Influences of forested watershed conditions on fluctuations in stream water temperature with special reference to watershed area and forest type

In order to gain insight into the effect of watershed conditions on fluctuations in stream water temperature, we statistically analyzed water temperature data for 1 year, using root mean square (Rms) and harmonic (A Amplitude, φ delay time) methods. The average values of delay time (days) between air and water temperatures (T _a and T _w) of small (< 0.5 ha), medium (0.5–100 ha) and large (> 100 ha) watersheds were 4.53 ± 0.82 days, 11.83 ± 3.88 days and 4.45 ± 1.52 days, respectively. Fluctuations in stream water temperature expressed by Rms (Rms T _w/Rms T _a) and harmonic methods (A −T _w/A −T _a) in the medium-sized watersheds with moderate slope gradients were 0.37 ± 0.09 and 0.56 ± 0.14, respectively. These values increased in the larger watersheds with low slope gradients, including five large rivers covered by various landscapes, with their averages of 0.53 ± 0.09 and 0.78 ± 0.09, respectively, indicating the influences of solar radiation and heat transfer processes. In the smaller watersheds with high slope gradients, these values were 0.73 ± 0.02 and 0.87 ± 0.03, respectively, suggesting that shorter passage time affected water temperatures. With respect to forest type, these values at badly managed hinoki forest watersheds (0.45 ± 0.04 and 0.73 ± 0.07) were larger than those at broadleaf forest (0.34 ± 0.04 and 0.51 ± 0.12) and well-managed hinoki forest (0.33 ± 0.04 and 0.51 ± 0.07) watersheds, indicating different proportions of flow paths.     

