidation of Cu and the elution of Cu from the alloy. CONCLUSION: Inclusion of Au as a component of alloy reduces the cytotoxicity of the Cu plate, possibly by reducing its oxidation.

Chemical constituents and diversity of Ligularia lankongensis in Yunnan Province of China.

The chemical constituents of the roots of Ligularia lankongensis collected at seven different places in Yunnan Province, China, were investigated together with the DNA sequence of the atpB- rbcL intergenic region. All the samples contained a new, highly oxygenated bisabolane-type sesquiterpene ( 1). Four other oxygenated bisabolanes ( 2 and the new 3, 4, and 5) were also obtained. Intraspecific diversity was observed in the composition of the compounds, but not in the DNA sequence.

Himeic acid A: a new ubiquitin-activating enzyme inhibitor isolated from a marine-derived fungus, Aspergillus sp.

A new ubiquitin-activating enzyme (E1) inhibitor, himeic acid A, was isolated from a culture of marine-derived fungus, Aspergillus sp. The structure was determined by spectroscopic analysis. The formation of an E1-ubiquitin (Ub) intermediate was 65% inhibited by himeic acid A at the concentration of 50 microM, while two new related compounds, himeic acids B and C, showed little inhibitory activity even at 100 microM.

Isolation of cytochrome P450 inhibitors from strawberry fruit, Fragaria ananassa.

A new glycoside, 2-beta-d-glucopyranosyloxy-4,6-dihydroxyisovalerophenone (3), was isolated from strawberry fruit along with kaempferol-3-beta-D-(6-O-trans-p-coumaroyl)glucopyranoside (1) and kaempferol-3-beta-D-(6-O-cis-p-coumaroyl)glucopyranoside (2). Compounds 1 and 2 inhibited activity of a drug-metabolizing enzyme, CYP3A4.