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This study investigated the effects of age, sex and breed on serum cystatin C (Cys-C) and creatinine in small breed dogs. This retrospective study included 250 dogs weighing less than 15 kg without azotemia. Serum Cys-C and creatinine concentrations were analyzed, along with their correlation with age, and the difference between sexes or dog breeds. Serum Cys-C concentration correlated with age (P0.001), and did not differ between sexes or dog breeds. By contrast, serum creatinine concentration did not correlate with age. Serum creatinine concentration was higher in males than females (P0.05), and was lower in Miniature Dachshunds and Chihuahuas, and was higher in Shiba Inus compared to the general study population (P0.001). Serum Cys-C concentration correlates with age, and might be more sensitive to aging-associated subclinical renal dysfunction than serum creatinine concentration in dogs. Unlike serum creatinine concentration, serum Cys-C concentration is not affected by sex or dog breed.

Xianghuangqi County of Inner Mongolia is a typical high-fluoride area in China. Present work discussed a possible role of phosphate-solubilizing bacteria (PSBs) in the formation of high-fluoride gr...

Ultrafine bubbles (UFBs) are defined as small gas-filled bubbles with a diameter smaller than 1 μm. UFBs are stable for several weeks in aqueous solutions due to their small size. Although the mechanism of the stability of UFBs remains under intensive investigation, industrial applications of UFBs have recently arisen in various fields such as agricultural and fishery industries and medical therapy. The relevance of ions (protons and hydroxide anions) in UFB solutions has been discussed; however, the mechanism underlying the behavior of UFBs is still ambiguous and there is little direct evidence of the effect of UFBs on biological materials. This study deals with gaseous UFBs in aqueous solutions. Using small- and wide-angle X-ray scattering, we have investigated the structures of UFBs (air-UFBs, O2-UFBs, and N2-UFBs) and their effect on protein and lipid membrane structures. X-ray scattering and modeling data suggest that UFBs present a dynamic diffusive boundary (interface) due to the continuous release and absorption of gas. UFBs were found to not affect the structures of proteins at all hierarchal structure levels (from quaternary to tertiary, to internal, to secondary), whereas they did influence the packing and fluctuation of the hydrocarbon chains in the liposomes but not their shapes.

Aspartic acid 103 (D103) of sensory rhodopsin II from Halobacterium salinarum (HsSRII, or also called phoborhodopsin) corresponds to D115 of bacteriorhodopsin (BR). This amino acid residue is functionally important in BR. This work reveals that a substitution of D103 with asparagine (D103N) or glutamic acid (D103E) can cause large changes in HsSRII photocycle. These changes include (1) shortened lifetime of the M intermediate in the following order: the wild-type > D103N > D103E; (2) altered decay pathway of a 13-cis O-like species. The 13-cis O-like species, tentatively named Px, was detected in HsSRII photocycle. Px appeared to undergo branched reactions at 0°C, leading to a recovery of the unphotolyzed state and formation of a metastable intermediate, named P370, that slowly decayed to the unphotolyzed state at room temperature. In wild-type HsSRII at 0°C, Px mainly decayed to the unphotolyzed state, and the decay reaction toward P370 was negligible. In mutant D103E at 0°C, Px decayed to P370, while the recovery of the unphotolyzed state became unobservable. In mutant D103N, the two reactions proceeded at comparable rates. Thus, D103 of HsSRII may play an important role in regulation of the photocycle of HsSRII.

This study evaluated the monitoring methods in asymptomatic dogs with high serum cystatin C (Cys-C) concentrations. Ten dogs with high serum Cys-C were divided into two groups based on the owner's choice; one receiving clinical pathology-based monitoring at an animal hospital specialised in chronic kidney disease, and the other receiving symptom-based monitoring at home, partly because they showed no clinical symptoms. The dogs that received the clinical pathology-based monitoring led to an early treatment intervention, resulted in a longer survival period than dogs received the symptom-based monitoring (P

Case summary A 12-year-old neutered female domestic shorthair cat was admitted for syncope. Clinical signs and electrocardiography revealed high-grade atrioventricular (AV) block. Treatment with cilostazol ameliorated the clinical signs and arrhythmia. However, the high-grade AV block recurred on several occasions. After 640 days, the cat presented again with clinical deterioration owing to reoccurrence of the arrhythmia and it died 11 days later. Histopathological examination revealed a loss of conduction cells within the His bundle. Relevance and novel information To our knowledge, this is the first report of high-grade AV block treated with cilostazol in a cat. Treatment with cilostazol prolonged survival for 650 days without pacemaker implantation. Histological findings suggested that the AV block was related to fibrosis of the impulse conduction system.

