Thyroglobulin may affect telomerase activity in thyroid follicular cells

Telomerase (TA) activity is known to be present in malignant tumor cells, but not in most somatic differentiated cells. TA shows relatively high activity in thyroid cancer cells, but reports vary. This fact prompted us to elucidate whether cell component inhibitors of TA in the thyroid follicles can modulate its activity. The activity of TA extracted from Hela cells was inhibited by mixing with the supernatant fraction of human thyroid tissue extract. To examine the effect of iodine, thyroid hormones (l-T3 and l-T4) and human thyroglobulin (hTg) contained in the thyroid follicles, l-T3, l-T4 and hTg were added to the TRAP assay system in vitro, using TA from Hela cells. Iodine, l-T3 and l-T4 did not affect TA activity, but hTg inhibited the TA activity in a dose-dependent manner (IC(50) of hTg: ca 0.45 microM: inhibiting concentration of hTg was from 0.15 microM to 3.0 microM). The hTg inhibition was not evident in the RT-PCR system, suggesting no effect of hTg on Taq DNA polymerase activity. The hTg inhibition of TA activity was attenuated by dNTP but not significantly by TS primer. These data suggest that hTg contained in thyroid follicular cells of various thyroid diseases may affect the TA activity measured in biopsied thyroid specimens, and that the reduction of the TA activity by hTg may induce slow progression and growth, and low grade malignancy of thyroid cancer, particularly differentiated carcinoma.     

Liquid chromatographic determination of acetylcholine based on pre‐column alkaline cleavage reaction and post‐column tris(2,2′‐bipyridyl)ruthenium(III) chemiluminescence detection

A method for the determination of acetylcholine (ACh) has been developed using liquid chromatography with chemiluminescence detection. This method is based on the pre‐column alkaline cleavage of ACh to form trimethylamine (TMA) and the post‐column tris(2,2′‐bipyridyl)ruthenium(III) chemiluminescence detection of TMA. ACh was converted to TMA with high yield at 180°C in the presence of lithium hydroxide, and the produced TMA was separated on a cation‐exchange/reversed‐phase dual‐functional column using a mixture of 0.2 m potassium phosphate buffer (pH 5.9) and acetonitrile (20:1, v/v) as the mobile phase. The eluate was online mixed with acidic tris(2,2′‐bipyridyl)ruthenium(III) solution, and the generated chemiluminescence was detected. The detection limit (signal‐to‐noise ratio = 3) for ACh was 0.80 nmol/mL, which corresponded to 1.1 pmol TMA per injection volume of 5 µL. This is simple and robust method that does not need an expensive device and unstable enzymes, and was applied to the determination of ACh in pharmaceutical formulations. Copyright © 2009 John Wiley & Sons, Ltd.     

Isolation and molecular characterization of multidrug-resistant Gram-negative bacteria from imported flamingos in Japan

Imported animals, especially those from developing countries, may constitute a potential hazard to native animals and to public health. In this study, a new flock of lesser flamingos imported from Tanzania to Hiroshima Zoological Park were screened for multidrug-resistant Gram-negative bacteria, integrons and antimicrobial resistance genes. Thirty-seven Gram-negative bacterial isolates were obtained from the flamingos. Seven isolates (18.9%) showed multidrug resistance phenotypes, the most common being against: ampicillin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and nalidixic acid. Molecular analyses identified class 1 and class 2 integrons, β-lactamase-encoding genes, bla _TEM-1 and bla _CTX-M-2 and the plasmid-mediated quinolone resistance genes, qnrS and qnrB. This study highlights the role of animal importation in the dissemination of multidrug-resistant bacteria, integrons and antimicrobial resistance genes from one country to another.     

Cryopreservation and low-temperature storage of seeds of Phaius tankervilleae

In this study we established reliable methods for conservation of seeds of Phaius tankervilleae as an orchid genetic resource. The seeds, which were dehydrated to 5% water content and preserved at 4°C, showed no decrease in viability and germinability after three months. After storage for six months, however, the seeds showed a drastic decrease in germinability, even though survival rate was high. For long-term preservation of seeds of P. tankervilleae, cryopreservation is applied to the freshly harvested seeds. When the seeds were cryopreserved by the vitrification method for up to 12 months there was no apparent deterioration effect of storage time. These results indicate that cryopreservation by the vitrification method is useful for long-term conservation of P. tankervilleae seeds, which are difficult to preserve for more than three months under dry and low-temperature conditions.     

