Novel urushiols with human immunodeficiency virus type 1 reverse transcriptase inhibitory activity from the leaves of Rhus verniciflua.

Two novel urushiols, 1 and 2, and two known urushiols, 3 and 4, were isolated from the leaves of Rhus verniciflua and were examined for their human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitory activity. The novel urushiols were found to be 1,2-dihydroxyphenyl-3-[7'(E),9'(Z),11'(Z)-pentadecatrienyl]-14'-ol (1) and 1,2-dihydroxyphenyl-3-[8'(Z),10'(E),12'(E)-pentadecatrienyl]-14'-ol (2) by spectroscopic analyses. The absolute configuration at C-14' in 1 and 2 was determined to be a racemic mixture of (R) and (S) isomers by ozonolysis. Compound 2 (IC50: 12.6 µM) showed the highest HIV-1 RT inhibitory activity among the four urushiols, being 2.5-fold more potent than the positive control, adriamycin (IC50: 31.9 µM). Although the known urushiols were isolated from the sap and leaves of R. verniciflua, 1 was exclusively present in the leaves, and higher amounts of 2 were found in the leaves than in the sap. Present findings indicate that the leaves of R. verniciflua represent a new biological resource from which novel and known urushiols may be prepared, and the possible use of novel urushiols as bioactive products.

fMRI evidence of improved visual function in patients with progressive retinitis pigmentosa by eye-movement training.

To evaluate changes in the visual processing of patients with progressive retinitis pigmentosa (RP) who acquired improved reading capability by eye-movement training (EMT), we performed functional magnetic resonance imaging (fMRI) before and after EMT. Six patients with bilateral concentric contraction caused by pigmentary degeneration of the retina and 6 normal volunteers were recruited. Patients were given EMT for 5 min every day for 8-10 months. fMRI data were acquired on a 3.0-Tesla scanner while subjects were performing reading tasks. In separate experiments (before fMRI scanning), visual performances for readings were measured by the number of letters read correctly in 5 min. Before EMT, activation areas of the primary visual cortex of patients were 48.8% of those of the controls. The number of letters read correctly in 5 min was 36.6% of those by the normal volunteers. After EMT, the activation areas of patients were not changed or slightly decreased; however, reading performance increased in 5 of 6 patients, which was 46.6% of that of the normal volunteers (p< 0.05). After EMT, increased activity was observed in the frontal eye fields (FEFs) of all patients; however, increases in the activity of the parietal eye fields (PEFs) were observed only in patients who showed greater improvement in reading capability. The improvement in reading ability of the patients after EMT is regarded as an effect of the increased activity of FEF and PEF, which play important roles in attention and working memory as well as the regulation of eye movements.

Ariakemicins A and B, novel polyketide-peptide antibiotics from a marine gliding bacterium of the genus Rapidithrix.

Ariakemicins A (1) and B (2), unusual linear hybrid polyketide-nonribosomal peptide antibiotics, were discovered from the fermentation extract of the marine gliding bacterium Rapidithrix sp. These metabolites were positional isomers with regard to a double bond and chromatographically inseparable, rendering the structure study on a mixture basis. The ariakemicins were composed of threonine, two omega-amino-(omega-3)-methyl carboxylic acids with diene or triene units, and delta-isovanilloylbutyric acid. The antibiotics selectively inhibited the growth of Gram-positive bacteria.

Noncontact backscatter-mode near-infrared time-resolved imaging system: Preliminary study for functional brain mapping.

To improve the spatial resolution and to obtain the depth information of absorbers buried in highly scattering material, we developed a noncontact backscatter-mode near-infrared time-resolved imaging system (noncontact B-TRIS) that is intended for functional human brain mapping. It consists of mode-locked Ti-sapphire lasers as light sources and a charge-coupled device camera equipped with a time-resolved intensifier as a detector. The system was tested with a white polyacetal phantom as a light-scattering medium and black polyacetal particles as absorbers. Illumination and detection of light through an objective lens system (phi = 150 mm) enabled us to capture images from an area whose diameter is about 70 mm without coming into contact with it. The scattering and absorption coefficients of the white phantom obtained by B-TRIS were similar to those obtained by a conventional time-resolved spectroscopy. Although the imaged diameter of an absorber buried within a phantom was considerably larger than the actual diameter, the center position of the absorber coincided with the actual position with accuracy <2 mm. Furthermore, the depth information can be also detected by the noncontact B-TRIS. These results suggest a potential of noncontact B-TRIS for imaging cognitive human brain function.

Vacuolar H(+)-ATPase and plasma membrane H(+)-ATPase contribute to the tolerance against high-pressure carbon dioxide treatment in Saccharomyces cerevisiae.