Previously, we have detected the expression of 2 lipocalin genes (lp1 and lp2) in the olfactory epithelium of the Japanese newt Cynops pyrrhogaster. Recombinant proteins of these genes (Cp-Lip1 and Cp-Lip2, respectively) exhibited high affinities to various odorants, suggesting that they work like the odorant-binding proteins (OBPs). However, the physiological functions of OBP generally remain inconclusive. Here, we examined the effect of Cp-Lip1 on the electrophysiological responses of newt olfactory receptor cells. We observed that the electro-olfactogram induced by the vapor of an odorant with high affinity to Cp-Lip1 appeared to increase in amplitude when a tiny drop of Cp-Lip1 solution was dispersed over the olfactory epithelium. However, the analysis was difficult because of possible interference by intrinsic components in the nasal mucus. We subsequently adopted a mucus-free condition by using suction electrode recordings from isolated olfactory cells, in which impulses were generated by puffs of odorant solution. When various concentration (0–5 µM) of Cp-Lip1 was mixed with the stimulus solution of odorants highly affinitive to Cp-Lip1, the impulse frequency increased in a concentration-dependent manner. The increase by Cp-Lip1 was seen more evidently at lower concentration ranges of stimulus odorants. These results strongly suggest that Cp-Lip1 broadens the sensitivity of the olfactory cells toward the lower concentration of odorants, by which animals can detect very low concentration of odorants.

An efficient, accurate HPLC-MS/TQMS method was introduced for the quantitative/qualitative simultaneous analysis of main ingredients, namely geniposide and genipingentiobioside, in the Gardenia fruit. The separation was successfully obtained using a C8 (100mm×2.1mm, 5μm, 30°C) column by gradient elution with ultrapure water as mobile phase, where flow rate was set to 0.2 ml/min and detection wavelength at 240 nm. The analytical method was validated and the quantification of active compounds, namely genipingentiobioside and gardenoside, was performed. Linearity, precision, repeatability, stability and recovery were also reported. The quantitative analysis revealed that both main ingredients as geniposide and genipingentiobioside have performed a good linear relationship in 0.1-100 mg/ml concentration range (r=1.00000 and r =0.99998). The average content was measured to be 4.842% with RSD 0.96% for geniposide and 1.1976% with RSD 0.47% for genipingentiobioside in the Gardenia fruit. Accordingly, this method would be feasible for the quantity and quality control of crude drugs.

Enantioselective aldol reactions of various isatins with ketones using newly designed amino amide organocatalysts were found to provide chiral 3-substituted 3-hydroxy-2-oxindoles in good to excellent yields and with excellent stereoselectivities (up to 99 %, up to 98 % ee, syn/anti = 99:1); one catalyst, 3i, proved particularly successful. One of the resulting oxindoles, 3-hydroxy-3-(2-oxocyclohexyl)-2-indolinone may serve as a synthetic intermediate for pharmaceutically important compounds and, in its own right, shows interesting anticonvulsant activities.

Camponotus japonicus uses basiconic antennal sensilla (s. basiconica) to sense a colony-specific blend of species-specific cuticular hydrocarbons (CHCs). The inner portion of the s. basiconica is filled with sensillar lymph and chemosensory proteins (CSPs) presumed to transport CHCs to olfactory neuron receptors. Although 12 CSPs have been found in C. japonicus antennae, we focused on CjapCSP1 and CjapCSP13. The molecular basis of CSP1 function was explored by observation of its structure in solution at pH 4.0 and 7.0 through circular dichroism (CD) and X-ray solution scattering. Although the secondary structure did not vary with pH change, the radius of gyration (Rg) was larger by 5.3% (0.74 A increase) at pH 4.0 than at pH 7.0. The dissociation constant (Kd) for CjapCSP1 measured with a fluorescent probe, 1-N-phenylnaphthylamine, was larger at pH 4.0 than at pH 7.0, suggesting that acidic pH triggers ligand dissociation. In contrast to CjapCSP1, the Rg of CjapCSP13 was slightly smaller at pH 4.0 than at pH 7.0. Western blotting and immunohistochemistry with protein-specific antisera revealed that both CjapCSP1 and CjapCSP13 are detected in the antennae, but differ in their specific internal localization. Binding to four compounds, including the ant CHC (z)-9-tricosene, was examined. Although both CjapCSP1 and CjapCSP13 bound to (z)-9-tricosene, CjapCSP13 bound with higher affinity than CjapCSP1 and showed different binding properties. CjapCSP1 and CjapCSP13 are synthesized by the same cells of the antenna, but function differently in CHC distribution due to differences in their localization and binding characteristics.

