Rapid Bactericidal Action of Propolis against Porphyromonas gingivalis.

Propolis, a resinous substance produced by bees, is used as a folk medicine for treatment of periodontal diseases. However, its mode of the action and the compounds responsible for its activities remain obscure. In the present study, we comprehensively investigated the antibacterial activities of ethanol-extracted propolis (EEP) and EEP-derived compounds toward Porphyromonas gingivalis, a keystone pathogen for periodontal diseases. Broth microdilution and agar dilution assays were used to determine the minimum inhibitory concentrations of EEP against a range of oral bacterial species, of which P. gingivalis showed a higher level of sensitivity than oral commensals such as streptococci. Its antibacterial activity toward P. gingivalis was maintained even after extensive heat treatment, demonstrating a high level of thermostability. EEP also induced death of P. gingivalis cells by increasing membrane permeability within 30 min. Spatiotemporal analysis based on high-speed atomic force microscopy revealed that EEP immediately triggered development of aberrant membrane blebs, followed by bleb fusion events on the bacterial surface. Furthermore, we isolated artepillin C, baccharin, and ursolic acid from EEP as antibacterial compounds against P. gingivalis. Of those, artepillin C and baccharin showed bacteriostatic activities with membrane blebbing, while ursolic acid showed bactericidal activity with membrane rupture. In particular, ursolic acid demonstrated a greater ability to affect bacterial membrane potential with increased membrane permeability, probably because of its highly lipophilic nature as compared with other compounds. Taken together, these findings provide mechanistic insight into the antibacterial activities of EEP and its exquisite membrane-targeting antibacterial compounds and imply the applicability of narrow-spectrum therapeutics with EEP for treatment of periodontitis. In addition, the advanced technology utilized in the present study to visualize the nanometer-scale dynamics of microorganisms will contribute to expanding our understanding of the activities of antimicrobials and the mechanism of drug resistance in bacteria.

Shell Evolution towards

The level structure of the neutron-rich ^{77}Cu nucleus is investigated through ß-delayed γ-ray spectroscopy at the Radioactive Isotope Beam Factory of the RIKEN Nishina Center. Ions of ^{77}Ni are produced by in-flight fission, separated and identified in the BigRIPS fragment separator, and implanted in the WAS3ABi silicon detector array, surrounded by Ge cluster detectors of the EURICA array. A large number of excited states in ^{77}Cu are identified for the first time by correlating γ rays with the ß decay of ^{77}Ni, and a level scheme is constructed by utilizing their coincidence relationships. The good agreement between large-scale Monte Carlo shell model calculations and experimental results allows for the evaluation of the single-particle structure near ^{78}Ni and suggests a single-particle nature for both the 5/2_{1}^{-} and 3/2_{1}^{-} states in ^{77}Cu, leading to doubly magic ^{78}Ni.

94 ß-Decay Half-Lives of Neutron-Rich _{55}Cs to _{67}Ho: Experimental Feedback and Evaluation of the r-Process Rare-Earth Peak Formation.

The ß-decay half-lives of 94 neutron-rich nuclei ^{144-151}Cs, ^{146-154}Ba, ^{148-156}La, ^{150-158}Ce, ^{153-160}Pr, ^{156-162}Nd, ^{159-163}Pm, ^{160-166}Sm, ^{161-168}Eu, ^{165-170}Gd, ^{166-172}Tb, ^{169-173}Dy, ^{172-175}Ho, and two isomeric states ^{174m}Er, ^{172m}Dy were measured at the Radioactive Isotope Beam Factory, providing a new experimental basis to test theoretical models. Strikingly large drops of ß-decay half-lives are observed at neutron-number N=97 for _{58}Ce, _{59}Pr, _{60}Nd, and _{62}Sm, and N=105 for _{63}Eu, _{64}Gd, _{65}Tb, and _{66}Dy. Features in the data mirror the interplay between pairing effects and microscopic structure. r-process network calculations performed for a range of mass models and astrophysical conditions show that the 57 half-lives measured for the first time play an important role in shaping the abundance pattern of rare-earth elements in the solar system.

Yrast 6⁺ seniority isomers of (136,138)Sn.