Chloroplasts autophagy during senescence of individually darkened leaves

We recently reported that autophagy plays a role in chloroplasts degradation in individually-darkened senescing leaves. Chloroplasts contain approximately 80% of total leaf nitrogen, mainly as photosynthetic proteins, predominantly ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco). During leaf senescence, chloroplast proteins are degraded as a major source of nitrogen for new growth. Concomitantly, while decreasing in size, chloroplasts undergo transformation to non-photosynthetic gerontoplasts. Likewise, over time the population of chloroplasts (gerontoplasts) in mesophyll cells also decreases. While bulk degradation of the cytosol and organelles is mediated by autophagy, the role of chloroplast degradation is still unclear. In our latest study, we darkened individual leaves to observe chloroplast autophagy during accelerated senescence. At the end of the treatment period chloroplasts were much smaller in wild-type than in the autophagy defective mutant, atg4a4b-1, with the number of chloroplasts decreasing only in wild-type. Visualizing the chloroplast fractions accumulated in the vacuole, we concluded that chloroplasts were degraded by two different pathways, one was partial degradation by small vesicles containing only stromal-component (Rubisco containing bodies; RCBs) and the other was whole chloroplast degradation. Together, these pathways may explain the morphological attenuation of chloroplasts during leaf senescence and describe the fate of chloroplasts.Key words: Arabidopsis, autophagy, chloroplast, dark treatment, leaf senescence, nutrients recyclingThe most abundant chloroplast protein is Rubisco, comprising approximately 50% of the soluble protein.¹ The amount of Rubisco decreases rapidly in the early phase of leaf senescence, and more slowly in the later phase. During senescence, chloroplasts gradually shrink and their numbers gradually decrease in mesophyll cells.^2,3 During leaf senescence, leaves lose approximately 75% of their Rubisco, while chloroplast numbers decrease by only about 15%.⁴ Previous studies showed chloroplasts localized within the central vacuole by electron microscopy, indicating chloroplast degradation in the highly hydrolytic vacuole.⁵ However, there was no direct evidence showing translocation of chloroplasts from the cytosol to the vacuole, and the mechanism of transportation was also unclear.Recent reverse genetic approaches are helping to elucidate the autophagy system in plants, which has a similar molecular mechanism as in yeast.^6–11 In Arabidopsis (Arabidopsis thaliana), atg mutants have phenotypically accelerated leaf senescence, insufficient root elongation in nutrient starvation condition and reduced seeds yields, therefore, autophagy is considered to be important for nutrient recycling especially nutrient starvation and senescence in plants.¹²In Arabidopsis, individually darkened rosette leaves (IDLs) exhibit enhanced senescence.¹³ Appling IDLs treatment as an experimental model of leaf senescence, we recently demonstrated that chloroplasts are degraded in two different pathways by autophagy, one for RCBs,^14,15 and one for whole chloroplast.¹⁶ Darkened leaves became pale in 3 to 5 days treatment, while illuminated parts normally grow in both wild-type and autophagy defective mutant, atg4a4b-1. Furthermore, genes specifically expressed during senescence, SAG12 and SEN1, were rapidly upregulated, meanwhile, photosynthetic genes, such as RBCS2B and CAB2B, were gradually downregulated. All analyzed ATG genes were also upregulated under IDL treatment, which suggests that autophagy is important in IDL senescence. It has been reported that approximately three quarter genes of upregulated in IDL were also upregulated in naturally senescing leaves, including the ATG genes.¹⁷ This suggests that the autophagy pathways used in IDLs are also used in naturally senescing leaves.Over the 5 day treatment period, chloroplasts of wild-type IDL shrink to approximately one third their original size. In atg4a4b-1, by contrast, chloroplasts shrinkage occurred immediately after the start of IDL treatment after which no further shrinkage was noted. While the shrunk chloroplasts in fixed cells of wild-type were still smooth and round, while wrinkly chloroplasts were observed in atg4a4b-1. At same time, in the living mesophyll cells of wild-type IDL, RCBs accumulated in the vacuole (Fig 1B). The shrinkage of chloroplasts may be due to the consumption of the chloroplast envelope by RCB formation. Immunological quantification of inner and outer envelope proteins might confirm this hypothesis. The chloroplast number was also gradually decreased in IDL of wild-type plants, but no decline in chloroplast number was noted in atg4a4b-1. Chloroplasts exhibiting chlorophyll auto-fluorescence were found in the vacuole of wild-type IDLs, but not in atg4a4b-1 IDLs. These results show that whole chloroplast degradation is also performed by autophagy. However, the transport pathway of whole chloroplasts into the vacuole remains unclear. The chloroplast, even in its shrunken state, is a large organelle, and the autophagosome, the carrier bodies of autophagy, which usually target small spherical organelles like mitochondria and peroxisomes, may be incapable of isolating large organelles. In the yeast autophagy system, specific cellular organelles and fractions are also transported via vacuolar membrane invagination using the microautophagy system.¹⁸ RCB uptake into the vacuole is termed macroautophagy, while larger organelles, such as chloroplasts, are engulfed in a process known as microautophagy. Whether there exists a molecular difference between these processes, or whether this is an arbitrary division based solely on the size of the consumed body is unclear.Open in a separate windowFigure 1Visualization of stroma-targeted DsRed and chlorophyll autofluorescence in living mesophyll cells of wild-type plants by laser-scanning confocal microscopy. A excised control leaf (A, Light) and an individually darkened leaf (B, IDL) from plants grown under 14 h-photoperiod condition and a leaf from whole-plant darkened condition (WD, C) for 5days were incubated with 1 µM concanamycin A in 10 mM MES-NaOH (pH 5.5) at 23C° for 20 h in darkness. Stroma-targeted DsRed appears green and chlorophyll fluorescence appears red. In merged images, overlap of DsRed and chlorophyll fluorescence appears yellow. Small vesicles with stromal-targeted DsRed, i.e. RCBs, can be found in the vacuole (A, B). In IDL (B), massive accumulation of stroma-targeted DsRed is entirely seen in the vacuolar lumen and chloroplasts losing DsRed fluorescence are found in some cells. Bars = 50 µm.Whole darkened plants exhibit retarded leaf aging, in contrast to the accelerated senescence in IDLs.¹³ Whole darkened plants suppress leaf senescence with the leaves retaining green color. After 5 days, in the mesophyll cells of whole darkened plants, any translocation of chloroplast components, stroma-targeted DsRed, RCBs, and whole chloroplasts, into the vacuole could hardly be detected (Fig. 1C). This suggests that autophagy is not induced by darkness alone, and is associated closely with senescence. ATG genes were downregulated in the whole darkened wild-type plants less than control plants during the treatment. Previous studies have shown that following about 5 day period of whole plant darkening, atg mutants lose their ability to protect themselves against photo-damage.⁷ Upon return to the light, these plant quickly undergo terminal photo-bleaching.Concentrations of chlorophyll, soluble protein, leaf nitrogen and Rubisco rapidly declined under IDL condition of both wild-type and atg4a4b-1. Considering the accumulated fluorescence of stroma-targeted Ds-Red in the vacuole and autophagy dependent size shrinkage of chloroplasts in IDL, in wild-type plants RCB autophagy appear to be responsible for a sizable proportion of chloroplast protein degradation. In atg4a4b-1 which cannot form RCBs, alternative degradation pathways must be upregulated, with chloroplast proteases the most likely candidates. Intriguingly, the decrease in Rubisco concentration proceeds at the almost identical rates in both wild-type and atg4a4b-1 plants, despite the different degradation pathways. It seems likely that the rate of Rubisco degradation may be regulated at an early step in the degradation pathway, by some, as yet unknown, factors.Chloroplasts appear to have the ability to control their volume during cell division, dividing and increasing their density up to the certain level,¹⁹ and transferring their cellular components between them via stromules.²⁰ How chloroplasts are able to regulate their volume remains unclear, but it seems likely that chloroplasts grow and divide, like any other bacteria, as long as sufficient resources remain in the environment, in this case the cell. Total chloroplast volume, therefore, may be limited by the availability of carbon, nitrogen, or other nutrients in the cell during leaf emergence. Chloroplasts may be also able to reduce and control their volumes during leaf senescence via multiple degradation pathways. Our next goal is to estimate the contribution of both RCBs and whole chloroplasts autophagy in chloroplast protein degradation during natural leaf senescence. Further investigations are required for understanding the specific molecular mechanisms of RCB production and whole chloroplast degradation.     

