Mechanism of proliferation arrest of embryonic cells of Xenopus by diterpene compounds

Three diterpene compounds isolated from the anti-cancer herbal medicine kansui, namely, kansuinin B, 20-OD-ingenol Z, and 20-OD-ingenol E, specifically inhibited the proliferation of isolated embryonic cells from Xenopus embryos. We conducted a cytologic study to determine the mechanism underlying the arrest of the cellular proliferation by these compounds. While kansuinin B and 20-OD-ingenol Z treatment decreased the cell numbers in the S phase and the M phase substages of the cell cycle, 20-OD-ingenol E inhibited mitosis.     

Inhibition by polyamines of lipid peroxide formation in rat liver microsomes.

Both NADPH- and ascorbic acid-dependent lipid peroxidations were inhibited by spermine, the degree of inhibition being greater with the former peroxidation. The effective concentration of spermine required for inhibition was higher when larger amounts of microsomes were used. However, the activities of NADPH-cytochrome c reductase and NADPH-peroxidase were not influenced by spermine. These results suggest that spermine inhibits lipid peroxidation by binding to phospholipids in the microsomes.     

Development of a biological mercury removal-recovery system

A mercury removal-recovery system was developed for collection of elemental mercury volatilized by biological mercuric ion reduction. Using the mercury removal-recovery system, removal of mercuric chloride from mercury-containing buffer without nutrients by resting cells of mercury-resistant bacterium, Pseudomonas putida PpY101/pSR134 was tested. Optimum temperature, pH, thiol compounds and cell concentration on removal of mercuric chloride were determined, and 92 to 98% of 40 mg Hg l^–1 was recovered in 24 h. The efficiency of mercuric chloride removal from river water and seawater was as high as that observed when using a buffered solution.     

A Conditional Knockout Toolkit for Caenorhabditis elegans Based on the Cre/loxP Recombination

Conditional knockout (cKO) based on site-specific recombination (SSR) technology is a powerful approach for estimating gene functions in a spatially and temporally specific manner in many model animals. In Caenorhabditis elegans (C. elegans), spatial- and temporal-specific gene functions have been largely determined by mosaic analyses, rescue experiments and feeding RNAi methods. To develop a systematic and stable cKO system in C. elegans, we generated Cre recombinase expression vectors that are driven by various tissue-specific or heat-shock promoters. Validation using Cre-mediated fluorescence protein inactivation or activation systems demonstrated successful Cre-dependent loxP excision. We established a collection of multi-copy Cre transgenic strains for each evaluated vector. To evaluate our Cre/loxP-based cKO system, we generated sid-1 deletion mutants harboring floxed sid-1 single-copy integration (SCI) using ultraviolet trimethylpsoralen (UV/TMP) methods. sid-1 mutants that were rescued by the floxed sid-1 SCI were then crossed with the Pdpy-7::Cre strain for cKO in the hypodermis. The sid-1 cKO animals were resistant to bli-3 RNAi, which causes the Bli-phenotyple in the hypodermis, but they were sensitive to unc-22 RNAi, which leads to twitching of the body wall muscle. Our system, which is based on the combination of a transgenic Cre collection, pre-existing deletion mutants, and UV/TMP SCI methods, provided a systematic approach for cKO in C. elegans.     

Respiratory chain of the alkalophilic bacterium Bacillus firmus RAB and its non-alkalophilic mutant derivative

The membrane-bound respiratory chain components of alkalophilic Bacillus firmus RAB were studied by difference spectroscopy and oxidation-reduction potentiometric titrations. Cytochromes with the following midpoint potentials were identified at pH 9.0: a-type cytochromes, +110 and +210 mV; b-type cytochromes, +20, -120, -280, and -400 mV; and cytochrome c, +60 mV. Only the higher-potential cytochrome a showed an upward shift in midpoint potential when titrated at pH 7.0. Parallel studies of a non-alkalophilic mutant derivative of B. firmus RAB, strain RABN, revealed the presence of only one species each of a-, b-, and c-type cytochromes which exhibited midpoint potentials of +110, -150, and +160 mV, respectively, at pH 7.0. Membranes of both strains were found to contain menaquinone. At pH 9.0, NADH caused the reduction of essentially all of the cytochromes that were seen in fully reduced preparations of wild-type B. firmus RAB membranes. By contrast, at pH 7.0, NADH failed to appreciably reduce the b-type cytochromes. These findings may relate to our recent proposal that an inadequacy in energy transduction (production of a proton motive force) by the alkalophilic respiratory chain at pH 7.0 is what precludes the growth of B. firmus RAB at a neutral pH.     

Isolation and Characterization of 2-Nitroimidazole Produced by Streptomyces Species as an Inhibitor of Both Carbonic Anhydrase and Shell Formation in the Barnacle Balanus amphitrite

Carbonic anhydrase is thought to be involved in the process of calcium carbonate deposition in calcified tissues of many organisms. Barnacles form hard calcified shells for protection against predation, and represent a class of marine-fouling animals. In order to inhibit barnacle growth by inhibiting shell formation, we searched for carbonic anhydrase inhibitors from microbial secondary metabolites. A simple assay for assessing carbonic-anhydrase-inhibiting activity was developed. Screening of many microorganisms isolated from soil with this assay resulted in a microbial strain that produced a carbonic anhydrase inhibitor. This strain was identified as Streptomyces eurocidicus mf294. The inhibitor was isolated through 4 purification steps and identified as 2-nitroimidazole on the basis of spectroscopic data. 2-Nitroimidazole inhibited barnacle carbonic anhydrase dose-dependently and complete inhibition was reached at the concentration of 1 x 10(-5) M. 2-Nitroimidazole did not affect settlement or metamorphosis of barnacle larvae, but inhibited shell formation at concentrations higher than 1 x 10(-4) M. These findings strongly support the idea that carbonic anhydrase is involved in calcification.     