Delayed γ-ray cascades, originating from the decay of (6⁺) isomeric states, in the very neutron-rich, semimagic isotopes (136,138)Sn have been observed following the projectile fission of a ²³8U beam at RIBF, RIKEN. The wave functions of these isomeric states are proposed to be predominantly a fully aligned pair of f(7/2) neutrons. Shell-model calculations, performed using a realistic effective interaction, reproduce well the energies of the excited states of these nuclei and the measured transition rates, with the exception of the B(E2;6⁺→4⁺) rate of ¹³6Sn, which deviates from a simple seniority scheme. Empirically reducing the νf(7/2)(2) orbit matrix elements produces a 41⁺ state with almost equal seniority 2 and 4 components, correctly reproducing the experimental B(E2;6⁺→4⁺) rate of ¹³6Sn. These data provide a key benchmark for shell-model interactions far from stability.

ß-Decay half-lives of 76,77Co, 79,80Ni, and 81Cu: experimental indication of a doubly magic 78Ni.

The half-lives of 20 neutron-rich nuclei with Z=27-30 have been measured at the RIBF, including five new half-lives of (76)Co(21.7(-4.9)(+6.5) ms), (77)Co(13.0(-4.3)(+7.2) ms), (79)Ni(43.0(-7.5)(+8.6) ms), (80)Ni(23.9(-17.2)(+26.0) ms), and (81)Cu(73.2 ± 6.8 ms). In addition, the half-lives of (73-75)Co, (74-78)Ni, (78-80)Cu, and (80-82)Zn were determined with higher precision than previous works. Based on these new results, a systematic study of the ß-decay half-lives has been carried out, which suggests a sizable magicity for both the proton number Z = 28 and the neutron number N=50 in (78)Ni.

Monopole-driven shell evolution below the doubly magic nucleus 132Sn explored with the long-lived isomer in 126Pd.

A new isomer with a half-life of 23.0(8) ms has been identified at 2406 keV in (126)Pd and is proposed to have a spin and parity of 10(+) with a maximally aligned configuration comprising two neutron holes in the 1h(11/2) orbit. In addition to an internal-decay branch through a hindered electric octupole transition, ß decay from the long-lived isomer was observed to populate excited states at high spins in (126)Ag. The smaller energy difference between the 10(+) and 7(-) isomers in (126)Pd than in the heavier N=80 isotones can be interpreted as being ascribed to the monopole shift of the 1h(11/2) neutron orbit. The effects of the monopole interaction on the evolution of single-neutron energies below (132)Sn are discussed in terms of the central and tensor forces.

1p3/2 proton-hole state in 132Sn and the shell structure along N = 82.

A low-lying state in 131In82, the one-proton hole nucleus with respect to double magic 132Sn, was observed by its γ decay to the Iπ=1/2- ß-emitting isomer. We identify the new state at an excitation energy of Ex=1353 keV, which was populated both in the ß decay of 131Cd83 and after ß-delayed neutron emission from 132Cd84, as the previously unknown πp3/2 single-hole state with respect to the 132Sn core. Exploiting this crucial new experimental information, shell-model calculations were performed to study the structure of experimentally inaccessible N=82 isotones below 132Sn. The results evidence a surprising absence of proton subshell closures along the chain of N=82 isotones. The consequences of this finding for the evolution of the N=82 shell gap along the r-process path are discussed.

Isomers in 128Pd and 126Pd: evidence for a robust shell closure at the neutron magic number 82 in exotic palladium isotopes.

The level structures of the very neutron-rich nuclei 128Pd and 126Pd have been investigated for the first time. In the r-process waiting-point nucleus 128Pd, a new isomer with a half-life of 5.8(8) µs is proposed to have a spin and parity of 8(+) and is associated with a maximally aligned configuration arising from the g(9/2) proton subshell with seniority υ=2. For 126Pd, two new isomers have been identified with half-lives of 0.33(4) and 0.44(3) µs. The yrast 2(+) energy is much higher in 128Pd than in 126Pd, while the level sequence below the 8(+) isomer in 128Pd is similar to that in the N=82 isotone 130Cd. The electric quadrupole transition that depopulates the 8(+) isomer in 128Pd is more hindered than the corresponding transition in 130Cd, as expected in the seniority scheme for a semimagic, spherical nucleus. These experimental findings indicate that the shell closure at the neutron number N=82 is fairly robust in the neutron-rich Pd isotopes.

Toxicity of Hydnora johannis Becca. dried roots and ethanol extract in rats.

