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The availability of inorganic phosphate (Pi) for ATP synthesis is thought to limit photosynthesis at elevated [CO2] when Pi regeneration via sucrose or starch synthesis is limited. We report here another mechanism for the occurrence of Pi-limited photosynthesis caused by insufficient capacity of chloroplast triose phosphate isomerase (cpTPI). In cpTPI-antisense transgenic rice (Oryza sativa) plants with 55%–86% reductions in cpTPI content, CO2 sensitivity of the rate of CO2 assimilation (A) decreased and even reversed at elevated [CO2]. The pool sizes of the Calvin–Benson cycle metabolites from pentose phosphates to 3-phosphoglycerate increased at elevated [CO2], whereas those of ATP decreased. These phenomena are similar to the typical symptoms of Pi-limited photosynthesis, suggesting sufficient capacity of cpTPI is necessary to prevent the occurrence of Pi-limited photosynthesis and that cpTPI content moderately affects photosynthetic capacity at elevated [CO2]. As there tended to be slight variations in the amounts of total leaf-N depending on the genotypes, relationships between A and the amounts of cpTPI were examined after these parameters were expressed per unit amount of total leaf-N (A/N and cpTPI/N, respectively). A/N at elevated [CO2] decreased linearly as cpTPI/N decreased before A/N sharply decreased, owing to further decreases in cpTPI/N. Within this linear range, decreases in cpTPI/N by 80% led to decreases up to 27% in A/N at elevated [CO2]. Thus, cpTPI function is crucial for photosynthesis at elevated [CO2].

Although N levels affect leaf photosynthetic capacity, the effects of N levels on the photochemistry of photosystems II and I (PSII and PSI, respectively) are not well-understood. In the present study, we examined this aspect in rice (Oryza sativa L. ‘Hitomebore’) plants grown under three different N levels at normal or high temperatures that can occur during rice culture and do not severely suppress photosynthesis. At both growth temperatures, the quantum efficiency of PSII [Y(II)] and the fraction of the primary quinone electron acceptor in its oxidized state were positively correlated with the amount of total leaf-N, whereas the quantum yields of non-photochemical quenching and donor-side limitation of PSI [Y(ND)] were negatively correlated with the amount of total leaf-N. These changes in PSII and PSI parameters were strongly correlated with each other. Growth temperatures scarcely affected these relationships. These results suggest that the photochemistry of PSII and PSI is coordinately regulated primarily depending on the amount of total leaf-N. When excess light energy occurs in low N-acclimated plants, oxidation of the reaction center chlorophyll of PSI is thought to be stimulated to protect PSI from excess light energy. It is also suggested that PSII and PSI normally operate at high temperature used in the present study. In addition, as the relationships between Y(II) and Y(ND) were found to be almost identical to those observed in osmotically stressed rice plants, common regulation is thought to be operative when excess light energy occurs due to different causes.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) limits the regeneration of ribulose 1,5-bisphosphate (RuBP) in the Calvin–Benson cycle. However, it does not always limit the rate of CO2 assimilation. In the present study, the effects of overproduction of GAPDH on the rate of CO2 assimilation under elevated [CO2] conditions, where the capacity for RuBP regeneration limits photosynthesis, were examined in transgenic rice (Oryza sativa). GAPDH activity was increased to 3.2- and 4.5-fold of the wild-type levels by co-overexpression of the GAPDH genes, GAPA and GAPB, respectively. In the transgenic rice plants, the rate of CO2 assimilation under elevated [CO2] conditions increased by approximately 10%, whereas that under normal and low [CO2] conditions was not affected. These results indicate that overproduction of GAPDH is effective in improving photosynthesis under elevated [CO2] conditions, although its magnitude is relatively small. By contrast, biomass production of the transgenic rice plants was not greater than that of wild-type plants under elevated [CO2] conditions, although starch content tended to increase marginally.