Three new species of bipolar budding yeasts of the genus Hanseniaspora and its anamorph Kloeckera isolated in Thailand

In the course of a survey of yeast biodiversity in the natural substrates in Thailand, eight strains were found to represent three hitherto undescribed species of Hanseniaspora/Kloeckera . They were isolated from insect frass, flower, lichen, rotted fruit and rotted wood. Based on the morphological and physiological characteristics, and sequences of D1/D2 domain, six strains represent a single species of the genus Hanseniaspora , described as Hanseniaspora thailandica sp. nov. (type BCC 14938^T=NBRC 104216^T=CBS 10841^T), and another strain as Hanseniaspora singularis sp. nov. (type BCC 15001^T=NBRC 104214^T=CBS 10840^T). A further strain, which belongs to Kloeckera and does not produce ascospores, is described as Kloeckera hatyaiensis sp. nov. (type BCC 14939^T=NBRC 104215^T=CBS 10842^T). Strains belonging to H. thailandica sp. nov. differed by 17–19 nucleotide substitutions from Hanseniaspora meyeri , the closest species. DNA reassociation between the two taxa showed 30–48% relatedness. Kloeckera hatyaiensis sp. nov. and H. singularis sp. nov. differed by eight and 16 nucleotide substitutions with one gap from the nearest species, Hanseniaspora clermontiae and Hanseniaspora valbyensis , respectively.     

Phenylethanoid oligoglycosides and acylated oligosugars with vasorelaxant activity from Cistanche tubulosa

The methanolic extract from the dried stems of Cistanche tubulosa (Schrenk) R. Wight was found to show an inhibitory effect on contractions induced by noradrenaline in isolated rat aortic strips. From the extract, new phenylethanoid oligoglycoside constituents, kankanosides F and G, and an acylated oligosugar, kankanose, were isolated together with 14 known compounds. The structures of these new compounds were determined on the basis of their chemical and physicochemical evidence. In addition, principal constituents, kankanoside F, kankanose, echinacoside, acteoside, and cistanoside F, showed vasorelaxant activity, and several structural requirements for the activity were clarified.     

Inhibitory effects of coumarin and acetylene constituents from the roots of Angelica furcijuga on D-galactosamine/lipopolysaccharide-induced liver injury in mice and on nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages

The methanolic extract (200 mg/kg, p.o. and i.p.), principal coumarin constituents (isoepoxypteryxin, anomalin, and praeroside IV), and a polyacetylene constituent (falcarindiol) (25 mg/kg, i.p.) from the roots of Angelica furcijuga protected the liver injury induced by D-galactosamine (D-GalN)/lipopolysaccharide (LPS) in mice. In in vitro experiments, coumarin constituents (hyuganins A-D, anomalin, pteryxin, isopteryxin, and suksdorfin) and polyacetylene constituents [(-)-falcarinol and falcarindiol] substantially inhibited LPS-induced NO and/or TNF-alpha production in mouse peritoneal macrophages, and isoepoxypteryxin inhibited D-GalN-induced cytotoxicity in primary cultured rat hepatocytes. Furthermore, hyuganin A, anomalin, and isopteryxin inhibited the decrease in cell viability by TNF-alpha in L929 cells.     

