Single-Molecule Observation of the Ligand-Induced Population Shift of Rhodopsin,A G-Protein-Coupled Receptor

Rhodopsin is a G-protein-coupled receptor, in which retinal chromophore acts as inverse-agonist or agonist depending on its configuration and protonation state. Photostimulation of rhodopsin results in a pH-dependent equilibrium between the active state (Meta-II) and its inactive precursor (Meta-I). Here, we monitored conformational changes of rhodopsin using a fluorescent probe Alexa594 at the cytoplasmic surface, which shows fluorescence increase upon the generation of active state, by single-molecule measurements. The fluorescence intensity of a single photoactivated rhodopsin molecule alternated between two states. Interestingly, such a fluorescence alternation was also observed for ligand-free rhodopsin (opsin), but not for dark-state rhodopsin. In addition, the pH-dependences of Meta-I/Meta-II equilibrium estimated by fluorescence measurements deviated notably from estimates based on absorption spectra, indicating that both Meta-I and Meta-II are mixtures of two conformers. Our observations indicate that rhodopsin molecules intrinsically adopt both active and inactive conformations, and the ligand retinal shifts the conformational equilibrium. These findings provide dynamical insights into the activation mechanisms of G-protein-coupled receptors.     

Single-Molecule Observation of the Ligand-Induced Population Shift of Rhodopsin,A G-Protein-Coupled Receptor

Rhodopsin is a G-protein-coupled receptor, in which retinal chromophore acts as inverse-agonist or agonist depending on its configuration and protonation state. Photostimulation of rhodopsin results in a pH-dependent equilibrium between the active state (Meta-II) and its inactive precursor (Meta-I). Here, we monitored conformational changes of rhodopsin using a fluorescent probe Alexa594 at the cytoplasmic surface, which shows fluorescence increase upon the generation of active state, by single-molecule measurements. The fluorescence intensity of a single photoactivated rhodopsin molecule alternated between two states. Interestingly, such a fluorescence alternation was also observed for ligand-free rhodopsin (opsin), but not for dark-state rhodopsin. In addition, the pH-dependences of Meta-I/Meta-II equilibrium estimated by fluorescence measurements deviated notably from estimates based on absorption spectra, indicating that both Meta-I and Meta-II are mixtures of two conformers. Our observations indicate that rhodopsin molecules intrinsically adopt both active and inactive conformations, and the ligand retinal shifts the conformational equilibrium. These findings provide dynamical insights into the activation mechanisms of G-protein-coupled receptors.     

Raman and Autofluorescence Spectrum Dynamics along the HRG-Induced Differentiation Pathway of MCF-7 Cells

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space.     

Differences in the heat stress associated with white sportswear and being semi-nude in exercising humans under conditions of radiant heat and wind at a wet bulb globe temperature of greater than 28 °C

This study investigated whether wearing common white sportswear can reduce heat stress more than being semi-nude during exercise of different intensities performed under radiant heat and wind conditions, such as a hot summer day. After a 20-min rest period, eight male subjects performed three 20 min sessions of cycling exercise at a load intensity of 20 % or 50 % of their peak oxygen uptake (VO_2peak) in a room maintained at a wet bulb globe temperature (WBGT) of 28.7?±?0.1 °C using two spot lights and a fan (0.8 m/s airflow). Subjects wore common white sportswear (WS) consisting of a long-sleeved shirt (45 % cotton and 55 % polyester) and short pants (100 % polyester), or only swimming pants (SP) under the semi-nude condition. The mean skin temperature \( \left(\overline{T} sk\right) \) was greater when subjects wore SP than WS under both the 20 % and 50 % exercise conditions. During the 50 % exercise, the rating of perceived exertion (RPE) and thermal sensation (TS), and the increases in esophageal temperature (ΔTes) and heart rate were significantly higher (P?<?0.001–0.05), or tended to be higher (P?<?0.07), in the WS than SP trials at the end of the third 20-min exercise session. The total sweat loss (m _sw,tot) was also significantly higher in the WS than in the SP trials (P?<?0.05). However, during the 20 % exercise, the m _sw,tot during exercise, and the ΔTes, RPE and TS at the end of the second and third sessions of exercise did not differ significant between conditions. The heat storage (S), calculated from the changes in the mean body temperature (0.9Tes + 0.1 \( \overline{T} sk \) ), was significantly lower in the WS trials than in the SP trials during the 20 min resting period before exercise session. However, S was similar between conditions during the 20 % exercise, but was greater in the WS than in the SP trials during 50 % exercise. These results suggest that, under conditions of radiant heat and wind at a WBGT greater than 28 °C, the heat stress associated with wearing common WS is similar to that of being semi-nude during light exercise, but was greater during moderate exercise, and the storage of body heat can be reduced by wearing WS during rest periods.     

