Specific recognition of the collagen triple helix by chaperone HSP47: minimal structural requirement and spatial molecular orientation

The unique folding of procollagens in the endoplasmic reticulum is achieved with the assistance of procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed "flying capstan model" (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319).     

Two types of X-ray-induced radioresistance in mice: Presence of 4 dose ranges with distinct biological effects

Preirradiation with 0.05 Gy of X rays 2 months before a second exposure to a mid-lethal dose significantly enhanced the survival rate in both female and male ICR strain mice. The radioresistance was observed between 2–2.5 months after exposure to 0.05 Gy. It did not appear within 1.5 months, and disappeared after 3 months. This radioresistance was induced only by whole-body preirradiation (not by partial irradiation of the head or the trunk). On the other hand, preirradiation with 0.30 Gy as well as 0.50 Gy resulted in radioresistance 2 weeks later, but not 2 months later. The radioresistance was induced by whole-body preirradiation or partial preirradiation of the trunk. No radioresistance was evident after exposure of intermediate preirradiation doses of 0.15 and 0.20 Gy administered before 2 months and 2–5 weeks, respectively. The present and previous results show that the biological effects of ionizing radiation may be distinguished with the following four radiation dose ranges; (1) below 0.025 Gy: no radioresistance after 2 months; (2) 0.05–0.10 Gy: significant radioresistance after 2–2.5 months; (3) 0.20 Gy: no radioresistance after 2–5 weeks; and (4) 0.30–0.50 Gy or more: significant radioresistance after 2 weeks. These results conflict with previous findings of the biological effects of ionizing radiation in which the radiation hazard increases in relation to increasing accumulated doses. Some stimulation, in addition to adaptation, by low dose irradiation may have occurred.     

Differential distribution of receptors for two fucose-binding lectins in embryos and adult tissues of the mouse

Ulex europaeus agglutinin-I (UEA-I) recognizes the Fuc alpha 1----2 Gal linkage. Receptors for UEA-I were not detected in mouse embryos until the 13th day of embryo-genesis, except for their temporary expression in early trophectoderm cells. In adult mice, UEA-I receptors were detected at various sites, including cells of the digestive tracts, the bronchial epithelium, Hassall's corpuscle of the thymus, and the skin. The fucose-binding protein of Lotus tetragonolobus (FBP) is another lectin that recognizes fucosyl residues. The distribution of FBP receptors was significantly different from that of UEA-I receptors. FBP receptors were first detected in late 8-cell embryos and were expressed in the embryonic ectoderm, visceral endoderm, and trophoblastic giant cells in egg-cylinders. At later stages, the distribution of FBP receptors became restricted to certain parts of the embryo. In the adult, the distribution of FBP receptors was more restricted than that of UEA-I receptors. Particularly in embryos before the 11th day of gestation, the distribution of FBP receptors resembled that of SSEA-1, which is defined by the Gal beta 1----4(Fuc alpha 1----3) GlcNAc linkage. From the specificity of FBP, we inferred that the disappearance of SSEA-1 and FBP receptors during embryogenesis is not the result of alpha 1----2 fucosylation of the terminal galactosyl residue in the determinant. The fact that the expression of two fucose-related cell-surface markers, i.e., UEA-I receptors and SSEA-1 (or FBP receptors), is developmentally regulated in an entirely different fashion is an excellent example illustrating the precise control of differentiation-dependent alterations in cell-surface carbohydrates.     

Polygonum sachalinense alters the balance between capacities of regeneration and carboxylation of ribulose‐1,5‐bisphosphate in response to growth CO2 increment but not the nitrogen allocation within the photosynthetic apparatus

The limiting step of photosynthesis changes depending on CO₂ concentration and, in theory, photosynthetic nitrogen use efficiency at a respective CO₂ concentration is maximized if nitrogen is redistributed from non‐limiting to limiting processes. It has been shown that some plants increase the capacity of ribulose‐1,5‐bisphoshate (RuBP) regeneration (evaluated as J_max) relative to the RuBP carboxylation capacity (evaluated as V_cmax) at elevated CO₂, which is in accord with the theory. However, there is no study that tests whether this change is accompanied by redistribution of nitrogen in the photosynthetic apparatus. We raised a perennial plant, Polygonum sachalinense, at two nutrient availabilities under two CO₂ concentrations. The J_max to V_cmax ratio significantly changed with CO₂ increment but the nitrogen allocation among the photosynthetic apparatus did not respond to growth CO₂. Enzymes involved in RuBP regeneration might be more activated at elevated CO₂, leading to the higher J_max to V_cmax ratio. Our result suggests that nitrogen partitioning is not responsive to elevated CO₂ even in species that alters the balance between RuBP regeneration and carboxylation. Nitrogen partitioning seems to be conservative against changes in growth CO₂ concentration.     

Interspecific difference in the photosynthesis–nitrogen relationship: patterns,physiological causes,and ecological importance

The photosynthesis–nitrogen relationship is significantly different among species. Photosynthetic capacity per unit leaf nitrogen, termed as photosynthetic nitrogen-use efficiency (PNUE), has been considered an important leaf trait to characterise species in relation to their leaf economics, physiology, and strategy. In this review, I discuss (1) relations between PNUE and species ecology, (2) physiological causes and (3) ecological implications of the interspecific difference in PNUE. Species with a high PNUE tend to have high growth rates and occur in disturbed or high productivity habitats, while those with a low PNUE occur in stressful or low productivity habitats. PNUE is an important leaf trait that correlates with other leaf traits, such as leaf mass per area (LMA) and leaf life span, irrespective of life form, phylogeny, and biomes. Various factors are involved in the interspecific difference. In particular, nitrogen allocation within leaves and the mesophyll conductance for CO₂ diffusion are important. To produce tough leaves, plants need to allocate more biomass and nitrogen to make thick cell walls, leading to a reduction in the mesophyll conductance and in nitrogen allocation to the photosynthetic apparatus. Allocation of biomass and nitrogen to cell walls may cause the negative relationship between PNUE and LMA. Since plants cannot maximise both PNUE and leaf toughness, there is a trade-off between photosynthesis and persistence, which enables the existence of species with various leaf characteristics on the earth.     