Archaerhodopsins (ARs) is one of the members of microbial rhodopsins. Threonine 164 (T164) and serine 165 (S165) residues of the AR from Halorubrum sp. ejinoor (HeAR) are fully conserved in ARs, although they are far from the proton transfer channel and the retinal Schiff base, and are likely involved in a hydrogen-bonding network at the end of the Helix E where most microbial rhodopsins assume a "bent structure". In the present work, T164 and/or S165 were replaced with an alanine (A), and the photocycles of the mutants were analyzed with flash photolysis. The amino acid replacements caused profound changes to the photocycle of HeAR including prolonged photocycle, accelerated decay of M intermediate and appearance of additional two intermediates which were evident in T164A- and T164A/S165A-HeAR photocyles. These results suggest that although T164 and S165 are located at the far end of the photoactive center, these two amino acid residues are important for maintaining the fast turnover of the HeAR photocycle. The underlying molecular mechanisms are discussed in relation to hydrogen-bonding networks involving these two amino acids. Present study may arouse our interests to explore the functional role of the well-conserved "bent structure" in different types of microbial rhodopsin.

Microbial rhodopsins are photoactive proteins that use a retinal molecule as the photoactive center. Because of structural simplicity and functional diversity, microbial rhodopsins have been an excellent model system for structural biology. In this study, a halophilic archaea that has three microbial rhodopsin-type genes in its genome was isolated from Ejinoor salt lake in Inner Mongolia of China. A sequence of 16S rRNA showed that the strain belongs to Halorubrum genus and named Halorubrum sp. ejinoor (He). The translated amino acid sequences of its microbial rhodopsin-type genes suggest that they are homologs of archaerhodopsin (HeAR), halorhodopsin (HeHR) and sensory rhodopsin II (HeSRII). The mRNAs of three types of genes were detected by RT-PCR and their amounts were investigated by Real-Time RT-PCR. The amount of mRNA of HeSRII was the smallest and the amounts of of HeAR and HeHR were 30 times and 10 times greater than that of HeSRII. The results of light-induced pH changes suggested the presence of a light-driven proton pump and a light-driven chloride ion pump in the membrane vesicles of He. Flash induced absorbance changes of the He membrane fraction indicated that HeAR and HeHR are photoactive and undergo their own photocycles. This study revealed that three microbial rhodopsin-type genes are all expressed in the strain and at least two of them, HeAR and HeHR, are photochemically and physiologically active like BR and HR of Halobacterium salinarum, respectively. To our knowledge, this is the first report of physiological activity of HR-homolog of Halorubrum species.

The catalytic activity of a simple amino alcohol that contains a bulky super silyl group [i.e., tris(trimethylsilyl)silyl (TTMSS)] bonded to the oxygen atom at the γ-position along with a primary amine moiety was examined in the enantioselective 1,3-dipolar cycloaddition of nitrones to α,β-unsaturated aldehydes. The organocatalyst successfully provided optically active isoxazolidines in good chemical yields (up to 86 %) with excellent diastereoselectivities (endo/exo, up to 96:4) and enantioselectivities (up to 97 % ee). Furthermore, the obtained isoxazolidines were easily converted into γ-amino diols that contain three contiguous stereogenic centers.

Photo-reaction pathways of a bacteriorhodopsin Y185F mutant were examined using in situ photo-irradiation solid-state NMR spectroscopy. (13)C CP MAS NMR spectra were recorded at -40 °C in the dark (D1), under irradiation with 520 nm light (L1), subsequently in the dark (D2), and again under irradiation with 520 nm light (L2). In the process from D1 to L1, the 13-cis, 15-syn (CS; bR548) state changed to a CS*- (13-cis, 15-syn) intermediate, which was highly stable at -40 °C, and the all-trans (AT; bR568) state transformed to an N-intermediate. Under the D2 conditions, the N-intermediate transformed to an O-intermediate, which was highly stable at -40 °C in the dark. During subsequent irradiation with 520 nm light (L2), the O-intermediate transformed to the N-intermediate through the AT state, whereas the CS*-intermediate did not change. The CS*-intermediate was converted to the AT state (or O-intermediate) after the temperature was increased to -20 °C. Upon subsequent increase of the temperature to 20 °C, the AT state (or O-intermediate) was converted to the CS state until reaching equilibrium. In this experiment, the chemical shift values of [20-(13)C, 14-(13)C]retinal provided the 13C[double bond, length as m-dash]C and 15C[double bond, length as m-dash]N configurations, respectively. From these data, the configurations of the AT and CS states and the CS*-, N-, and O-intermediates were determined to be (13-trans, 15-anti), (13-cis, 15-syn), (13-cis, 15-syn), (13-cis, 15-anti), and (13-trans, 15-anti), respectively. (13)C NMR signals of the CS*- and O-intermediates were observed for the first time for the Y185F bR mutant by in situ photo-irradiation solid-state NMR spectroscopy and the configuration of the CS*-intermediate was revealed to be significantly twisted from that of the CS state although both were assigned as (13-cis, 15-syn) configurations.