As a non-thermal sterilization process, high-pressure carbon dioxide treatment (HPCT) is considered to be promising. The main sterilizing effect of HPCT is thought to be acidification in cytoplasm of microorganisms. We investigated the tolerance mechanism of Saccharomyces cerevisiae to HPCT with special reference to vacuolar and plasma membrane H(+)-ATPases. HPCT was imposed at 35 degrees C, 4 to 10 MPa, for 10 min. slp1 mutant defective in vacuole morphogenesis was more sensitive to HPCT than its isogenic parent. Concanamycin A, a specific inhibitor of vacuolar H(+)-ATPase (V-ATPase), at 10 microM rendered the parent more HPCT-sensitive to the level of slp1. To confirm further the contribution of V-ATPase to the tolerance against HPCT in S. cerevisiae, we compared vma1 mutant of V-ATPase with its isogenic parent for their HPCT sensitivity. vma1 mutant was more sensitive to HPCT than its parent. Addition of 10 microM vanadate, an inhibitor of plasma membrane H(+)-ATPase (P-ATPase), to the wild type strains also increased the inactivation ratio. These results clearly show that V- and P-ATPases contribute to the tolerance against HPCT in S. cerevisiae by accumulating excess H(+) from cytoplasm to vacuole and excluding H(+) outside of the cell, respectively.

Aglycon specificity profiling of alpha-glucosidases using synthetic probes.

We designed and synthesized hydrogen bond based probes 1-8 with the exception of known glycosidase inhibition mechanisms, and aglycon specificity of 11 different sources of alpha-glucosidases were investigated using their probes. Probe 4 (2,6-anhydro-1-deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-D-glycero-D-ido-heptitol) showed a potent inhibition of S. cerevisiae alpha-glucosidase among all alpha-glucosidases. Probe 4 was found to be a competitive inhibitor for S. cerevisiae alpha-glucosidase with Ki 0.13 mM.

Circulatory basis of fMRI signals: relationship between changes in the hemodynamic parameters and BOLD signal intensity.

Blood oxygenation level-dependent functional magnetic resonance imaging (BOLD-fMRI) is widely used as a tool for functional brain mapping. During brain activation, increases in the regional blood flow lead to an increase in blood oxygenation and a decrease in paramagnetic deoxygenated hemoglobin (deoxy-Hb), causing an increase in the MR signal intensity at the site of brain activation. However, not a few studies using fMRI have failed to detect activation of areas that ought to have been activated. We assigned BOLD-positive (an increase in the signal intensity), BOLD-negative (a decrease in the signal intensity), and BOLD-silent (no change) brain activation to respective circulatory conditions through a description of fMRI signals as a function of the concentration of oxygenated Hb (oxy-Hb) and deoxy-Hb obtained with near-infrared optical imaging (NIOI). Using this model, we explain the sensory motor paradox in terms of BOLD-positive, BOLD-negative, and BOLD-silent brain activation.

Absorber's effect projected directly above improves spatial resolution in near infrared backscattered imaging.

The spatial resolution of near infrared spectroscopic imaging in brain function mapping studies needs to be improved. Most near infrared spectroscopic imaging systems use optical fibers that are arranged like a lattice. The light source and the detector have one-on-one correspondence at intervals of about 3 cm. In this study, we apply several detectors against one source to improve spatial resolution. We assume that a local absorber within a strong scattering medium is a model for local cerebral activation. When we calculate absorbance, which is used to spectroscopically calculate hemoglobin concentration, the peak position shifts away from above the position where an absorber is located. As far as absorbance is calculated, we cannot obtain spatial information about the absorber even if many detectors are used against one source. When we calculate the difference between detected light intensities, however, we demonstrate that an absorber projected onto the measuring surface has an influence directly above it. We predict this property from a light diffusion equation and also prove it experimentally through measurements of a uniform resinous phantom with an absorber. This is one of the basic principles supporting the achievement of higher spatial resolutions with near infrared spectroscopic imaging.

Mepanipyrim, a novel inhibitor of pharmacologically induced Golgi dispersion.

Mepanipyrim inhibited retrograde Golgi-to-ER trafficking induced by brefeldin A (BFA), nordihydroguaiaretic acid, clofibrate, and arachidonyltrifluoromethyl ketone in NRK and other types of cells, but did not inhibit anterograde trafficking of Golgi-resident proteins translocated to ER by BFA and newly synthesized VSV-G. However, mepanipyrim did not block the TGN38 dispersion induced by any of these compounds. Mepanipyrim acted on the Golgi, and swollen vesicular Golgi structures were formed and similar structures accumulated during rebuilding of the Golgi after BFA removal. These actions of mepanipyrim were readily reversed after its removal. Mepanipyrim did not stabilize microtubules, but prevented nocodazole-induced fragmentation and dispersion of the Golgi. These results suggest that the mepanipyrim-sensitive molecules participated in stabilizing the Golgi and its anchoring in the perinuclear region, and equally importantly, that the novel action of mepanipyrim may be used as a pharmacological tool for investigating membrane transport, Golgi membrane dynamics, and differentiation of the Golgi from TGN.