In mature leaves acclimated to low N levels and in senescent leaves, photosystems II and I (PSII and PSI, respectively) show typical responses to excess light energy. As CO2 assimilation is not transiently suppressed in these situations, the behavior of PSII and PSI is likely caused by endogenous biochemical changes in photosynthesis. In this study, this subject was studied in rice (Oryza sativa L.). Analysis was performed on mature and senescent leaves of control and N-deficient plants. Total leaf-N, Rubisco and chlorophyll (Chl) levels and their ratios were determined as biochemical parameters of photosynthesis. Total leaf-N, Rubisco and Chl levels decreased in the mature leaves of N-deficient plants and senescent leaves. The percentage of Rubisco-N in the total leaf-N decreased in these leaves, whereas that of Chl-N tended to remain almost constant in mature leaves but increased in senescent leaves. Changes in PSII and PSI parameters were best accounted for by the Rubisco-N percentage, strongly suggesting that the behavior of PSII and PSI is modulated depending on changes in N partitioning to Rubisco in mature leaves acclimated to low N levels and in senescent leaves. It is likely that a decrease in N partitioning to Rubisco leads to a decrease in Rubisco capacity relative to other photosynthetic capacities that inevitably generate excess light energy and that the operation of PSII and PSI is modulated in such situations.

Environmental stress increases the risk of electron accumulation in photosystem I (PSI) of chloroplasts, which can cause oxygen (O2) reduction to superoxide radicals and decreased photosynthetic ability. We used three Arabidopsis thaliana lines: wild-type (WT) and the mutants pgr5hope1 and paa1-7/pox1. These lines have different reduced states of iron/sulfur (Fe/S) signals, including Fx, FA/FB, and ferredoxin, the electron carriers at the acceptor side of PSI. In the dark, short-pulse light was repetitively illuminated to the intact leaves of the plants to provide electrons to the acceptor side of PSI. WT and pgr5hope1 plants showed full reductions of Fe/S during short-pulse light and PSI inactivation. In contrast, paa1-7/pox1 showed less reduction of Fe/S and its PSI was not inactivated. Under continuous actinic-light illumination, pgr5hope1 showed no P700 oxidation with higher Fe/S reduction due to the loss of photosynthesis control and PSI inactivation. These results indicate that the accumulation of electrons at the acceptor side of PSI may trigger the production of superoxide radicals. P700 oxidation, responsible for the robustness of photosynthetic organisms, participates in reactive oxygen species suppression by oxidizing the acceptor side of PSI.

PSI photoinhibition is usually avoided through P700 oxidation. Without this protective mechanism, excess light represents a potentially lethal threat to plants. PGR5 is suggested to be a major component of cyclic electron transport around PSI and is important for P700 oxidation in angiosperms. The known Arabidopsis PGR5 deficient mutant, pgr5-1, is incapable of P700 oxidation regulation and has been used in numerous photosynthetic studies. However, here it was revealed that pgr5-1 was a double mutant with exaggerated PSI photoinhibition. pgr5-1 significantly reduced growth compared to the newly isolated PGR5 deficient mutant, pgr5hope1. The introduction of PGR5 into pgr5-1 restored P700 oxidation regulation, but remained a pale-green phenotype, indicating that pgr5-1 had additional mutations. Both pgr5-1 and pgr5hope1 tended to cause PSI photoinhibition by excess light, but pgr5-1 exhibited an enhanced reduction in PSI activity. Introducing AT2G17240, a candidate gene for the second mutation into pgr5-1 restored the pale-green phenotype and partially restored PSI activity. Furthermore, a deficient mutant of PGRL1 complexing with PGR5 significantly reduced PSI activity in the double-deficient mutant with AT2G17240. From these results, we concluded that AT2G17240, named PSI photoprotection 1 (PTP1), played a role in PSI photoprotection, especially in PGR5/PGRL1 deficient mutants.

