Photochemistry and Photoinduced Proton-Transfer by Pharaonis Phoborhodopsin

Phoborhodopsin (pR or sensory rhodopsin II, sRII) is a photoreceptor of the negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. The photocycle of ppR is essentially as follows: ppR(498) ppR_K(540) ppR_KL(512) ppR_L(488) ppR_M(390) ppR_O(560) ppR (numbers in parenthesis denote the maximum absorbance). The photocycle is very similar to that of bacteriorhodopsin, but the rate of initial pigment recovery is about two-orders of magnitude slower. By low-temperature spectroscopy, two K-intermediates were found but the L intermediate was not detected. The lack of L indicates extraordinary stability of K at low temperature. ppR_M is photoactive similar to M of bR. The ground state ppR contains only all-trans retinal whereas ppR_M and ppR_O contain 13-cis and all-trans, respectively. ppR has the ability of lightinduced proton transport from the inside to the outside. Proton uptake occurs at the formation of ppR_O and the release at its decay. ppR associates with its transducer and this complex transmits a signal to the cytoplasm. The proton transport ability is lost when the complex forms, but the proton uptake and release still occur, suggesting that the proton movement is non-electrogenic (release and uptake occur from the same side). The stoichiometry of the complex between ppR and the transducer is 1 : 1. ppR or pR has absorption maximum at 500 nm, which is blue-shifted from those of other archaeal rhodopsins. The molecular mechanism of this color regulation is not yet solved.     

Indispensability of transmembrane domains of Golgi UDP-galactose transporter as revealed by analysis of genetic defects in UDP-galactose transporter-deficient murine had-1 mutant cell lines and construction of deletion mutants

UDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galactosyltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters. We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations. Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein.     

Sleep patterns and sleep disorders among female shift workers in a computer factory of Japan

Using a repeated questionnaire survey for female workers in a computer factory of Japan, consisting of 45 daytime workers and 84 weekly rotating shift workers (of whom 40 and 44 were respectively engaged in early-shift work and late-shift work during the survey week), the present study aimed to clarify the effects of shift work on their sleep patterns and sleep disorders. The time of retiring, time of rising, duration of sleep, and self-evaluated sleep quality based on four sleep-related problems were investigated for four days (Sunday-Monday, Tuesday-Wednesday, Thursday-Friday, and Saturday-Sunday) in a week. The intergroup comparison revealed that the earlier the rising time and the shorter the duration of sleep, the lower the sleep quality; the lowest sleep quality was observed in the early-shift workers.     

The carbohydrate recognition by cytokines modulates their physiological activities

A variety of cytokines have been reported to be able to recognize specific carbohydrate moieties. To date, the role of carbohydrate recognition in cytokine function has been analyzed for several cytokines, including fibroblast growth factor (FGF), tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2. The FGF family and their receptors have been found to recognize a heparan sulfate proteoglycan, which generates rigid complexes that induce signal transduction. We have found that IL-2 recognizes a high-mannose type glycan on the alpha subunit of the IL-2 receptor as well as a peptide portion of this subunit. Blocking this carbohydrate-IL-2 interaction diminished IL-2-induced signaling and T-cell proliferation. We have also shown that TNF-alpha recognizes the second mannose 6-phosphate diester of the glycan portion of glycosylphosphatidylinositol (GPI)-anchored glycoproteins. Blocking this GPI-anchored glycan-TNF-alpha interaction abrogates TNF-alpha-induced apoptosis. We aim to increase the number of cytokines which modulate their functions through the unique carbohydrate recognition, and open the way to systematically elucidate the biological functions of cytokine-carbohydrate interaction in immune system.     

p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload

The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38alpha is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38alpha in hearts. First, we generated mice with floxed p38alpha alleles and crossbred them with mice expressing the Cre recombinase under the control of the alpha-myosin heavy-chain promoter to obtain cardiac-specific p38alpha knockout mice. These cardiac-specific p38alpha knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38alpha plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.     

Six1 controls patterning of the mouse otic vesicle

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.     

Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice

Interleukin-17 is a T cell-derived proinflammatory cytokine. This cytokine is suspected to be involved in the development of rheumatoid arthritis (RA) because this cytokine expression is augmented in synovial tissues of RA patients. The pathogenic roles of IL-17 in the development of RA, however, still remain to be elucidated. In this study, effects of IL-17 deficiency on collagen-induced arthritis (CIA) model were examined using IL-17-deficient mice (IL-17(-/-) mice). We found that CIA was markedly suppressed in IL-17(-/-) mice. IL-17 was responsible for the priming of collagen-specific T cells and collagen-specific IgG2a production. Thus, these observations suggest that IL-17 plays a crucial role in the development of CIA by activating autoantigen-specific cellular and humoral immune responses.     

Fatigue complaints among female shift workers in a computer factory of Japan

For female workers in a computer factory in Japan, consisting of 41 daytime workers and 74 weekly-rotating shift workers (of whom, 37 each were engaged in, respectively, early-shift work and late-shift work during the survey week), within-day variations in the number of fatigue complaints were elucidated. Based on a repeated questionnaire survey, changes of fatigue complaints in a day were evaluated at three occasions, i.e., just before work, just after work, and before retiring, for three working days and one off day. The occasions of fatigue feelings differed among the three work groups: the complaints were significantly more frequent before work in the early-shift workers, after work in the late-shift workers, and before retiring in the daytime workers. Feeling of fatigue before and after work may be disadvantageous to safety and efficiency of work.     

Tyr-199 and charged residues of pharaonis Phoborhodopsin are important for the interaction with its transducer

pharaonis Phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a retinal protein in Natronobacterium pharaonis and is a receptor of negative phototaxis. It forms a complex with its transducer, pHtrII, in membranes and transmits light signals by protein-protein interaction. Tyr-199 is conserved completely in phoborhodopsins among a variety of archaea, but it is replaced by Val (for bacteriorhodopsin) and Phe (for sensory rhodopsin I). Previously, we (Sudo, Y., M. Iwamoto, K. Shimono, and N. Kamo, submitted for publication) showed that analysis of flash-photolysis data of a complex between D75N and the truncated pHtrII (t-Htr) give a good estimate of the dissociation constant K(D) in the dark. To investigate the importance of Tyr-199, K(D) of double mutants of D75N/Y199F or D75N/Y199V with t-Htr was estimated by flash-photolysis and was approximately 10-fold larger than that of D75N, showing the significant contribution of Tyr-199 to binding. The K(D) of the D75N/t-Htr complex increased with decreasing pH, and the data fitted well with the Henderson-Hasselbach equation with a single pK(a) of 3.86 +/- 0.02. This suggests that certain deprotonated carboxyls at the surface of the transducer (possibly Asp-102, Asp-104, and Asp-106) are needed for the binding.     

Differences between maize and rice in N-use efficiency for photosynthesis and protein allocation

The N-use efficiency for photosynthesis was higher in a C(4) plant, maize, than in a C(3) plant, rice, including rbcS antisense rice with optimal ribulose-1,5-bisphosphate carboxylase (Rubisco) content for CO(2)-saturated photosynthesis, even when photosynthesis was measured under saturating CO(2) conditions. The N cost for the C(4) cycle enzymes in maize was not large, and the lower amount of Rubisco allowed a greater N investment in the thylakoid components. This greater content of the thylakoid components as well as the CO(2) concentrating mechanism may support higher N-use efficiency for photosynthesis in maize.