Molecular properties of rod and cone visual pigments from purified chicken cone pigments to mouse rhodopsin in situ.

We have investigated the molecular properties of rod and cone visual pigments to elucidate the differences in the molecular mechanism(s) of the photoresponses between rod and cone photoreceptor cells. We have found that the cone pigments exhibit a faster pigment regeneration and faster decay of meta-II and meta-III intermediates than the rod pigment, rhodopsin. Mutagenesis experiments have revealed that the amino acid residues at positions 122 and 189 in the opsins are the determinants for these differences. In order to study the relationship between the molecular properties of visual pigments and the physiology of rod photoreceptors, we used mouse rhodopsin as a model pigment because, by gene-targeting, the spectral properties of the pigment can be directly correlated to the physiology of the cells. In the present paper, we summarize the spectroscopic properties of cone pigments and describe our studies with mouse rhodopsin utilizing a high performance charge coupled device (CCD) spectrophotometer.     

Combination of hTERT and bmi-1, E6, or E7 induces prolongation of the life span of bone marrow stromal cells from an elderly donor without affecting their neurogenic potential

Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro. Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.     

An acidic protein, YBAP1, mediates the release of YB-1 from mRNA and relieves the translational repression activity of YB-1

Eukaryotic Y-box proteins are nucleic acid-binding proteins implicated in a wide range of gene regulatory mechanisms. They contain the cold shock domain, which is a nucleic acid-binding structure also found in bacterial cold shock proteins. The Y-box protein YB-1 is known to be a core component of messenger ribonucleoprotein particles (mRNPs) in the cytoplasm. Here we disrupted the YB-1 gene in chicken DT40 cells. Through the immunoprecipitation of an epitope-tagged YB-1 protein, which complemented the slow-growth phenotype of YB-1-depleted cells, we isolated YB-1-associated complexes that likely represented general mRNPs in somatic cells. RNase treatment prior to immunoprecipitation led to the identification of a Y-box protein-associated acidic protein (YBAP1). The specific association of YB-1 with YBAP1 resulted in the release of YB-1 from reconstituted YB-1-mRNA complexes, thereby reducing the translational repression caused by YB-1 in the in vitro system. Our data suggest that YBAP1 induces the remodeling of YB-1-mRNA complexes.     

Generation of knock-in mice carrying third cones with spectral sensitivity different from S and L cones

Red-green color vision in primates is unique in the sense that it is mediated by two photoreceptor cells that are indistinguishable in all aspects except for their visual pigments. In order to generate an animal model for investigation of the interaction between red-green inputs at the molecular level, we applied knock-in technology and X-chromosome inactivation machinery to make a mouse model with cone cells possessing visual pigments with different spectral sensitivities. We introduced a S308A point mutation into the Green opsin gene allele on the X-chromosome. This manipulation generated a 24 nm red-shift of absorption maximum in the cone pigment with negligible functional differences in other molecular properties. Amplitudes of responses in ERG and ganglion cell recordings of homozygotes were similar to those of wild-types, although the spectral sensitivities differed. Heterozygotes showed variable spectral sensitivities of ganglion cell responses due to the different integration of the native and the S308A cone inputs on the dendritic fields. In situ hybridization experiments showed that cone cells with respective pigments formed patch-like clusters of specific L cone-types, approximately 30 mum in diameter, which were randomly distributed in the dorsal region of the retinas. Since the patch-like clustering was arranged by X-inactivation, such clustering could be present in the peripheral retinas of New World monkeys with polymorphic L pigments, indicating that our mice would be a suitable model to study evolution of the mammalian color vision system.     

Variation in the activity of some enzymes of photorespiratory metabolism in C4 grasses