AIM OF THE STUDY: Hydnora johannis Becca. (Hydnoraceae) commonly is used for the treatment of dysentery, diarrhoea, cholera and swelling tonsillitis in the folk medicine of Sudan and other African countries. This study evaluates the toxicological effects of Hydnora johannis roots on Wistar rats. MATERIALS AND METHODS: Rats were randomized into control, groups fed with 2, 10, 20% of dried roots for 8 weeks and other groups given ethanol extract (50, 100, 200 and 400mg/kg/day) through oral and intramuscularly administration for 2 weeks. Toxicity was evaluated using biochemical and histopathological assays. RESULTS: Alterations in the levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, cholesterol and urea were observed. Histopathological analysis revealed that the toxic effect were mainly on the liver, kidney and spleen on all treated groups. However, the impact of the dried roots was mild compared to the ethanol extract. Remarkably, there was a drop in cholesterol level in all treatment groups suggesting the antiartherogenic effect of Hydnora johannis roots. CONCLUSION: The results from this study suggest that the powder preparation as well as ethanolic extract of Hydnora johannis roots induced toxic effect on Wistar rats. The observed toxic effect might be due to the dose and/or frequency of administration. Although in traditional medicine the extract is administrated in low dose, the results suggest the necessity of standardization of the drug.

The influence of temperature and humidity on oviposition and hatchability of Amblyomma lepidum (Dönitz, 1808) (Acarina: Ixodidae) under laboratory conditions.

The effect of temperature and humidity on the oviposition and hatchability of Ablyomma lepidum was studied. Above 90% of adult ticks applied on calves succeeded to attach and feed through 6-13 days. The development process was studied under three levels of temperature: 27, 35 and 40 degrees C, each level with five sets of humidity. Temperature rather than humidity affected all developmental parameters. It was found that high temperature of 40 degrees C, even at high humidity 75.5-97.8% significantly affected pre-oviposition, oviposition, pre-hatching periods, egg mass weight and egg conversion ratio (p

Antitumor properties and modulation of antioxidant enzymes' activity by Aloe vera leaf active principles isolated via supercritical carbon dioxide extraction.

The aim of this study was to evaluate the potential anticancer properties and modulatory effect of selected Aloe vera (A. vera) active principles on antioxidant enzyme activities. Thus, three anthraquinones (Namely: aloesin, aloe-emodin and barbaloin) were extracted from A. vera leaves by supercritical fluid extraction and subsequently purified by high performance liquid chromatography. Additionally, the N-terminal octapeptide derived from verectin, a biologically active 14 kDa glycoprotein present in A. vera, was also tested. In vivo, active principles exhibited significant prolongation of the life span of tumor-transplanted animals in the following order: barbaloin> octapeptide> aloesin > aloe-emodin. A. vera active principles exhibited significant inhibition on Ehrlich ascite carcinoma cell (EACC) number, when compared to positive control group, in the following order: barbaloin> aloe-emodin > octapeptide > aloesin. Moreover, in trypan blue cell viability assay, active principles showed a significant concentration-dependent cytotoxicity against acute myeloid leukemia (AML) and acute lymphocytes leukemia (ALL) cancerous cells. Furthermore, in MTT cell viability test, aloe-emodin was found to be active against two human colon cancer cell lines (i.e. DLD-1 and HT2), with IC(50) values of 8.94 and 10.78 microM, respectively. Treatments of human AML leukemic cells with active principles (100 microg ml(-1)) resulted in varying intensities of internucleosomal DNA fragmentation, hallmark of cells undergoing apoptosis, in the following order: aloe-emodin> aloesin> barbaloin> octapeptide. Intererstingly, treatment of EACC tumors with active principles resulted in a significant elevation activity of key antioxidant enzymes (SOD, GST, tGPx, and LDH). Our data suggest that the tested A. vera compounds may exert their chemo-preventive effect through modulating antioxidant and detoxification enzyme activity levels, as they are one of the indicators of tumorigenesis. These findings are discussed in the light of the potential of A. vera plant extracts for developing efficient, specific and non-toxic anticancer drugs that are affordable for developing countries.

Fast-scanning atomic force microscopy reveals the molecular mechanism of DNA cleavage by ApaI endonuclease.

Newly developed fast-scanning atomic force microscopy (AFM) allows the dissection of molecular events such as DNA-enzyme reactions at the single-molecule level. With this novel technology, a model is proposed of the DNA cleavage reaction by a type IIP restriction endonuclease ApaI. Detailed analyses revealed that ApaI bound to DNA as a dimer and slid along DNA in a one-dimensional diffusion manner. When it encountered a specific DNA sequence, the enzyme halted for a moment to digest the DNA. Immediately after digestion, the ApaI dimer separated into two monomers, each of which remained on the DNA end and then dissociated from the DNA end. Thus, fast-scanning AFM is a powerful tool to aid the understanding of protein structures and dynamics in biological reactions at the single-molecule level in sub-seconds.