Nordihydroguaiaretic acid, of a new family of microtubule-stabilizing agents, shows effects differentiated from paclitaxel.

Nordihydroguaiaretic acid (NDGA) protected microtubules in NRK cells from depolymerization caused by structurally and functionally diverse drugs such as nocodazole, colchicine, vinblastine, and ilimaquinone. Hitherto reported drugs, although structurally unrelated to paclitaxel, stabilize microtubules in a way similar to that of paclitaxel and compete for paclitaxel binding to tubulin. However, NDGA had activity toward microtubules different from the effects of paclitaxel. In NRK cells, paclitaxel caused microtubule bundle formation in the presence and absence of microtubule-depolymerizing drugs. However, microtubule bundle did not form, and microtubules radiated from the microtubule-organizing center, in cells treated with NDGA. Acceleration of tubulin polymerization in vitro by paclitaxel was strong but that by NDGA was weak. Microtubules polymerized in vitro in the presence of paclitaxel, but not those polymerized in the presence of NDGA, resisted the effects of cold. NDGA seemed to bind to tubulin, but did not compete for [3H]paclitaxel binding to tubulin. These observations indicate that NDGA belongs to a novel family of microtubule-stabilizing drugs.

A novel action of terpendole E on the motor activity of mitotic Kinesin Eg5.

To reveal the mechanism of mitosis, the development of M phase-specific inhibitors is an important strategy. We have been screening microbial products to find specific M phase inhibitors that do not directly target tubulins, and rediscovered terpendole E (TerE) as a novel Eg5 inhibitor. TerE did not affect microtubule integrity in interphase, but induced formation of a monoastral spindle in M phase. TerE inhibited both motor and microtubule-stimulated ATPase activities of human Eg5, but did not affect conventional kinesin from either Drosophila or bovine brain. Although terpendoles have been reported as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT), the Eg5 inhibitory activity of TerE was independent of ACAT inhibition. Taken together, we demonstrate that TerE is a novel Eg5 inhibitor isolated from a fungal strain.

Glycon specificity profiling of alpha-glucosidases using monodeoxy and mono-O-methyl derivatives of p-nitrophenyl alpha-D-glucopyranoside.

Hydrolysis of probe substrates, eight possible monodeoxy and mono-O-methyl analogs of p-nitrophenyl alpha-D-glucopyranoside (pNP alpha-D-Glc), modified at the C-2, C-3, C-4, and C-6 positions, was studied as part of investigations into the glycon specificities of seven alpha-glucosidases (EC 3.2.1.20) isolated from Saccharomyces cerevisiae, Bacillus stearothermophilus, honeybee (two enzymes), sugar beet, flint corn, and Aspergillus niger. The glucosidases from sugar beet, flint corn, and A. niger were found to hydrolyze the 2-deoxy analogs with substantially higher activities than against pNP alpha-D-Glc. Moreover, the flint corn and A. niger enzymes showed hydrolyzing activities, although low, for the 3-deoxy analog. The other four alpha-glucosidases did not exhibit any activities for either the 2- or the 3-deoxy analogs. None of the seven enzymes exhibited any activities toward the 4-deoxy, 6-deoxy, or any of the methoxy analogs. The hydrolysis results, with the deoxy substrate analogs, demonstrated that alpha-glucosidases having remarkably different glycon specificities exist in nature. Further insight into the hydrolysis of deoxyglycosides was obtained by determining the kinetic parameters (k(cat) and K(m)) for the reactions of sugar beet, flint corn, and A. niger enzymes.

Possible implication of Golgi-nucleating function for the centrosome.

The Golgi apparatus breaks down at mitosis, resulting in the dispersal of Golgi-resident proteins. In NRK cells, however, subsets of both TGN38 and golgin-97, but not ManII and GM130, remained associated with the centrosome throughout the cell cycle. This centrosome association of TGN38 and golgin-97 was not disrupted by treatment with brefeldin A, additional inducers of retrograde trafficking and inhibitors of either kinases or protein phosphatases. Anchoring of the Golgi apparatus within the juxtanuclear region depends on microtubules; the association of TGN38 and golgin-97 subsets with the centrosome, however, was insensitive to nocodazole treatment. Drugs such as PDMP, which block Golgi dispersal both by nocodazole, despite microtubule depolymerization, and by inducers of retrograde trafficking, strengthened the microtubule-nucleating activity of the centrosome. These observations cumulatively suggest the centrosome is implicated in nucleation of the Golgi apparatus through interactions with Golgi-resident proteins, such as TGN38 and golgin-97.