Structure and biosensor characteristics of complex between glucose oxidase and plasma-polymerized nanothin film

The structure and biosensor characteristics of complex between glucose oxidase (GOD) and plasma-polymerized nanothin film (PPF), in which the thickness is several nanometers, were investigated by atomic force microscopy (AFM) and electrochemical measurement. The GOD molecules were densely adsorbed onto the PPF surface treated by nitrogen plasma and the individual GOD molecules were observed. Subsequently, the GOD densely packed array on the PPF surface was subsequently treated by plasma polymerization (overcoating). AFM image showed that the thicker film gave the smoother surface, indicating that the GOD adsorbed on the surface was embedded more deeply in PPF. The amperometric biosensor characteristics of the GOD-PPF complex on a platinum electrode showed the current increment due to the enzymatic reaction with glucose addition, indicating that enzyme activity was retained although the enzyme has been exposed to the plasma gas related to diffusion of the substrate. This means that under mild exposure to organic plasma, the enzyme does not become seriously dysfunctional. Amperometric biosensor characteristics were strongly affected by monomer and thickness of PPF overcoating related with the diffusion of the substrate (glucose). Considering that the film deposition was performed using microfabrication-compatible organic plasma, our new method for protein architecture has a great potential of enabling high throughput production of bioelectronic devices.     

ATR homolog Mec1 controls association of DNA polymerase zeta-Rev1 complex with regions near a double-strand break

DNA polymerase zeta (Polzeta) and Rev1 contribute to the bypassing of DNA lesions, termed translesion DNA synthesis (TLS). Polzeta consists of two subunits, one encoded by REV3 (the catalytic subunit) and the other encoded by REV7. Rev1 acts as a deoxycytidyl transferase, inserting dCMP opposite lesions. Polzeta and Rev1 have been shown to operate in the same TLS pathway in the budding yeast Saccharomyces cerevisiae. Here, we show that budding yeast Polzeta and Rev1 form a complex and associate together with double-strand breaks (DSBs). As a component of the Polzeta-Rev1 complex, Rev1 plays a noncatalytic role in the association with DSBs. In budding yeast, the ATR-homolog Mec1 plays a central role in the DNA-damage checkpoint response. We further show that Mec1-dependent phosphorylation promotes the Polzeta-Rev1 association with DSBs. Rev1 association with DSBs requires neither the function of the Rad24 checkpoint-clamp loader nor the Rad6-Rad18-mediated ubiquitination of PCNA. Our results reveal a novel role of Mec1 in the localization of the Polzeta-Rev1 complex to DNA lesions and highlight a linkage of TLS polymerases to the checkpoint response.     

Self-aggregation of synthetic zinc chlorophyll derivative possessing a perfluoroalkyl group in a fluorinated solvent

Zinc 3(1)-hydroxy-13(1)-oxo-chlorin possessing a perfluoroheptyl group on the 17-propionate was prepared by modifying chlorophyll a. The synthetic compound self-aggregated in 0.1% (v/v) THF and HCFC225cb (CClF2CF2CHClF) to give similar large oligomers to natural light-harvesting antennae of green photosynthetic bacteria and their models. Visible absorption, circular dichroism, and fluorescence emission spectra showed that F-F interaction between the perfluoroheptyl side chain and the fluorinated solvent (HCFC225cb) stabilized the supramolecular structure. The core part of the supramolecular structure was constructed by a special bond of Zn...O3(1)-H...O=C13(1) and pi-pi stacking, and the 17-propionates were at the peripheral part.     

Characterization of acatalasemic erythrocytes treated with low and high dose hydrogen peroxide. Hemolysis and aggregation

The effects of hydrogen peroxide on normal and acatalasemic erythrocytes were examined. Severe hemolysis of acatalasemic erythrocytes and a small tyrosine radical signal (g = 2.005) associated with the formation of ferryl hemoglobin were observed upon the addition of less than 0.25 mM hydrogen peroxide. However, when the concentration of hydrogen peroxide was increased to 0.5 mM, acatalasemic erythrocytes became insoluble in water and increased the tyrosine radical signal. Polymerization of hemoglobin and aggregation of the erythrocytes were observed. On the other hand, normal erythrocytes exhibited only mild hemolysis by the addition of hydrogen peroxide under similar conditions. From these results, the scavenging of hydrogen peroxide by hemoglobin generates the ferryl hemoglobin species (H-Hb-Fe(IV)=O) plus protein-based radicals (*Hb-Fe(IV)=O). These species induce hemolysis of erythrocytes, polymerization of hemoglobin, and aggregation of the acatalasemic erythrocytes. A mechanism for the onset of Takarara disease is proposed.     

Identification of neurite outgrowth-promoting domains of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, and involvement of phosphatidylinositol 3-kinase and protein kinase C signaling pathways in neuritogenesis

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that is exclusively expressed in the central nervous system. We report that the recombinant ectodomain of NGC core protein enhances neurite outgrowth from rat neocortical neurons in culture. Both protein kinase C (PKC) inhibitors and phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated the NGC-mediated neurite outgrowth in a dose-dependent manner, suggesting that NGC promotes neurite outgrowth via PI3K and PKC pathways. The active sites of NGC for neurite outgrowth existed in the epidermal growth factor (EGF)-like domain and acidic amino acid (AA)-domain of the NGC ectodomain. The EGF-domain caused cells to extend preferentially one neurite from a soma, whereas the AA-domain caused several neurites to develop. The EGF-domain also enhanced neurite outgrowth from GABA-positive neurons, but the AA-domain did not. These results suggest that the EGF-domain and AA-domain have distinct functions in terms of neuritogenesis. From these findings, NGC can be considered to be involved in neuritogenesis in the developing central nervous system.