Radical scavenging glycoprotein inhibiting cyclooxygenase-2 and thromboxane A2 synthase from aloe vera gel.

An active glycoprotein fraction containing 58 % protein was isolated from Aloe vera gel by precipitation with 55 % ammonium sulfate followed by gel permeation using DEAE-Sephacel A-25, Sepharose 6B and Sephadex G-50 columns in a yield of 3 x 10 -3 %. The glycoprotein fraction showed a single band corresponding to a subunit of verectin at the same position when stained with both Coomassie brilliant blue and periodic acid-Schiff reagents on 18 % SDS-PAGE. The molecular weight (14 kDa) was confirmed by Sephadex G-50 column chromatography. The glycoprotein fraction showed a radical scavenging activity against superoxide anion generated by the xanthine-xanthine oxidase system as well as inhibition of cyclooxygenase-2 and reduction of thromboxane A 2 synthase level in vitro.

Antioxidant, free radical scavenging and anti-inflammatory effects of aloesin derivatives in Aloe vera.

Antioxidant components in Aloe vera were examined for lipid peroxidation using rat liver microsomal and mitochondrial enzymes. Among the aloesin derivatives examined, isorabaichromone showed a potent antioxidative activity. The DPPH radical and superoxide anion scavenging activities were determined. As one of the most potent components, isorabaichromone together with feruloylaloesin and p-coumaroylaloesin showed potent DPPH radical and superoxide anion scavenging activities. Electron spin resonance (ESR) using the spin trapping method suggested that the potent superoxide anion scavenging activity of isorabaichromone may have been due to its caffeoyl group. As A. vera has long been used to promote wound healing, the inhibitory effects of aloesin derivatives for cyclooxygenase (Cox)-2 and thromboxane (Tx) A 2 synthase were examined and the participation of p-coumaroyl and feruloyl ester groups in the aloesin skeleton was demonstrated. These findings may explain, at least in part, the wound healing effects of A.vera. Abbreviations. ADP:adenosine diphosphate ASA:ascorbic acid BHT:butylated hydroxytoluene BSA:bovine serum albumin DMPO:5,5-dimethyl-1-pyrroline N-oxide DPPH:1,1-diphenyl-2-picrylhydrazyl EDTA:edetic acid HEPES: N-(2-hydroxyethyl)-piperazine- N-2'-ethane-sulfonic acid NADH:reduced nicotinamide adenine dinucleotide NADPH:reduced nicotinamide adenine dinucleotide phosphate NBT:nitroblue tetrazolium Pg:prostaglandin SOD:superoxide dismutase TBA:thiobarbituric acid TCA:trichloroacetic acid XOD:xanthine oxidase

Expression of Sprouty genes 1, 2 and 4 during mouse organogenesis.

We demonstrate that Sprouty genes 1, 2 and 4 are expressed in several developing organs of the craniofacial area and trunk, including the brain, cochlea, nasal organs, teeth, salivary gland, lungs, digestive tract, kidneys and limb buds. In organs such as the semicircular canal, Rathke's pouch, nasal organs, the follicle of vibrissae and teeth, Sprouty1 and Sprouty2 are expressed in the epithelium and Sprouty4 in the mesenchyme or neuronal tissue, while in the lung Sprouties1, 2 and 4 are all expressed mainly in the epithelial tissue. In the kidney, Sprouty1 is prominent in the ureteric bud whereas Sprouty2 and 4 are expressed in both the ureteric bud and the kidney mesenchyme and glomeruli deriving from it. The expression profiles suggest roles for these Sprouties in the epithelial-mesenchymal interactions that govern organogenesis.

NADH dehydrogenases: from basic science to biomedicine.