Molecular evidence of sorbitol dehydrogenase in tomato, a non-Rosaceae plant

The enzyme NAD-dependent sorbitol dehydrogenase (SDH) is well characterized in the Rosaceae family of fruit trees, which synthesizes sorbitol as a translocatable photosynthate. Expressed sequence tags of SDH-like sequences have also been generated from various non-Rosaceae species that do not synthesize sorbitol as a primary photosynthetic product, but the physiological roles of the encoded proteins in non-Rosaceae plants are unknown. Therefore, we isolated an SDH-like cDNA (SDL) from tomato (Lycopersicon esculentum Mill.). Genomic Southern blot analysis suggested that SDL exists in the tomato genome as a single-copy gene. Northern blot analysis showed that SDL is ubiquitously expressed in tomato plants. Recombinant SDL protein was produced and purified for enzymatic characterization. SDL catalyzed the interconversion of sorbitol and fructose with NAD (H). SDL showed highest activity for sorbitol among the several substrates tested. SDL showed no activity with NADP+. Thus, SDL was identified as a SDH, although the Km values and substrate specificity of SDL were significantly different from those of SDH purified from the Japanese pear (Pyrus pyrifolia), a Rosaceae fruit tree. In addition, tomato was transformed with antisense SDL to evaluate the contribution of SDL to SDH activity in tomato. The transformation decreased SDH activity to approximately 50% on average. Taken together, these results provide molecular evidence of SDH in tomato, and SDL was renamed LeSDH.     

PINK1 controls mitochondrial localization of Parkin through direct phosphorylation

PTEN-induced putative kinase 1 (PINK1) and Parkin, encoded by their respective genes associated with Parkinson’s disease (PD), are linked in a common pathway involved in the protection of mitochondrial integrity and function. However, the mechanism of their interaction at the biochemical level has not been investigated yet. Using both mammalian and Drosophila systems, we here demonstrate that the PINK1 kinase activity is required for its function in mitochondria. PINK1 regulates the localization of Parkin to the mitochondria in its kinase activity-dependent manner. In detail, Parkin phosphorylation by PINK1 on its linker region promotes its mitochondrial translocation, and the RING1 domain of Parkin is critical for this occurrence. These results demonstrate the biochemical relationship between PINK1, Parkin, and the mitochondria and thereby suggest the possible mechanism of PINK-Parkin-associated PD pathogenesis.     

Purification and properties of cytochrome P-450 from homogenates of human fetal livers

A form of cytochrome P-450, namely P-450HFLa of human fetal livers, was purified to a specific content of 12.6 nmol/mg protein. The cytochrome P-450 preparation was electrophoretically homogeneous and had an apparent monomeric molecular weight of 51,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cytochrome showed catalytic activities as oxidations of N-methylaniline, ethylmorphine, N,N-dimethylaniline, N,N-dimethylnitrosamine, benzphetamine, aminopyrine, aniline, p-nitroanisole, and 7-ethoxycoumarin to various extents. In fetal liver homogenate, the amount of cytochrome P-450 that reacted with the antiserum to P-450HFLa accounted for more than 36% of the total cytochrome P-450 in three different fetal livers. On the other hand, the amount of P-450HFLa was less than 5% of the total cytochrome P-450 in adult liver microsomes.     

Changes in Inner and Outer Retinal Layer Thicknesses after Vitrectomy for Idiopathic Macular Hole: Implications for Visual Prognosis

Purpose

To investigate sequential post-operative thickness changes in inner and outer retinal layers in eyes with an idiopathic macular hole (MH).

Methods

Retrospective case series. Twenty-four eyes of 23 patients who had received pars plana vitrectomy (PPV) for the closure of MH were included in the study. Spectral domain optical coherence tomography C-scan was used to automatically measure the mean thickness of the inner and outer retinal layers pre-operatively and up to 6 months following surgery. The photoreceptor outer segment (PROS) length was measured manually and was used to assess its relationship with best-corrected visual acuity (BCVA).

Results

Compared with the pre-operative thickness, the inner layers significantly thinned during follow-up (P = 0.02), particularly in the parafoveal (P = 0.01), but not perifoveal, area. The post-operative inner layer thinning ranged from the ganglion cell layer to the inner plexiform layer (P = 0.002), whereas the nerve fiber layer was unaltered. Outer layer thickness was significantly greater post-operatively (P = 0.002), and especially the PROS lengthened not only in the fovea but also in the parafovea (P < 0.001). Six months after surgery, BCVA was significantly correlated exclusively with the elongated foveal PROS (R = 0.42, P = 0.03), but not with any of the other thickness parameters examined.

Conclusions

Following PPV for MH, retinal inner layers other than the nerve fiber layer thinned, suggestive of subclinical thickening in the inner layers where no cyst was evident pre-operatively. In contrast, retinal outer layer thickness significantly increased, potentially as a result of PROS elongation linking tightly with favorable visual prognosis in MH eyes.