BACKGROUND AND AIMS: Photorespiration occurs in C4 plants, although rates are small compared with C3 plants. The amount of glycine decarboxylase in the bundle sheath (BS) varies among C4 grasses and is positively correlated with the granal index (ratio of the length of appressed thylakoid membranes to the total length of all thylakoid membranes) of the BS chloroplasts: C4 grasses with high granal index contained more glycine decarboxylase per unit leaf area than those with low granal index, probably reflecting the differences in O2 production from photosystem II and the potential photorespiratory capacity. Thus, it is hypothesized that the activities of peroxisomal enzymes involved in photorespiration are also correlated with the granal development. METHODS: The granal development in BS chloroplasts was investigated and activities of the photorespiratory enzymes assayed in 28 C4 grasses and seven C3 grasses. KEY RESULTS: The NADP-malic enzyme grasses were divided into two groups: one with low granal index and the other with relatively high granal index in the BS chloroplasts. Both the NAD-malic enzyme and phosphoenolpyruvate carboxykinase grasses had high granal index in the BS chloroplasts. No statistically significant differences were found in activity of hydroxypyruvate reductase between the C3 and C4 grasses, or between the C4 subtypes. The activity of glycolate oxidase and catalase were smaller in the C4 grasses than in the C3 grasses. Among the C4 subtypes, glycolate oxidase activities were significantly smaller in the NADP-malic enzyme grasses with low granal index in the BS chloroplasts, compared with in the C4 grasses with substantial grana in the BS chloroplasts. CONCLUSIONS: There is interspecies variation in glycolate oxidase activity associated with the granal development in the BS chloroplasts and the O2 production from photosystem II, which suggests different potential photorespiration capacities among C4 grasses.     

Exogenous NO suppresses flow-induced endothelium-derived NO production because of depletion of tetrahydrobiopterin

Exogenous nitric oxide (NO) suppresses endothelium-derived NO production. We were interested in determining whether this is also the case in flow-induced endothelium-derived NO production. If so, then is the mechanism because of intracellular depletion of tetrahydrobiopterin [BH4; a cofactor of NO synthase (NOS)], which results in superoxide production by uncoupled NOS? Isolated canine femoral arteries were perfused with 100 microM S-nitroso-N-acetylpenicillamine (SNAP; an NO donor) and/or 64 microM BH4. Perfusion of SNAP suppressed flow-induced NO production, which was evaluated as a change in the slope of the linear relationship between perfusion rate and NO production rate (P < 0.02 vs. control; n = 7). Subsequent BH4 perfusion returned the slope to the control level. Concomitant perfusion of SNAP and BH4 retained the control-level NO production (n = 7). Concomitant perfusion of SNAP and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; 1 mM; a membrane-permeable superoxide scavenger) also retained the control-level NO production (n = 7), whereas perfusion of Tiron after SNAP could not return the NO production to the control level (P < 0.02 vs. control; n = 7). We also found a significant decrease in BH4 concentration in the endothelial cells after SNAP perfusion. In conclusion, these results indicate that exogenous NO suppresses the flow-induced, endothelium-derived NO production by superoxide released from uncoupled NOS because of intracellular BH4 depletion.     

Functional changes in adenylyl cyclases and associated decreases in relaxation responses in mesenteric arteries from diabetic rats

To assess the functional change in adenylyl cyclases (AC) associated with the diabetic state, we investigated AC-mediated relaxations and cAMP production in mesenteric arteries from rats with streptozotocin (STZ)-induced diabetes. The relaxations induced by the water-soluble forskolin (FSK) analog NKH477, which is a putative AC5 activator, but not by the beta-adrenoceptor agonist isoproterenol (Iso) and the AC activator FSK, were reduced in intact diabetic mesenteric artery. In diabetic rats, however, Iso-, FSK-, and NKH477-induced relaxations were attenuated in the presence of inhibitors of nitric oxide synthase and cyclooxygenase. To exclude the influence of phosphodiesterase (PDE), we also examined the relaxations induced by several AC activators in the presence of 3-isobutyl-1-methylxanthine (IBMX; a PDE inhibitor). Under these conditions, the relaxation induced by Iso was greatly impaired in STZ-diabetic rats. This Iso-induced relaxation was significantly attenuated by pretreatment with SQ-22536, an AC inhibitor, in mesenteric rings from age-matched controls but not in those from STZ-diabetic rats. Under the same conditions, the relaxations induced by FSK or NKH477 were impaired in STZ-diabetic rats. Neither FSK- nor A-23187 (a Ca2+ ionophore)-induced cAMP production was significantly different between diabetics and controls. However, cAMP production induced by Iso or NKH477 was significantly impaired in diabetic mesenteric arteries. Expression of mRNAs and proteins for AC5/6 was lower in diabetic mesenteric arteries than in controls. These results suggest that AC-mediated relaxation is impaired in the STZ-diabetic rat mesenteric artery, perhaps reflecting a reduction in AC5/6 activity.     

VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis

Vascular endothelial growth factor receptor-2 (VEGFR-2) activation by VEGF-A is essential in vasculogenesis and angiogenesis. We have generated a pan-phosphorylation site map of VEGFR-2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C-terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR-2-expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T-cell-specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF-A-induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd-deficient mice, indicating a critical role of Y951-TSAd signaling in pathological angiogenesis.