Fragility of photosystem I has been observed in transgenic rice plants that overproduce Rubisco activase (RCA). In this study, we examined the effects of RCA overproduction on the sensitivity of PSI to photoinhibition in three lines of plants overexpressing RCA (RCA-ox). In all the RCA-ox plants the quantum yield of PSI [Y(I)] decreased whilst in contrast the quantum yield of acceptor-side limitation of PSI [Y(NA)] increased, especially under low light conditions. In the transgenic line with the highest RCA content (RCA-ox 1), the quantum yield of PSII [Y(II)] and CO2 assimilation also decreased under low light. When leaves were exposed to high light (2000 μmol photon m−2 s−1) for 60 min, the maximal activity of PSI (Pm) drastically decreased in RCA-ox 1. These results suggested that overproduction of RCA disturbs PSI electron transport control, thus increasing the susceptibility of PSI to photoinhibition. When flavodiiron protein (FLV), which functions as a large electron sink downstream of PSI, was expressed in the RCA-ox 1 background (RCA-FLV), PSI and PSII parameters, and CO2 assimilation were recovered to wild-type levels. Thus, expression of FLV restored the robustness of PSI in RCA-ox plants.

Background Exposure to radiation during catheter ablation procedures poses a risk to the heath of both the patient and electrophysiology laboratory staff. Recently, the feasibility and effectiveness of zero‐fluoroscopy ablation have been reported. However, studies on the outcomes of zero‐fluoroscopy ablation in Japan remain limited. This study investigated the outcomes of zero‐fluoroscopy ablation for cardiac arrhythmias at a Japanese institute. Methods and Results We present a retrospective analysis of the safety, efficacy, and feasibility data from 221 consecutive patients who underwent zero‐fluoroscopy ablation. Of these patients, 181 had atrial fibrillation, 17 had paroxysmal supraventricular tachycardia, 13 had atrial tachycardia, 6 had ventricular tachycardia, and 4 had ventricular premature contractions. We performed zero‐fluoroscopy ablation using three‐dimensional electro‐anatomical mapping systems and intracardiac echocardiography imaging. Ultrasound‐guided sheath insertion was performed on all cases. Our experience includes exclusively endocardial cardiac ablations. The mean follow‐up was 24 months. The recurrence rates were 25.4% for atrial fibrillation, 5.9% for paroxysmal supraventricular tachycardia, 15.4% for atrial tachycardia, 33.3% for ventricular tachycardia, and 25% for ventricular premature contraction. Complications occurred in two patients (0.9%), and there was no occurrence of death. A fluoroscopic guide was used in three cases for the confirmation of vascular access (one case) and for complications (two cases). Conclusions Zero‐fluoroscopy ablation was routinely performed without compromising on safety and efficacy. This approach may eliminate the exposure to radiation for all individuals involved in this procedure., Zero‐fluoroscopy ablation is routinely performed without compromising safety and efficacy. This approach may eliminate the exposure to radiation for all individuals involved in this procedure.

Upon exposure to environmental stress, the primary electron donor in photosystem I (PSI), P700, is oxidized to suppress the production of reactive oxygen species that could oxidatively inactivate the function of PSI. The illumination of rice leaves with actinic light induces intrinsic fluctuations in the opening and closing of stomata, causing the net CO2 assimilation rate to fluctuate. We examined the effects of these intrinsic fluctuations on electron transport reactions. Under atmospheric O2 conditions (21 kPa), the effective quantum yield of photosystem II (PSII) (Y(II)) remained relatively high while the net CO2 assimilation rate fluctuated, which indicates the function of alternative electron flow. By contrast, under low O2 conditions (2 kPa), Y(II) fluctuated. These results suggest that photorespiration primarily drove the alternative electron flow. Photorespiration maintained the oxidation level of ferredoxin (Fd) throughout the fluctuation of the net CO2 assimilation rate. Moreover, the relative activity of photorespiration was correlated with both the oxidation level of P700 and the magnitude of the proton gradient across the thylakoid membrane in 21 kPa O2 conditions. These results show that photorespiration oxidized P700 by stimulating the proton gradient formation when CO2 assimilation was suppressed by stomatal closure.