This review article is concerned with two on-going research projects in our laboratory, both of which are related to the study of the NADH dehydrogenase enzyme complexes in the respiratory chain. The goal of the first project is to decipher the structure and mechanism of action of the proton-translocating NADH-quinone oxidoreductase (NDH-1) from two bacteria, Paracoccus denitrificans and Thermus thermophilus HB-8. These microorganisms are of particular interest because of the close resemblance of the former (P. denitrificans) to a mammalian mitochondria, and because of the thermostability of the enzymes of the latter (T. thermophilus). The NDH-1 enzyme complex of these and other bacteria is composed of 13 to 14 unlike subunits and has a relatively simple structure relative to the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I), which is composed of at least 42 different subunits. Therefore, the bacterial NDH-I is believed to be a useful model for studying the mitochondrial complex I, which is understood to have the most intricate structure of all the membrane-associated enzyme complexes. Recently, the study of the NADH dehydrogenase complex has taken on new urgency as a result of reports that complex I defects are involved in many human mitochondrial diseases. Thus the goal of the second project is to develop possible gene therapies for mitochondrial diseases caused by complex I defects. This project involves attempting to repair complex I defects in the mammalian system using Saccharomyces cerevisiae NDI1 genes, which code for the internal, rotenone-insensitive NADH-quinone oxidoreductase. In this review, we will discuss our progress and the data generated by these two projects to date. In addition, background information and the significance of various approaches employed to pursue these research objectives will be described.

[Clinical effectiveness of intra-arterial chemotherapy combined with degradable starch microspheres (DSM) for liver metastases and prediction of effectiveness by diagnostic imaging].

The aim of this study was to evaluate the clinical effectiveness of intra-arterial chemotherapy combined with degradable starch microspheres (DSM) for liver metastases and the possibility of predicting the effectiveness of the chemotherapy by pretreatment diagnostic imaging. The subjects were 67 patients with metastatic liver cancer, treated with Seldinger method via the left brachial artery, and tumor selective hepatic injection using a micro-catheter. The early response rate was 38.7% for colorectal cancer, 42.8% for gastric cancer, 16.7% for bile tract cancer and 80% for uterine cancer. The relationship between effectiveness and the tumor occupation rate in the liver estimated from pretreatment CT images was not significant, but the degree of tumor stain in the early phase of contrast enhancement CT correlated well with early responsiveness of the liver metastases for this treatment. This suggests the possibility of pretreatment prediction of the effectiveness of intra-arterial chemotherapy combined with DSM for metastatic liver tumors.

Simultaneous determination of glycyrrhizin, glycyrrhetic acid and glycyrrhetic acid mono-glucuronide in Shakuyaku-kanzo-to incubated with rat feces by semi-micro high-performance liquid chromatography.

A method for semi-micro high-performance liquid chromatography (HPLC) has been established for the simultaneous determination of glycyrrhizin (GL), glycyrrhetic acid (GA) and glycyrrhetic acid mono-glucuronide (GAMG) in incubation mixtures of rat feces with Shakuyaku-kanzo-to decoction (combination of licorice root and peony root). The analysis could be accomplished within 20 min with a TSKgel ODS-80TsQA (150 x 2.0 mm i.d.) column by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile at a flow rate of 0.2 ml x min(-1), a thermostatic oven at 25 degrees C, and detection at 254 nm. The detection limits of these compounds were 0.1-0.85 pmol per injection (5 microl). The concentrations of GL and its metabolites in the incubation mixture after continuous consumption of Shakuyaku-kanzo-to were significantly different compared with those of untreated control. GL-hydrolysis of rat feces was enhanced by pre-consumption of Shakuyaku-kanzo-to.

Candida tropicalis Etr1p and Saccharomyces cerevisiae Ybr026p (Mrf1'p), 2-enoyl thioester reductases essential for mitochondrial respiratory competence.

We report here on the identification and characterization of novel 2-enoyl thioester reductases of fatty acid metabolism, Etr1p from Candida tropicalis and its homolog Ybr026p (Mrf1'p) from Saccharomyces cerevisiae. Overexpression of these proteins in S. cerevisiae led to the development of significantly enlarged mitochondria, whereas deletion of the S. cerevisiae YBR026c gene resulted in rudimentary mitochondria with decreased contents of cytochromes and a respiration-deficient phenotype. Immunolocalization and in vivo targeting experiments showed these proteins to be predominantly mitochondrial. Mitochondrial targeting was essential for complementation of the mutant phenotype, since targeting of the reductases to other subcellular locations failed to reestablish respiratory growth. The mutant phenotype was also complemented by a mitochondrially targeted FabI protein from Escherichia coli. FabI represents a nonhomologous 2-enoyl-acyl carrier protein reductase that participates in the last step of the type II fatty acid synthesis. This indicated that 2-enoyl thioester reductase activity was critical for the mitochondrial function. We conclude that Etr1p and Ybr026p are novel 2-enoyl thioester reductases required for respiration and the maintenance of the mitochondrial compartment, putatively acting in mitochondrial synthesis of fatty acids.