To clarify the effect of Fe3+ concentration on magnetite formation, Magnetospirillum magnetotacticum MS-1 was cultured at various initial concentrations of Fe3+ and the yield and magnetic properties of the cells were investigated. Although total cell yields (g/l) were not dependent on the initial concentration of Fe3+ (0–68 µM) in the growth medium, the percentage of magnetic cells increased as the initial Fe3+ concentration increased. The coercivity (H c) and saturation magnetization (M s) of dried MS-1 cells increased and the blocking temperature (T B) decreased as the initial Fe3+ concentration increased. These values are similar to those reported previously. The Verwey transition temperature (T V) was almost identical for all initial Fe3+ concentrations (95–97 K), except for medium without added Fe3+ (0 µM; 88 K). Fe3+ concentration strongly affects the magnetic properties of MS-1 cells, especially in the lower concentration range, suggesting that the Fe3+ concentration should be considered when evaluating the magnetic properties of MS-1 cells.

To predict the propagation velocity of different concentrations of bovine serum albumin (BSA) in a Rayleigh-type biosensor, we derived a new calculation method for a Rayleigh surface acoustic wave (SAW) device. The method can calculate propagation velocities of leaky Rayleigh waves and Rayleigh waves when liquid is placed in the groove of a Rayleigh-type SAW device. Different concentration of BSA proteins were used to evaluate the effect of these concentrations. In this study, we used different concentrations of BSA and a Rayleigh-type SAW device consisted of grooves of 0.25 mm width and 0.26 mm height and grooves of 0.40 mm width and 0.25 mm height, quartz as the substrate and interdigital transducers (IDT) with a 25.00 mm distance. The frequency of the Rayleigh-type SAW device was set to 157.6 MHz. Our data suggests that the propagation velocity of SAWs depends mainly upon BSA protein concentration and the size of groove. At the same concentration of BSA, the propagation acoustic wave velocity of BSA depends on the size of groove. The results showed the 0.40 mm width-0.25 mm height groove was more suitable than the 0.25 mm width-0.26 mm height groove for studying the propagation velocity of wave with liquid in a Rayleigh-type SAW device. With the same groove, the propagation velocity decreased when the concentration of BSA protein increased.

This study examined the predictive value of serum cystatin C (Cys-C) concentration, measured during routine periodic health examinations, in the renal prognosis of dogs. A cohort of 140 dogs weighing15 kg whose serum Cys-C concentrations were measured during periodic health examinations from December 2013 to March 2016 were prospectively studied, with renal disease-related death the predicted end point. Of the 140 dogs, nine died from renal diseases during the follow-up period (539 ± 249 days). Serum Cys-C concentrations were higher in the dogs that subsequently died of renal disease than in the censored group (0.8 ± 0.25 vs. 0.3 ± 0.1 mg/dl, respectively; P .01). Dogs with high serum Cys-C concentrations (0.55 mg/dl) had a shorter (P .01) renal disease-specific survival period than those with low serum Cys-C concentrations (≤0.55 mg/dl). In conclusion, high serum Cys-C concentrations in periodic health examinations in dogs15 kg predicted poorer prognosis for renal function.

The enantioselective Diels-Alder reaction of 1,2-dihydropyridines with aldehydes using an easily prepared optically active β-amino alcohol catalyst was found to provide optically active isoquinuclidines, an efficient synthetic intermediate of pharmaceutically important compounds such as oseltamivir phosphate, with a satisfactory chemical yield and enantioselectivity (up to 96%, up to 98% ee). In addition, the obtained highly optically pure isoquinuclidine was easily converted to an optically active piperidine having four successive carbon centers.