Cloning and expression of medaka cholesterol side chain cleavage cytochrome P450 during gonadal development

Cholesterol side chain cleavage cytochrome P450 (P450scc, Cyp11a) is responsible for the first step in steroidogenesis, catalyzing the conversion of cholesterol to prognenolone. To investigate the differentiation of steroid‐producing cells and the function of sex steroids during gonadal differentiation in the teleost fish, medaka (Oryzias latipes), we isolated the full length cDNA of medaka P450scc and analyzed the expression pattern of P450scc mRNA during gonadal development using in situ hybridization. At hatching, and just after the initiation of morphological sex differentiation, we did not detect any P450scc expression in both sexes. In male gonads, expression of P450scc was detected in the interstitial somatic cells 15 days after hatching following the formation of the seminiferous tubule precursor, and was maintained in the interstitial somatic cells throughout testicular development. In the female gonad, expression of P450scc was initially detected in interstitial somatic cells 5 days after hatching. Subsequently, the expression of P450scc was continuously detected in the interstitial somatic cells of the developing ovary. This expression pattern of P450scc differed from that of female specific steroidogenic enzyme P450arom. Both P450scc and P450arom expressing cells, only P450scc expressing cells, and only P450arom expressing cells were observed. Our results suggest that expression of steroidogenic enzymes is regulated by various mechanisms during ovarian development.     

A novel ryanodine receptor expressed in pancreatic islets by alternative splicing from type 2 ryanodine receptor gene

Cyclic ADP-ribose (cADPR), a potent Ca²⁺ mobilizing intracellular messenger synthesized by CD38, regulates the opening of ryanodine receptors (RyRs). Increases in intracellular Ca²⁺ concentrations in pancreatic islets, resulting from Ca²⁺ mobilization from RyRs as well as Ca²⁺ influx from extracellular sources, are important in insulin secretion by glucose. In the present study, by screening a rat islet cDNA library, we isolated a novel RyR cDNA (the islet-type RyR), which is generated from the RyR2 gene by alternative splicing of exons 4 and 75. When the expression vectors for the islet-type and the authentic RyRs were transfected into HEK293 cells, the islet-type RyR2 as well as the authentic one showed high affinity [³H]ryanodine binding. Intracellular Ca²⁺ release in the islet-type RyR2-transfected cells was enhanced in the presence of cADPR but not in the authentic RyR2-transfected cells. The islet-type RyR2 mRNA was expressed in a variety of tissues such as in pancreatic islets, cerebrum, and cerebellum, whereas the authentic RyR2 mRNA was predominantly expressed in heart and aorta. These results suggest that the islet-type RyR2 may be an intracellular target for cADPR signaling.     

Role of Phosphatidylinositol 3-Kinase in Friend Spleen Focus-Forming Virus-Induced Erythroid Disease