Photorespiration coupled with CO2 assimilation is thought to act as a defense system against photoinhibition caused by osmotic stress. In the present study, we examined whether such a mechanism is operative for the protection of photosystem I (PSI) in rice (Oryza sativa L.) including transgenic plants with decreased and increased Rubisco content (RBCS-antisense and RBCS-sense plants, respectively). All plants were hydroponically grown and moderate osmotic stress was imposed using hydroponic culture solutions containing poly(ethylene glycol) (PEG) at 16% or 20% (w/v) for 2 d. In wild-type plants, the rates of CO2 assimilation (A) were significantly decreased by the PEG treatment, whereas the photorespiration activity estimated from the rates of electron transport in photosystem II (PSII) and A were not affected. The maximal quantum efficiency of PSII (F v/F m) and the maximal activity of PSI (P m) were also not affected. In RBCS-antisense plants, A and the estimated photorespiration activity were considerably lower than those in wild-type plants in the presence or absence of the PEG treatment. P m and both F v/F m and P m decreased in the 16% PEG-treated and 20% PEG-treated RBCS-antisense plants, respectively. Thus, the decrease in Rubisco content led to the photoinhibition of PSI and PSII, indicating the importance of photorespiration coupled with CO2 assimilation for the protection of PSI from moderate PEG-induced osmotic stress. It was also shown that PSI was more sensitive to osmotic stress than PSII. In the PEG-treated wild-type and RBCS-antisense plants, osmotic-stress responses of the photosynthetic electron transport reactions upstream of PSI led to the oxidation of P700, which is thought to prevent PSI from over-reduction. Although such a defense system operated, it was not sufficient for the protection of PSI in RBCS-antisense plants. In addition, there were no large differences in the parameters measured between wild-type and RBCS-sense plants, as overproduction of Rubisco did not increase photorespiration activity.

Overproduction of Rubisco did not proportionately increase the rate of CO2 assimilation (A) in rice probably because the capacity of regeneration of the substrate of Rubisco, ribulose-1,5-bisphosph...

Flavodiiron protein (FLV) mediates photoreduction of O2 to H2O. It is conserved from cyanobacteria to gymnosperms but not in angiosperms. The introduction of a moss (Physcomitrella patens) FLV (PpFLV) gene into Arabidopsis (Arabidopsis thaliana) made photosystem I (PSI) resistant to fluctuating light. Here, we used the same strategy with three rice (Oryza sativa) genotypes. PpFLV in the wild-type rice background functioned as an efficient PSI electron sink and increased resistance to PSI photodamage under fluctuating light. The introduction of PpFLV into the PGR5-RNAi mutant [defective in PROTON GRADIENT REGULATION5 (PGR5)-dependent cyclic electron transport around PSI, CET-PSI], the crr6 mutant [defective in chloroplast NAD(P)H-dehydrogenase-like complex (NDH)-dependent CET-PSI], and the PGR5-RNAi crr6 double mutant (double defective in CET-PSI activity) alleviated PSI photodamage under fluctuating light. Furthermore, PpFLV substituted for the function of PGR5- and NDH-dependent CET-PSI without competing for CO2 assimilation under constant light, as there was no difference in CO2 assimilation per Rubisco content and biomass production was recovered to the wild-type level. Thus, the exogenous FLV system could act not only as a safety valve under fluctuating light, but also generate a proton motive force for balancing the ATP/NADPH production ratio during steady-state photosynthesis.

The vacuolar lytic degradation of proteins releases free amino acids that plants can use instead of sugars for respiratory energy production. Autophagy is a major cellular process leading to the transport of proteins into the vacuole for degradation. Here, we examine the contribution of autophagy to the amino acid metabolism response to sugar starvation in mature leaves of Arabidopsis thaliana. During sugar starvation arising from the exposure of wild-type (WT) plants to darkness, autophagic transport of chloroplast stroma, which contains most of the proteins in a leaf, into the vacuolar lumen was induced within 2 d. During this time, the level of soluble proteins, primarily Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), decreased and the amount of free amino acid increased. In dark-treated autophagy-defective (atg) mutants, the decrease of soluble proteins was suppressed, which resulted in the compromised release of basic amino acids, branched-chain amino acids (BCAAs) and aromatic amino acids. The impairment of BCAA catabolic pathways in the knockout mutants of the electron transfer flavoprotein (ETF)/ETF:ubiquinone oxidoreductase (etfqo) complex and the electron donor protein isovaleryl-CoA dehydrogenase (ivdh) caused a reduced tolerance to dark treatment similar to that in the atg mutants. The enhanced accumulation of BCAAs in the ivdh and etfqo mutants during the dark treatment was reduced by additional autophagy deficiency. These results indicate that vacuolar protein degradation via autophagy serves as an adaptive response to disrupted photosynthesis by providing substrates to amino acid catabolic pathways, including BCAA catabolism mediated by IVDH and ETFQO.