Bacteriorhodopsin (BR) is a light driven proton pump firstly found in the membrane of halophilic archaea. BR absorbs wide range of visible light with high quantum efficiency. To elucidate the sensitizing effect of BR, we tested the photocatalytic degradation of phenol with BR-sensitized TiO2 nanoparticles and compared with that of Alizarin red (AR). When the concentration of BR was 13.1 mg/L (4.9×10-7 mol/L), 23.8% of phenol was degradated after 2 hours of light irradiation. The degradation percentage was 3.5 times higher than that with TiO2 nanoparticles alone and similar to that sensitized by AR (20.1%). The possibilities and approaches to increase the sensitizing performance of BR were discussed.

Salt lake environments are economically and ecologically important issues. Like any other ecosystems in the world, salt lake ecosystems are facing various kinds of pollutions. However, little information is available on the biodegradation of organic pollutants by halophilic microorganisms in such environments. In the present work we isolated a strain of phenol-degradating bacteria from a salt lake in Inner Mongolia. Sequence of 16S rRNA suggested that the strain belong toPropionibacteriumgenus. The strain can grow with phenol as a sole carbon source under 10% (w/v) salt concentration. PCR amplifications with the primers for multicomponent phenol hydroxylase or catechol 1, 2 dioxygenase gene were applied to genome DNA of the strain, suggesting that phenol might be transformed to catechol by multicomponent phenol hydroxylase and further degradated through ortho-ring cleavage pathway. When the primers for catechol 1, 2 dioxygenase were applied to the DNA obtained from the lake water, at least two types of catechol 1, 2 dioxygenase genes sequences were obtained, suggesting the presence of the bacteria with diverse aromatic compound-degradating genes.

We prepared a silk fibroin compact in which Ag nanoparticles (Ag NPs) were dispersed, by adding only silver nitrate (AgNO3) to a silk fibroin solution without using any reducing agent. FT-IR and DSC revealed that the formation of Ag NPs distorted the conformation of the silk fibroin. However, the Ag NPs did not affect the thermal stability of the silk fibroin compact. An antimicrobial test against Escherichia coli (E. coli) was performed using the silk fibroin compact in which Ag NPs were dispersed. The compact showed sufficient antimicrobial activity because the average survival of E. coli was 1.6% after 8 h incubation even when the silver content was 0.01 wt%. The addition of Ag NPs into silk fibroin compact can provide color and an antimicrobial function without spoiling the function of the silk fibroin compact.

Microbial rhodopsins are photoactive proteins that use retinal molecule as the photoactive center. Because of the structural simplicity and functional diversity, microbial rhodopsins have been an excellent model system for structural biology. In the past decades microbial rhodopisns were identified from diverse environments including salt lakes, fresh water, sea water, human and plant tissues as fungal pathogens. In the present work, we tried to identify microbial rhodopsin genes from salt lake in Inner Mongolia, China. Two bacteriorhodopsin-like genes were identified. The retrieved sequences were analyzed by comparing with four most widely studied haloarchaeal rhodopsins.

Magnetotactic bacteria are the bacteria which synthesize nano-sized magnetic particles within their cell body. In the present study, the cells of a magnetotactic bacteria M. magnetotacticum MS-1 was fractionated and the iron amount of the cell fractions were measured. In order to study the relationship between the process of the biosynthesis of magnetosome and the intracellular transport of iron, MS-1 cells were cultured under iron limited culture conditions and the amount of iron in the cell fractions were compared with those under optimal culture conditions. The whole amount of iron was measured colorimetrically. To estimate the number of ferric ion, the number of spin was determined from the ESR spectrum. The cell fractions obtained after removing magnetosome (non-magnetic fractions) contained 30-40 % of the total iron in the intact cells. There was a good correlation between the number of spin and the iron amount of non-magnetic fractions under optimal culture conditions. Under iron limited conditions, the iron amount decreased not only in the fraction containing magnetosome but also in the non-magnetic fractions. The number of spin in the soluble fraction decreased remarkably. So, the correlation between the number of spin and the amount of iron was not observed under iron limited conditions. These results imply that the ratio of ferrous ion to whole amount of iron increased in the non-magnetic fractions under iron limited conditions as compared with that under optimal conditions.