Light interception in species with different functional groups coexisting in moorland plant communities

Competition for light is one of the most essential mechanisms affecting species composition. It has been suggested that similar light acquisition efficiency (Φ_mass, absorbed photon flux per unit aboveground mass) may contribute to species coexistence in multi-species communities. On the other hand, it is known that traits related with light acquisition vary among functional groups. We studied whether Φ_mass was similar among species with different functional groups coexisting in moorland communities. We conducted stratified clipping in midsummer when the stand biomass reached a maximum. Light partitioning among species was estimated using a model accounting for both direct and diffuse light. Evergreen species were found to have a significantly lower Φ_mass than deciduous species, which resulted from their lower absorbed photon flux per unit leaf area and lower specific leaf area. Shrubs had a smaller leaf mass fraction, but their Φ_mass was not lower than that of herbs because they had a higher leaf position due to the presence of wintering stems. Species with vertical leaves had a higher Φ_mass than those with horizontal leaves despite vertical leaves being a decided disadvantage in terms of light absorption. This higher Φ_mass was achieved by a greater leaf height in species with vertical leaves. Our results clearly demonstrate that light acquisition efficiency was different among the functional groups. However, the trend observed is not necessarily the same as that expected based on prior knowledge, suggesting that disadvantages in some traits for light acquisition efficiency are partly compensated for by other traits.     

Inhibition of the interactions of cofilin, destrin, and deoxyribonuclease I with actin by phosphoinositides

Cofilin is a widely distributed actin-modulating protein that has the ability to bind along the side of F-actin and to depolymerize F-actin in a pH-dependent manner. We found that phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) inhibited both actions of cofilin in a dose-dependent manner, while inositol 1,4,5-triphosphate (IP3), 1-oleoyl-2-acetylglycerol (OAG), phosphatidylserine (PS), or phosphatidylcholine (PC) had little or no effect on them. Gel filtration analyses showed that PIP2 bound to cofilin and thereby inhibited the binding of cofilin to G-actin. Destrin is a mammalian, pH-independent actin-depolymerizing protein. The actin-depolymerizing activity of destrin was also inhibited by PI, PIP, and PIP2, but not by IP3, OAG, PS, or PC. In addition, we found further that an actin-depolymerizing activity of bovine pancreas deoxyribonuclease I, a G-actin-sequestering protein, was inhibited by PIP and PIP2, but not by PI, IP3, OAG, PS, or PC. These results together with previous findings (Lassing, I., and Lindberg, U. (1985) Nature 314, 472-474; Janmey, P. A., and Stossel, T. P. (1987) Nature 325, 362-364) suggest that the sensitivity to polyphosphoinositides may be a common feature in vitro among actin-binding proteins that can bind to G-actin and regulate the state of actin polymerization.     

Mutational analysis of an actin-binding site of cofilin and characterization of chimeric proteins between cofilin and destrin.

Cofilin and destrin are two related low molecular weight mammalian actin-binding proteins. Cofilin is an F-actin side-binding and pH-dependent actin-depolymerizing protein, and destrin is a pH-independent actin-depolymerizing protein. We have introduced a few point mutations within an actin-binding sequence of cofilin. Biochemical analyses of these mutant proteins have clearly shown that Lys112 and Lys114 of cofilin are crucially but differently involved in its interaction with actin and phosphatidylinositol 4,5-bisphosphate. This is the first example among actin-binding proteins whose point mutations inactivate their interaction with actin in vitro. We have also made and characterized a series of chimeric proteins between cofilin and destrin to identify the regions responsible for the pH dependence and the F-actin side binding activity of cofilin. Our results suggest that a central region consisting of 42 amino acid residues and a carboxyl-terminal quarter of cofilin are both involved in regulation of the pH-dependent actin depolymerizing activity and the activity to bind along F-actin.     

Fluorescence microscopic observations of catecholamines in cultures of the sympathetic chains

Summary A histochemical technique for the demonstration of catecholamines developed by Falck et al. has been successfully applied to the sympathetic chains of rats and mice maintained in vitro. Catecholamines were localized in the nerve fibers, showing identical green fluorescence as in tissue sections of healthy rats. The cultures 8 days in vitro exhibited positive reaction in a few terminals, whereas sister cultures 1 month in vitro showed strong fluorescence reaction in thicker proximal axons and networks of nerve fibers as well. Reactivity of neuron somas became positive after 1 month of cultivation. Application of reserpine in amount of 0.00025 mg/ml for 2 hours resulted in complete disappearance of fluorescence. Furthermore, cultures of spinal ganglia from fetal rat produced no fluorescence reaction with this technique. Therefore, the reaction is specific for sympathetic nervous tissue and reliable for the differentiation of sympathetic neurons from other types of nerve cells.This work was supported by research grant NBO 3173 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service, and research grant No. 355 from the National Multiple Sclerosis Society, New York.