It is of interest how photosynthetic electron transport (PET) reactions respond to excess light energy caused by the combination of drought stress and high temperatures. Since such information is scarcely available for photosystem I (PSI), this question was explored in rice (Oryza sativa L.) plants subjected to drought stress, using culture solutions that contain poly(ethylene glycol) at different concentrations under two day/night temperature regimes. At 27/22 &deg, C (day/night), drought stress led to the oxidation of the reaction center of the chlorophyll of PSI (P700), and also led to decreases in the quantum efficiencies of photosystem II (PSII) and PSI, and a reduction of the primary quinone electron acceptor of PSI. Such drought stress responses were wholly stimulated at 35/30 &deg, C. These parameters were strongly correlated with each other and were minimally affected by temperature. These results indicate that the drought stress responses of the respective PET reactions are closely associated with each other in the oxidization of P700 and that such responses are stimulated at high temperatures. The underlying mechanisms of these phenomena were discussed. While P700 oxidation is thought to suppress reactive oxygen species (ROS) production, PSI photoinhibition was observed under severe stress conditions, implying that P700 oxidation is not sufficient for the protection of PSI under drought stress.

In flowering plants, the tapetum, the innermost layer of the anther, provides both nutrient and lipid components to developing microspores, pollen grains, and the pollen coat. Though the programmed cell death of the tapetum is one of the most critical and sensitive steps for fertility and is affected by various environmental stresses, its regulatory mechanisms remain mostly unknown. Here we show that autophagy is required for the metabolic regulation and nutrient supply in anthers and that autophagic degradation within tapetum cells is essential for postmeiotic anther development in rice. Autophagosome-like structures and several vacuole-enclosed lipid bodies were observed in postmeiotic tapetum cells specifically at the uninucleate stage during pollen development, which were completely abolished in a retrotransposon-insertional OsATG7 (autophagy-related 7)-knockout mutant defective in autophagy, suggesting that autophagy is induced in tapetum cells. Surprisingly, the mutant showed complete sporophytic male sterility, failed to accumulate lipidic and starch components in pollen grains at the flowering stage, showed reduced pollen germination activity, and had limited anther dehiscence. Lipidomic analyses suggested impairment of editing of phosphatidylcholines and lipid desaturation in the mutant during pollen maturation. These results indicate a critical involvement of autophagy in a reproductive developmental process of rice, and shed light on the novel autophagy-mediated regulation of lipid metabolism in eukaryotic cells.

During leaf senescence, Rubisco is gradually degraded and its components are recycled within the plant. Although Rubisco can be mobilized to the vacuole by autophagy via specific autophagic bodies, the importance of this process in Rubisco degradation has not been shown directly. Here, we monitored Rubisco autophagy during leaf senescence by fusing synthetic green fluorescent protein (sGFP) or monomeric red fluorescent protein (mRFP) with Rubisco in Arabidopsis (Arabidopsis thaliana). When attached leaves were individually exposed to darkness to promote their senescence, the fluorescence of Rubisco-sGFP was observed in the vacuolar lumen as well as chloroplasts. In addition, release of free-sGFP due to the processing of Rubisco-sGFP was observed in the vacuole of individually darkened leaves. This vacuolar transfer and processing of Rubisco-sGFP was not observed in autophagy-deficient atg5 mutants. Unlike sGFP, mRFP was resistant to proteolysis in the leaf vacuole of light-grown plants. The vacuolar transfer and processing of Rubisco-mRFP was observed at an early stage of natural leaf senescence and was also obvious in leaves naturally covered by other leaves. These results indicate that autophagy contributes substantially to Rubisco degradation during natural leaf senescence as well as dark-promoted senescence.