Infection of erythroid cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia in mice due to expression of its unique envelope glycoprotein, gp55. Erythroid cells expressing SFFV gp55 proliferate in the absence of their normal regulator, erythropoietin (Epo), because of interaction of the viral envelope protein with the erythropoietin receptor and a short form of the receptor tyrosine kinase Stk (sf-Stk), leading to constitutive activation of several signal transduction pathways. Our previous in vitro studies showed that phosphatidylinositol 3-kinase (PI3-kinase) is activated in SFFV-infected cells and is important in mediating the biological effects of the virus. To determine the role of PI3-kinase in SFFV-induced disease, mice deficient in the p85α regulatory subunit of class IA PI3-kinase were inoculated with different strains of SFFV. We observed that p85α status determined the extent of erythroid hyperplasia induced by the sf-Stk-dependent viruses SFFV-P (polycythemia-inducing strain of SFFV) and SFFV-A (anemia-inducing strain of SFFV) but not by the sf-Stk-independent SFFV variant BB6. Our data also indicate that p85α status determines the response of mice to stress erythropoiesis, consistent with a previous report showing that SFFV uses a stress erythropoiesis pathway to induce erythroleukemia. We further showed that sf-Stk interacts with p85α and that this interaction depends upon sf-Stk kinase activity and tyrosine 436 in the multifunctional docking site. Pharmacological inhibition of PI3-kinase blocked proliferation of primary erythroleukemia cells from SFFV-infected mice and the erythroleukemia cell lines derived from them. These results indicate that p85α may regulate sf-Stk-dependent erythroid proliferation induced by SFFV as well as stress-induced erythroid hyperplasia.The Friend spleen focus-forming virus (SFFV) is a highly pathogenic retrovirus that induces rapid erythroblastosis in susceptible strains of mice (for a review, see reference 42). Friend SFFV is a replication-defective virus with deletions in its env gene, giving rise to a unique glycoprotein, SFFV gp55. This unique glycoprotein confers pathogenicity to the virus; a vector encoding SFFV gp55 alone is sufficient to induce erythroblastosis in susceptible strains of mice (49). The Fv-2 gene encodes one of the key susceptibility factors for SFFV-induced erythroid disease (18, 37), as follows: the receptor tyrosine kinase Stk/RON, a member of the Met family of receptor tyrosine kinases (11-12). Susceptibility to SFFV-induced disease is associated with expression of a short form of the receptor tyrosine kinase Stk, termed sf-Stk, that is transcribed from an internal promoter within the Stk gene of Fv-2-susceptible (Fv-2^ss) mice but not Fv-2-resistant (Fv-2^rr) mice (37) and is abundantly expressed in erythroid cells (11). Infection of erythroid cells with the polycythemia-inducing strain of SFFV (SFFV-P) induces erythropoietin (Epo)-independent proliferation and differentiation, whereas erythroid cells infected with the anemia-inducing strain of SFFV (SFFV-A) proliferate in the absence of Epo but still require Epo for differentiation (42). Previous studies demonstrated that this Epo-independent erythroblastosis is due to the cell surface interaction of the SFFV envelope protein with the Epo receptor (EpoR) and sf-Stk (31). While interaction with the EpoR appears to be responsible mainly for the induction of Epo-independent differentiation (52), Epo-independent erythroid cell proliferation depends upon activation of sf-Stk. We recently demonstrated that sf-Stk covalently interacts with SFFV-P gp55 in hematopoietic cells that express the EpoR and that this interaction induces sf-Stk activation (31). Furthermore, exogenous expression of sf-Stk, but not a kinase-inactive mutant of sf-Stk, in bone marrow cells from sf-Stk null mice can restore Epo-independent erythroid colony formation in response to SFFV infection (5, 41). Thus, the SFFV envelope glycoprotein induces Epo-independent proliferation of erythroid cells mainly by activating sf-Stk. While sf-Stk is a key susceptibility factor for erythroblastosis induced by both SFFV-P and SFFV-A (18), it is not required for the induction of erythroblastosis by the SFFV mutant BB6, which encodes an envelope glycoprotein, gp42, that is deleted in the membrane-proximal extracellular domain (19) and does not induce sf-Stk activation (31). gp42 of SFFV-BB6 appears to exert its biological effects on erythroid cells by efficiently interacting with the EpoR (9). Compared with wild-type SFFV, SFFV-BB6 causes a relatively indolent and slowly developing disease in mice (19).A number of signaling pathways normally activated in erythroid cells after erythropoietin (Epo) binds to its cell surface receptor (40) are constitutively activated in erythroid cells infected with SFFV. These include JAK/STAT, Ras/Raf/mitogen-activated protein kinase (MAPK), Jun N-terminal kinase, and the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathways (24, 25, 28-30, 32). SFFV gp55 is thought to activate these pathways by interacting with either the EpoR or sf-Stk (17, 31, 43). In several in vitro systems, class IA PI3-kinase has been shown to be activated by Epo through the EpoR (8, 20, 21) or by SFFV through sf-Stk (5, 14). We and others have shown that the PI3-kinase pathway is important for the induction of Epo independence by SFFV (5, 29). The class IA subclass of PI3-kinase is a heterodimer comprising the p110 (α, β, δ) catalytic unit and one of five regulatory subunits (85α, p55α, p50α, 85β, and 55γ) (15). The first 3 regulatory subunits are all splice variants of the same gene (pik3r1). Deletion of pik3r1, which encodes p85α, p55α, and p50α, is lethal (6, 7), and these regulatory subunits of PI3-kinase are required for normal murine fetal erythropoiesis in mice (10).To determine the role of p85α in SFFV-induced erythroleukemia, we used a distinct nonlethal pik3r1 knockout mouse which lacks only the p85α regulatory subunit of PI3-kinase (45, 47), allowing the study of SFFV-induced erythroleukemia in adult mice. Our results indicate that p85α regulates SFFV-induced erythroid hyperplasia induced in vivo by sf-Stk-dependent, but not sf-Stk-independent, isolates of the virus as well as stress-induced erythropoiesis and suggest that this regulation may occur through the interaction of sf-Stk with p85α.