In order to analyze the magnetic behaviors of iron complexes biologically synthesized in magnetic bacteria MS-1, we performed FMR (Ferromagnetic Resonance) measurements for each fraction of the cell. We observed FMR spectra from the ferric iron (Fe 3þ ) compounds distributed in each fraction of the MS-1 cell. In particular, the magnetosome fraction yielded an anisotropic FMR signal, whereas other fractions were simple FMR spectra of a Gaussian type. Upon counting the numbers of spins in various cell fractions, we compared them with the iron population as determined by the 1.10phenanthroline method. We found a good correlation between the number of spins and the iron population in several cell fractions. We concluded that the cell fractions, other than those fractions containing magnetite, consist mostly of ferric irons rather than ferrous irons. [doi:10.2320/matertrans.M2009177]

Magnetotactic bacteria have one or more chains of magnetosome, consisting of nano-sized magnetic crystal covered with a phospholipid bilayer and use it to sense the geomagnetic fields. In order to elucidate the molecular process to make magnetosome from the iron compounds found in the bacteria, laser Raman spectroscopic measurements were performed with the magnetotactic bacterium, Magnetospirillum magnetotacticum MS-1 and the fractions separated from it. The clear Raman signals were observed at 662 cm-1 and 740 cm-1. The former was observed in whole cell and magnetosome fraction, but not in membrane and cytoplasmic fraction and assigned to the Raman signal of magnetite. The Raman signal of the latter was observed not only in the magnetosome fraction, but also in the cytoplasmic fraction and membrane fraction. This signal was assumed to ferrihydrite. Based on the results, the pathway of the magnetosome synthesis and possible roles of ferrihydrite in the magnetotactic bacteria were discussed.

Pharaonis phoborhodopsin (ppR), also called pharaonis sensory rhodopsin II, NpSRII, is a photoreceptor for the photophobic response of Natronomonas pharaonis. Tryptophan 182 (W182) of bacteriorhodopsin (bR) is near the chromophore retinal and has been suggested to interact with retinal during the photoreaction and also to be involved in the hydrogen-bonding network around the retinal. W182 of bR is conserved in ppR as tryptophan 171 (W171). To elucidate whether W171 of ppR interacts with retinal during the photoreaction and/or is involved in the hydrogen-bonding network as in bR, we formed W171-substituted mutants of ppR, W171A and W171T. Our low-temperature spectroscopic study has revealed that the substitution of W171 to Ala or Thr resulted in the stabilization of M- and O-intermediates. The stability of M and absorption spectral changes during the M-decay were different depending on the substituted residue. These findings suggest that W171 in ppR interacts with retinal and the degree of the interaction depends on the substituted residues, which might be rate determining in the M-decay. In addition, the involvement of W171 in the hydrogen-bonding network is suggested by the O-decay. We also found that glycerol slowed the decay of M and not of O.

Bleached rhodopsin regenerates by way of the Schiff base formation between the 11-cis retinal and opsin. Recovery of human vision from light adapted states follows biphasic kinetics and each adaptive phase is assigned to two distinct classes of visual pigments in cones and rods, respectively, suggesting that the speed of Schiff base formation differs between iodopsin and rhodopsin. Matsumoto and Yoshizawa predicted the existence of a β-ionone ring-binding site in rhodopsin, which has been proven by structural studies. They postulated that rhodopsin regeneration starts with a non-covalent binding of the β-ionone ring moiety of 11-cis-retinal, followed by the Schiff base formation. Recent physiological investigation revealed that non-covalent occupation of the β-ionone ring binding site transiently activates the visual transduction cascade in the dark. In order to understand the role of non-covalent binding of 11-cis-retinal to opsin during regeneration, we studied the kinetics of rhodopsin regeneration from opsin and 11-cis-retinal and found that the Schiff base formation is accelerated ∼10(7) times compared to that between retinal and free amine. According to Cordes and Jencks, Schiff base formation in solution exhibits a bell-shaped pH dependence. However, we discovered that the rhodopsin formation is independent of pH over a wide pH range, suggesting that aqueous solvents do not have access to the Schiff base milieu during its formation. According to Hecht et al. the regeneration of iodopsin must be significantly faster than that of rhodopsin. Does this suggest that the Schiff base formation in iodopsin is favored due to its structural architecture? The iodopsin structure once solved would answer such a question as how molecular fine-tuning of retinal proteins realizes their dark adaptive functions. In contrast, bacteriorhodopsin does not require occupancy of a distinct β-ionone ring-binding site, enabling an aldehyde without the cyclohexene ring to form a pigment. Studies of regeneration reaction of other retinal proteins, which are scarcely available, would clarify the molecular structure-phenotype relationships and their physiological roles.