The PGR5 (PROTON GRADIENT REGULATION 5) gene that is required for PSI cyclic electron transport in Arabidopsis was knocked down in rice (Oryza sativa). In three PGR5 knockdown (KD) lines, the PGR5 protein level was reduced to 5-8% of that in the wild type, resulting in a 50% reduction in PGRL1 (PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE 1) protein levels. In ruptured chloroplasts, ferredoxin-dependent plastoquinone reduction activity was partially impaired; the phenotype was mimicked by addition of antimycin A to wild-type chloroplasts. As occurred in the Arabidopsis pgr5 mutant, non-photochemical quenching of Chl fluorescence (NPQ) induction was impaired in the leaves, but the electron transport rate (ETR) was only mildly affected at high light intensity. The P700(+) level was reduced even at low light intensity, suggesting that the PGR5 function was severely disturbed as in the Arabidopsis pgr5 mutant and that the other alternative routes of electrons could not compensate the stromal redox balance. The amplitude of the light-dark electrochromic shift (ECS) signal (ECSt), which reflects the total size of the proton motive force in steady-state photosynthesis, was reduced by 13-25% at approximately the growth light intensity. The CO(2) fixation rate was only slightly reduced in the PGR5 KD lines. Despite the drastic reduction in NPQ and P700(+) levels, total biomass was only slightly reduced in PGR5 KD lines grown at 370 µmol photons m(-2) s(-1). These results suggest that CO(2) fixation and growth rate are very robust in the face of alterations in the fundamental reactions of photosynthesis under constant light conditions in rice.

Objective: To describe changes of intraocular pressure (IOP) in horses affected by uveitis. Procedure: Study of medical records of 9 racehorses with unilateral uveitis (4 acute and 5 chronic) where IOPs were measured using a handheld applanation tonometer. Results: At first tonometry of the 9 horses, IOPs of the affected eyes (16.5 ± 5.1 mmHg) were significantly lower than those of the contralateral eyes (23.9 ± 4.7 mmHg). Two of the acute cases had lower IOPs in the affected eyes compared to the normal eyes, and changes in the IOP were monitored during treatment. Although both cases improved clinically, IOP recovered only in 1 case. In 4 of the chronically affected cases, uveitic eyes had developed phthisis bulbi and 3 of these eyes showed lowered IOP whereas the rest had IOP equivalent to the contralateral eye. The other chronic case, which had been affected by nonulcerative keratouveitis, had lowered IOP in the affected eye for 6 months, however, IOP difference between the both eyes got smaller. Conclusions: IOP can decrease with variable extent and duration in equine uveitis patients as well as in dogs [7]. However, IOP values of the affected equine eyes in this study were often higher than those of uveitic canine eyes [7]. Minor changes of IOP in uveitic eyes could be detected more accurately by tonometry than by observing or palpating the globes. IOP difference greater than 5 mmHg in each individual was considered to be a useful indicator in diagnosis of equine uveitis. Although further study is required, tonometry is a useful method to understand equine uveitis in detail, and should be included in complete ophthalmic examination especially in cases where the problem is longstanding and/or clinical progression is minor.

Autophagy is an intracellular process leading to vacuolar or lysosomal degradation of cytoplasmic components in eukaryotes. Establishment of proper methods to monitor autophagy was a key step in uncovering its role in organisms, such as yeast (Saccharomyces cerevisiae), mammals, and Arabidopsis (Arabidopsis thaliana), in which chloroplastic proteins were found to be recycled by autophagy. Chloroplast recycling has been predicted to function in nutrient remobilization for growing organs or grain filling in cereal crops. Here, to develop our understanding of autophagy in cereals, we established monitoring methods for chloroplast autophagy in rice (Oryza sativa). We generated transgenic rice-expressing fluorescent protein (FP) OsAuTophaGy8 (OsATG8) fusions as autophagy markers. FP-ATG8 signals were delivered into the vacuolar lumen in living cells of roots and leaves mainly as vesicles corresponding to autophagic bodies. This phenomenon was not observed upon the addition of wortmannin, an inhibitor of autophagy, or in an ATG7 knockout mutant. Markers for the chloroplast stroma, stromal FP, and FP-labeled Rubisco were delivered by a type of autophagic body called the Rubisco-containing body (RCB) in the same manner. RCB production in excised leaves was suppressed by supply of external sucrose or light. The release of free FP caused by autophagy-dependent breakdown of FP-labeled Rubisco was induced during accelerated senescence in individually darkened leaves. In roots, nongreen plastids underwent both RCB-mediated and entire organelle types of autophagy. Therefore, our newly developed methods to monitor autophagy directly showed autophagic degradation of leaf chloroplasts and root plastids in rice plants and its induction during energy limitation.