Apis cerana japonica discriminates between floral color phases of the oriental orchid, Cymbidium floribundum

Foragers of the Japanese honeybee (Apis cerana japonica) were attracted by flowers of an oriental orchid (Cymbidium floribundum) and were observed to carry the pollinia on their scutella. After the removal of pollinia from the flowers, their labial color changed from white to reddish brown. Both artificial removal of pollinia and ethrel treatment of the flowers also induced this labial color change. Labia in color-changed flowers showed a decreased reflectance of wavelengths less than 670 nm compared to control intact flower. Both reflectance irradiance spectra and ultraviolet photographs showed that only the nectar guide in white (unchanged) flowers reflected ultraviolet light, and that this reflectance decreased with labial color change. Dual choice experiments showed that the honeybee foragers preferentially visited flowers having white labia rather than reddish brown. We suggest that Japanese honeybees discriminate between the floral phases of C. floribundum using color vision.     

Isolation of a new heterolobosean amoeba from a rice field soil: Vrihiamoeba italica gen. nov., sp. nov.

A heterolobosean amoeba strain 6_5F was isolated from an Italian rice field soil. Although 18S rRNA gene sequence analysis demonstrated that the new isolate was closely related to Stachyamoeba sp. ATCC 50324, further molecular analysis and morphological observation showed distinct differences amongst the two. The 5.8S rRNA gene was successfully amplified and sequenced for strain 6_5F but not for strain ATCC 50324. Trophozoites of strain ATCC 50324 transform into flagellate forms in the late stage of incubation before encystment, while strain 6_5F do not show flagellate forms under different conditions of the flagellation test. Light and electron microscopic observation showed the structural difference of cysts of strain 6_5F from strain ATCC 50324 and also from the type strain Stachyamoeba lipophora. The results show that the strain 6_5F is distinct from Stachyamoeba spp. and we propose a new genus and species for this isolate, Vrihiamoeba italica gen. nov., sp. nov.     

Determining important regulatory relations of amino acids from dynamic network analysis of plasma amino acids

The changes in the concentrations of plasma amino acids do not always follow the flow-based metabolic pathway network. We have previously shown that there is a control-based network structure among plasma amino acids besides the metabolic pathway map. Based on this network structure, in this study, we performed dynamic analysis using time-course data of the plasma samples of rats fed single essential amino acid deficient diet. Using S-system model (conceptual mathematical model represented by power-law formalism), we inferred the dynamic network structure which reproduces the actual time-courses within the error allowance of 13.17%. By performing sensitivity analysis, three of the most dominant relations in this network were selected; the control paths from leucine to valine, from methionine to threonine, and from leucine to isoleucine. This result is in good agreement with the biological knowledge regarding branched-chain amino acids, and suggests the biological importance of the effect from methionine to threonine.     

Synthesis,in vitro and in vivo studies of Gd-DTPA-XDA-D1-Glc(OH) complex as a new potential MRI contrast agent

A new type of dendritic molecules Gd-DTPA-XDA-D1-Glc(OH), which work as a functionalized ligand coordinating gadolinium(III) ion at the center of their frameworks with two glucose moieties on the molecular surfaces, were readily synthesized with high yield. The structures were established by IR, ¹H, ¹³C NMR, and mass spectral studies. Its bio-distribution patterns were evaluated on rats.     

Preparation and characterization of novel 4-bromo-3,4-dimethyl-1-phenyl-2-phospholene 1-oxide and the analogous phosphorus heterocycles or phospha sugars

4-Bromo-3,4-dimethyl-1-phenyl-2-phospholene 1-oxide (3c) was first synthesized from 3,4-dimethyl-1-phenyl-2-phospholene 1-oxide (2c) by a bromo-radical substitution reaction occurred at C-4 position by N-bromosuccinimide and 2,2′-azobisisobutyronitrile. The novel phospha sugar analogue 3c exerted high anti-proliferative effect on U937 cells evaluated by MTT in vitro methods and was much more efficient than that of Gleevec®, which is known as a molecule targeting chemotherapeutical agent. The substitution of 2-phospholenes at C-3 and C-4 position with methyl groups as well as 4-bromo substituent suggests a good anti-proliferative effect.     

ISOLATION AND REGENERATION OF HAPLOID PROTOPLASTS FROM BANGIA ATROPURPUREA (RHODOPHYTA) WITH MARINE BACTERIAL ENZYMES1

Three kinds of enzymes, agarase, β-1,4-mannanase, and β-1,3-xylanase, required for isolation of protoplasts from the red alga Bangia atropurpurea (Roth) C. Ag. were prepared from bacterial culture fluids of Vibrio sp. PO-303, Vibrio sp. MA-138, and Alcaligenes sp. XY-234, respectively, isolated from the sea environment. The optimal pH of all enzymes was around 7.5. Suitable conditions for protoplast isolation from B. atropurpurea were examined. The pretreatment of the fronds with pa-pain solution (20 mM Mes buffer, pH 7.5, containing 2% papain and 0.5 M mannitol) contributed to successful protoplast isolation. When razor-cut fragments of the fronds (about 200 mg in fresh weight) immersed in 20 mM Mes buffer, 7.5, containing 0.5 M mannitol and one unit each of agarase, β-1,4-mannanase, and β-1,3-xylanase were incubated at 22°C for 90 min with gentle agitation, 5.7 × 10⁶ protoplasts were released from them. Many protoplasts regenerated into fronds of regular or irregular shape.