Local light stimulation of melanophores of a teleost, Zacco temmincki

Responses of melanophores of the teleost, Zacco temmincki, to local light stimulation were examined in preparations of isolated scales. The melanophores induced the aggregation of melanosomes in darkness and their dispersion in light. Local illumination of a melanophore in the melanosome-dispersed state inhibited centripetal migration of melanosomes only in the stimulated area. Local illumination of a pigment-free branch of a melanophore with aggregated melanosomes generally brought about pigment dispersion into the stimulated area. However, when that area was at a significant distance from the edge of the central melanosome mass, the melanosomes never migrated into the irradiated area. Local illumination of the centrosphere of a cell inhibited the full aggregation of melanosomes in the dispersed and aggregated state. The degree of the inhibition depended on the size of the irradiated area. The results suggest that photoreceptive sites are distributed over the whole of a cell, and that the movements of melanosomes are regulated locally in a very precise manner.     

Analysis of the thromboxane/prostacyclin balance in human urine by gas chromatography/selected ion monitoring: abnormalities in diabetics

We microanalyzed 2,3-dinor-6-keto-prostaglandin F_1α (2,3-dinor-6-keto-PGF_1α 1) and 11-dehydrothromboxane B₂ (11-dehydro-TXB₂, 2) in human urine. Samples containing a [²H₄]-analogue as an internal standard were extracted by chromatography using Sep Pak tC18 and silica gel. The compounds were then analysed by means of the lactone ring opening reaction and dimethylisopropylsilylation. The conversion of 1 to 1-methyl ester (ME)-propylamide (PA)-9,12,15-dimethylisopropylsilyl (DMIPS) ether derivative and of 2 to 1-ME-6-methoxime (MO)-9,12,15-tris-DMIPS ether derivative was followed by gas chromatography/selected ion monitoring (GC/SIM). Interfering substances from the urine matrix were eliminated during GC/SIM analysis using a DB-5 column. We were able to detect 1 (222–1031 pg/mg creatinine) and 2 (18–155 pg/mg creatinine) in human urine. Furthermore, the thromboxane/prostacyclin (IX/PGI) ratio in the urine of diabetics was higher than that of healthy volunteers. This method can be used to determine the TX/PGI balance in human urine.     

Idiotypic diversity and variable region gene usage by mouse anti-HLA-DQ3 mAb

The anti-HLA-DQ3 monoclonal antibodies (mAb) KS13, SO1, SO2, SO3, SO4, and SO5 recognize spatially close but distinct antigenic determinants, since they crossinhibit each other in their binding to HLA-DQ3 antigens, but do not share idiotopes recognized in their antigen combining site by syngeneic and anti-id antisera and mAb. Furthermore, mAb SO1, SO3, SO4, and SO5 react also with HLA-DQ allospecificities other than HLA-DQ3. Sequence analysis of the heavy (V _H ) and light (V _L ) chain variable region of the six mAb revealed preferential usage of V _H 36–60 and V _K 12/13 gene families. However, the individual V _H and V _L germline gene usage by the six mAb is diverse and the utilization of D, J _H , and J _L gene segments is heterogeneous. The diverse usage of V _H and V _L gene segments and heterogeneous amino acid sequences of V _H and V _L CDR, together with the heterogeneous idiotypic profile, may reflect the complexity of the determinants recognized by the six mAb on HLA-DQ3 antigens. The results we have presented provide for the first time information about the structural basis of the diversity of antibodies recognizing human histocompatibility antigens.The nucleotide sequence data reported in this Papershave been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L20499, L20957, L20961, L24557, L24558 and L20962, respectively, for V _H region genes, and L20956, L20958, L24555, L24556, L20959, and L20960, respectively, for V _L region genes     

Difference in superoxide toxicity between 4,7-dicyanobenzofurazan and paraquat.

The O2-. production by aerobically cultured Escherichia coli in the presence of benzofurazan (1), 4,7-dimethylbenzofurazan (2), 4,7-dibromobenzofurazan (3), 4-bromo-6-cyanobenzofurazan (4), and 4,7-dicyanobenzofurazan (5) was examined by using the cytochrome c reduction method in order to elucidate the mechanism of cytotoxicity of benzofurazans. Adding compound 5 to E. coli cell suspension caused cytochrome c reduction, which was completely inhibited by superoxide dismutase. The rate of cytochrome c reduction was in the order of 1 = 2 = 3 less than 4 less than 5, which correlates well with that of the reduction potentials of these benzofurazans. Adding glucose to the E. coli cell suspension-compound 5-cytochrome c system accelerated the rate of cytochrome c reduction. The formation of 4,7-dicyanobenzofurazan anion radical in the cell suspension-compound 5-glucose system in the absence of O2 was followed by ESR spectroscopy. The ESR signal of the anion radical disappeared when O2 was added. Compound 5 was shown to have an approximately 10-fold greater increasing effect on the flux of O2-. by E. coli than paraquat (PQ) by the cytochrome c reduction method. The results were confirmed by the electrochemical method with an oxygen electrode. However, compound 5 had a bacteriostatic, but not lethal, effect, while PQ had both effects. The effect of compound 5 and PQ on lethality of E. coli showed a dramatic difference when E. coli was exposed to these two compounds and washed prior to testing the effects of that exposure. This difference probably arose because compound 5 readily leaked from the cells during dilution and plating. Also, the reduced form of compound 5 exits from the cells more readily than the reduced form of PQ and then generates O2-. in the medium by autoxidation. This suggests the importance of the intracellular production of O2-., rather than the extracellular production of O2-., for lethal effect.     