Much of the nitrogen in leaves is distributed to chloroplasts, mainly in photosynthetic proteins. During leaf senescence, chloroplastic proteins, including Rubisco, are rapidly degraded, and the released nitrogen is remobilized and reused in newly developing tissues. Autophagy facilitates the degradation of intracellular components for nutrient recycling in all eukaryotes, and recent studies have revealed critical roles for autophagy in Rubisco degradation and nitrogen remobilization into seeds in Arabidopsis (Arabidopsis thaliana). Here, we examined the function of autophagy in vegetative growth and nitrogen usage in a cereal plant, rice (Oryza sativa). An autophagy-disrupted rice mutant, Osatg7-1, showed reduced biomass production and nitrogen use efficiency compared with the wild type. While Osatg7-1 showed early visible leaf senescence, the nitrogen concentration remained high in the senescent leaves. (15)N pulse chase analysis revealed suppression of nitrogen remobilization during leaf senescence in Osatg7-1. Accordingly, the reduction of nitrogen available for newly developing tissues in Osatg7-1 likely led its reduced leaf area and tillers. The limited leaf growth in Osatg7-1 decreased the photosynthetic capacity of the plant. Much of the nitrogen remaining in senescent leaves of Osatg7-1 was in soluble proteins, and the Rubisco concentration in senescing leaves of Osatg7-1 was about 2.5 times higher than in the wild type. Transmission electron micrographs showed a cytosolic fraction rich with organelles in senescent leaves of Osatg7-1. Our results suggest that autophagy contributes to efficient nitrogen remobilization at the whole-plant level by facilitating protein degradation for nitrogen recycling in senescent leaves.

Conventionally, perturbative and non-perturbative calculations are performed independently. In this paper, valleys in the configuration space in quantum mechanics are investigated as a way to treat them in a unified manner. All the known results of the interplay of them are reproduced naturally. The prescription for separating the non-perturbative contribution from the perturbative is given in terms of the analytic continuation of the valley parameter. Our method is illustrated on a new series of examples with the asymmetric double-well potential. We obtain the non-perturbative part explicitly, which leads to the prediction of the large order behavior of the perturbative series. We calculate the first 200 perturbative coefficients for a wide range of parameters and confirm the agreement with the prediction of the valley method., 13 pages, 4 eps figures, LaTeX, gzipped tar file

We study the path-integral formalism in the imaginary-time to show its validity in a case with a metastable ground state. The well-known method based on the bounce solution leads to the imaginary part of the energy even for a state that is only metastable and has a simple oscillating behavior instead of decaying. Although this has been argued to be the failure of the Euclidean formalism, we show that proper account of the global structure of the path-space leads to a valid expression for the energy spectrum, without the imaginary part. For this purpose we use the proper valley method to find a new type of instanton-like configuration, the ``valley instantons''. Although valley instantons are not the solutions of equation of motion, they have dominant contribution to the functional integration. A dilute-gas approximation for the valley instantons is shown to lead to the energy formula. This method extends the well-known imaginary-time formalism so that it can take into account the global behavior of the theory.