Canonical heterotrimeric G proteins in eukaryotes are major components that localize at plasma membrane and transmit extracellular stimuli into the cell. Genome of a seed plant Arabidopsis thaliana encodes at least one Gα (GPA1), one Gβ (AGB1), and 3 Gγ (AGG1, AGG2 and AGG3) subunits. The loss-of-function mutations of G protein subunit(s) cause multiple defects in development as well as biotic and abiotic stress responses. However, it remains elusive how these subunits differentially express these defects. Here, we report that Arabidopsis heterotrimeric G protein subunits differentially respond to the endoplasmic reticulum (ER) stress. An isolated homozygous mutant of AGB1, agb1-3, was more sensitive to the tunicamycin-induced ER stress compared to the wild type and the other loss-of-function mutants of G protein subunits. Moreover, ER stress responsive genes were highly expressed in the agb1-3 plant. Our results indicate that AGB1 positively contributes to ER stress tolerance in Arabidopsis.

The G protein messages in Octopus vulgaris photoreceptor were isolated and characterized with molecular biological techniques. Four classes of G protein α subunit cDNA were isolated from an octopus eye cDNA library: OvGαi, OvGαo, OvGαq and OvGαs. Northern blot analysis of octopus tissues showed abundant expression of OvGαq in the eye, and specific expression of OvGαo in neural tissues. OvGαi was expressed in all tissues studied. In situ hybridization revealed that OvGαq message was expressed in almost all photoreceptor cells. Based on these results, a possible phototransduction pathway in the octopus visual cells is discussed.

In vertebrate visual pigments, a glutamic acid serves as a negative counterion to the positively charged chromophore, a protonated Schiff base of retinal. When photoisomerization leads to the Schiff base deprotonating, the anionic glutamic acid becomes protonated, forming a neutral species that activates the visual cascade. We show that in octopus rhodopsin, the glutamic acid has no anionic counterpart. Thus, the “counterion” is already neutral, so no protonated form of an initially anionic group needs to be created to activate. This helps to explain another observation—that the active photoproduct of octopus rhodopsin can be formed without its Schiff base deprotonating. In this sense, the mechanism of light activation of octopus rhodopsin is simpler than for vertebrates, because it eliminates one of the steps required for vertebrate rhodopsins to achieve their activating state.

Isomeric octopus pigment analogues from 9-cis and 11-cis 10-fluoro retinal have been prepared and their photochemical as well as other properties examined. While most of the properties of the fluor...

G protein-coupled receptor kinases (GRKs) play an important role in stimulus-dependent receptor phosphorylation and desensitization of the receptors. Mammalian rhodopsin kinase (RK) and beta-adrenergic receptor kinase (betaARK) are the most studied members among known GRKs. In this work, we purified RK from octopus photoreceptors for the first time from invertebrate tissues. The molecular mass of the purified enzyme was 80 kDa as estimated by SDS-polyacrylamide gel electrophoresis, and this was 17 kDa larger than that of the vertebrate enzymes. Unlike vertebrate RK, octopus RK (ORK) was directly activated by betagamma-subunits of a photoreceptor G protein. We examined the effects of various known activators and inhibitors of GRKs on the activity of the purified ORK and found that their effects were different from those on either bovine RK or betaARK. To analyze the primary structure of the enzyme, we cloned the cDNA encoding ORK from an octopus retinal cDNA library. The deduced amino acid sequence of the cDNA was highly homologous to betaARK over the entire molecule, including a pleckstrin homology domain located in the C-terminal region, and homology to RK was significantly lower. Furthermore, Western blot analysis of various octopus tissues with an antibody against the purified ORK showed that ORK is expressed solely in the retina, which confirmed the identity of the enzyme as rhodopsin kinase. Thus, ORK appears to represent a unique subgroup in the GRK family, which is distinguished from vertebrate RK.

Femtosecond pump−probe spectroscopy of halorhodopsin from Halobacterium halobium and rhodopsin from octopus (paroctopus defleini) has been studied in a wide spectral range extending from 400 to 1000 nm. There are five common features to halorhodopsin and rhodopsin in the transient absorption and gain spectra. A comparative description of the primary photochemistries in the retinal proteins including bacteriorhodopsin is presented to explain the present experimental results systematically together with the previous studies. In the model there is a branching from the Franck−Condon state into coherent and incoherent channels. The former is the rapid process of direct formation of the first intermediate (bathorhodopsin for rhodopsin and hRK intermediate for halorhodopsin), and the latter is the thermalization of the excited state in the retinoid proteins, from which slow formation of the first intermediate takes place.

We determined the structure and site of fatty acid incorporated in octopus rhodopsin using a combination of fluorescence label and enzymatic cleavage methods in conjunction with fast-atom bombardment (FAB) mass spectrometry. A single peptide containing two adjacent cysteines, Cys337 and Cys338, was successfully isolated using the fluorescence from a dye conjugated to Cys345. The FAB mass spectrometric analysis of the peptide (323Phe-340Phe) revealed that two palmitoyl groups are linked to Cys337 and Cys338 via thioester bonds in octopus rhodopsin as in bovine rhodopsin.