Callus regeneration from cotyledon protoplasts ofChamaecyparis obtusa (Hinoki cypress)

Summary Protoplasts were isolated from cotyledons of 1- to 1.5-mo.-old seedlings ofChamaecyparis obtusa using 1% driselase or 0.25% pectolyase Y-23 in combination with 1% cellulase RS in 0.6M mannitol solution. Cell division and colony formation were induced efficiently in liquid Murashige and Skoog’s (MS) medium containing 0.6M mannitol and 10 to 30 μM 2,4-dichlorophenoxyacetic acid or 1 μM of naphthaleneacegic acid at the cell density of 1 to 2×10³ ml. Continued callus proliferations was observed by transferring tissue to fresh medium of the same composition as the induction medium without mannitol. Campbell and Durzan’s medium and ammonium nitrate-free MS medium were less effective than MS medium. High concentration of benzyladenine (1 or 10 μM) was inhibitory to cell division.     

Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

The stability of Watson–Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na⁺ or K⁺ in the presence or absence of Mg²⁺). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°₃₇ and T_m values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg²⁺, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR–Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.     

Expression of Arabidopsis thaliana xylose isomerase gene and its effect on ethanol production in Flammulina velutipes

To improve the pentose fermentation rate in Flammulina velutipes, the putative xylose isomerase (XI) gene from Arabidopsis thaliana was cloned and introduced into F. velutipes and the gene expression was evaluated in transformants. mRNA expression of the putative XI gene and XI activity were observed in two transformants, indicating that the putative gene from A. thaliana was successfully expressed in F. velutipes as a xylose isomerase. In addition, ethanol production from xylose was increased in the recombinant strains. This is the first report demonstrating the possibility of using plant genes as candidates for improving the characteristics of F. velutipes.     

DMSO Represses Inflammatory Cytokine Production from Human Blood Cells and Reduces Autoimmune Arthritis

Dimethyl sulfoxide (DMSO) is currently used as an alternative treatment for various inflammatory conditions as well as for cancer. Despite its widespread use, there is a paucity of data regarding its safety and efficacy as well as its mechanism of action in human cells. Herein, we demonstrate that DMSO has ex-vivo anti-inflammatory activity using Escherichia coli- (E. coli) and herpes simplex virus-1 (HSV-1)-stimulated whole human blood. Specifically, we found that between 0.5%– 2%, DMSO significantly suppressed the expression of many pro-inflammatory cytokines/chemokines and prostaglandin E₂ (PGE₂). However, a significant reduction in monocyte viability was also observed at 2% DMSO, suggesting a narrow window of efficacy. Anti-inflammatory concentrations of DMSO suppressed E. coli-induced ERK1/2, p38, JNK and Akt phosphorylation, suggesting DMSO acts on these signaling pathways to suppress inflammatory cytokine/chemokine production. Although DMSO induces the differentiation of B16/F10 melanoma cells in vitro, topical administration of DMSO to mice subcutaneously implanted with B16 melanoma cells was ineffective at reducing tumor growth, DMSO was also found to block mouse macrophages from polarizing to either an M1- or an M2-phenotype, which may contribute to its inability to slow tumor growth. Topical administration of DMSO, however, significantly mitigated K/BxN serum-induced arthritis in mice, and this was associated with reduced levels of pro-inflammatory cytokines in the joints and white blood cell levels in the blood. Thus, while we cannot confirm the efficacy of DMSO as an anti-cancer agent, the use of DMSO in arthritis warrants further investigation to ascertain its therapeutic potential.     

Wasabi leaf extracts attenuate adipocyte hypertrophy through PPARγ and AMPK

Hypertrophy of adipocytes in obese adipose tissues causes metabolic abnormality by adipocytokine dysregulation, which promotes type 2 diabetes mellitus, hypertension, and dyslipidemia. We investigated the effects of wasabi (Wasabia japonica Matsum) leaf extracts on metabolic abnormalities in SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP/ZF), which are a model of metabolic syndrome. Male SHRSP/ZF rats aged 7 weeks were divided into two groups: control and wasabi leaf extract (WLE) groups, which received water or oral treatment with 4 g/kg/day WLE for 6 weeks. WLE improved the body weight gain and high blood pressure in SHRSP/ZF rats, and the plasma triglyceride levels were significantly lower in the WLE group. Adipocyte hypertrophy was markedly prevented in adipose tissue. The expression of PPARγ and subsequent downstream genes was suppressed in the WLE group adipose tissues. Our data suggest that WLE inhibits adipose hypertrophy by suppressing PPARγ expression in adipose tissue and stimulating the AMPK activity by increased adiponectin.