In invertebrate photoreceptors, illuminated rhodopsin activates multiple G proteins, which are assumed to initiate multiple phototransduction cascades. In this paper, we focused on one of the phototransduction cascades, which utilizes rhodopsin, a Gq-like G protein, and phospholipase C (PLC). A Gq-like G protein from octopus photoreceptors was successfully purified to apparent homogeneity as an active form by simple two-step chromatography. The purified G protein had an alpha beta gamma-trimeric structure consisting of 44-kDa alpha, 37-kDa beta, and 9-kDa gamma subunits. The 44-kDa alpha subunit was assigned to the Gq class by western blot with antiserum against mammalian Gq alpha and by partial amino acid sequencing of its proteolytic fragments. Light-dependent binding of GTP gamma S was observed when the purified octopus Gq was reconstituted with octopus rhodopsin that had been integrated into phospholipid vesicles. Octopus Gq activated PLC beta 1 purified from bovine brain dose-dependently in the presence of A1F4-. Finally, light- and GTP-dependent activation of PLC beta 1 was observed in a reconstitution system consisting of octopus rhodopsin, Gq, and bovine PLC beta 1.

Phoborhodopsin from Halobacterium salinarum (salinarum phoborhodopsin, spR also called HsSR II) is a photoreceptor for the negative phototaxis of the bacterium. A unique feature of spR is the formation of a shorter wavelength photoproduct, P480, observed at liquid nitrogen temperature beside the K intermediate. Formation of similar photoproduct has not been reported in the other microbial rhodopsins. This photoproduct showed its maximum absorbance wavelength (λ(max)) at 482 nm and can thermally revert back to spR above -160 °C. It was revealed that P480 is a photoproduct of K intermediate by combination of an irradiation and warming experiment. Fourier transform infrared (FTIR) difference spectrum of P480 from spR in C-C stretching vibration region showed similar features with that of K intermediate, suggesting that P480 has a 13-cis-retinal chromophore. The appearance of a broad positive band at 1214 cm(-1) in the P480-spR spectrum suggested that configuration around C9═C10 likely be different between P480 and K intermediate. Vibrational bands in HOOP region (1035 to 900 cm(-1)) suggested that the chromophore distortion in K intermediate was largely relaxed in P480. The amount of P480 formed by the irradiation was greatly decreased by amino acid replacement of S201 with T, suggesting S201 was involved in the formation of P480. According to the crystal structure of pharaonis phoborhodopsin (ppR), a homologue of spR found in Natronomonas pharaonis, S201 should locate near the C14 of retinal chromophore. Thus, the interaction between S201 and C14 might be the main factor affecting formation of P480.

GTP-binding protein (G protein) linking to metabotropic glutamate receptor of bovine retinal on-bipolar cell was studied by use of pharmacologically selective ligands, 2-amino-4-phosphonobutyric acid (APB) on bacterial toxin-catalyzed ADP-ribosylation and GTP gamma S-binding. In contrast to the electrophysiological findings reported, G protein coupling to APB-sensitive glutamate receptor served as a substrate for pertussis toxin but did not for cholera toxin. Several glutamate analogues effective on on-bipolar cell, as well as APB, increased GTP gamma S binding to retinal membranes devoid of rod outer segments. The enhancement of GTP gamma S binding by APB was completely abolished when the membranes were pretreated with pertussis toxin and NAD. These results suggest that, in retinal on-bipolar cell, the G protein which couples metabotropic glutamate receptor to hyperpolarizing response of the cell is sensitive to pertussis toxin.

The infrared difference spectra between octopus rhodopsin and lumirhodopsin have been observed at 210 K under irradiation of blue and orange light under neutral conditions. The CN stretching frequencies of the protonated Schiff-base linkage shift upon H/D exchange. The H/D shifts observed in this study for octopus rhodopsin and lumirhodopsin are 25 and 23 cm −1 , respectively, whereas those for bovine rhodopsin and lumirhodopsin have been reported to be about 30 and 4cm −1 , respectively. These results indicate that environments around the Schiff-base linkage differ between octopus and bovine lumirhodopsin, whereas those of octopus rhodopsin and lumirhodopsin are similar. The relationship between the H/D shift of the CN stretching band and the hydrogen bond involving the protonated Schiff base is discussed.