Path-integral approach in imaginary and complex time has been proven successful in treating the tunneling phenomena in quantum mechanics and quantum field theories. Latest developments in this field, the proper valley method in imaginary time, its application to various quantum systems, complex time formalism, asympton theory for the large order analysis of the perturbation theory, are reviewed in a self-contained manner., Comment: 100 pages, LaTeX, PTPTeX.sty, 36 eps figures, To be published in Progress of Theoretical Physics Suppliment

Objective—To examine the distribution of water in hoof wall specimens of horses via nuclear magnetic resonance (NMR) microscopy and determine changes in water distribution during hydration. Sample—4 hoof wall specimens (2 obtained from the dorsum and 1 each obtained from the lateral quarter and lateral heel regions) of the stratum medium of healthy hooves of 1 horse. Procedures—Equine hoof wall specimens were examined via NMR microscopy. Proton density–weighted 3-D images were acquired. Changes during water absorption were assessed on sequential images. Results—The inner zone of the stratum medium had higher signals than did the outer zone. Areas of high signal intensity were evident in transverse images; these corresponded to the distribution of horn tubules. During water absorption, the increase in signal intensity started at the bottom of a specimen and extended to the upper region; it maintained the localization pattern observed before hydration. The relationship between the local maximal signals in areas corresponding to the horn tubules and minimal signal intensities in areas corresponding to the intertubular horn was similar and maintained approximately a linear distribution. Conclusions and Clinical Relevance—Based on the premise that signal intensity reflects water content, hydration in the equine hoof wall during water absorption occurred concurrently in the tubules and intertubular horn, and there was maintenance of the original water gradients. This technique can be applied for the assessment of pathophysiologic changes in the hoof wall on the basis of its hydration properties.

Seedling roots display not only gravitropism but also hydrotropism, and the two tropisms interfere with one another. In Arabidopsis (Arabidopsis thaliana) roots, amyloplasts in columella cells are rapidly degraded during the hydrotropic response. Degradation of amyloplasts involved in gravisensing enhances the hydrotropic response by reducing the gravitropic response. However, the mechanism by which amyloplasts are degraded in hydrotropically responding roots remains unknown. In this study, the mechanistic aspects of the degradation of amyloplasts in columella cells during hydrotropic response were investigated by analyzing organellar morphology, cell polarity and changes in gene expression. The results showed that hydrotropic stimulation or systemic water stress caused dramatic changes in organellar form and positioning in columella cells. Specifically, the columella cells of hydrotropically responding or water-stressed roots lost polarity in the distribution of the endoplasmic reticulum (ER), and showed accelerated vacuolization and nuclear movement. Analysis of ER-localized GFP showed that ER redistributed around the developed vacuoles. Cells often showed decomposing amyloplasts in autophagosome-like structures. Both hydrotropic stimulation and water stress upregulated the expression of AtATG18a, which is required for autophagosome formation. Furthermore, analysis with GFP-AtATG8a revealed that both hydrotropic stimulation and water stress induced the formation of autophagosomes in the columella cells. In addition, expression of plastid marker, pt-GFP, in the columella cells dramatically decreased in response to both hydrotropic stimulation and water stress, but its decrease was much less in the autophagy mutant atg5. These results suggest that hydrotropic stimulation confers water stress in the roots, which triggers an autophagic response responsible for the degradation of amyloplasts in columella cells of Arabidopsis roots.

Autophagy is an intracellular process facilitating the vacuolar degradation of cytoplasmic components and is important for nutrient recycling during starvation. We previously demonstrated that chloroplasts can be partially mobilized to the vacuole by autophagy via spherical bodies named Rubisco-containing bodies (RCBs). Although chloroplasts contain approximately 80% of total leaf nitrogen and represent a major carbon and nitrogen source for new growth, the relationship between leaf nutrient status and RCB production remains unclear. We examined the effects of nutrient factors on the appearance of RCBs in leaves of transgenic Arabidopsis (Arabidopsis thaliana) expressing stroma-targeted fluorescent proteins. In excised leaves, the appearance of RCBs was suppressed by the presence of metabolic sugars, which were added externally or were produced during photosynthesis in the light. The light-mediated suppression was relieved by the inhibition of photosynthesis. During a diurnal cycle, RCB production was suppressed in leaves excised at the end of the day with high starch content. Starchless mutants phosphoglucomutase and ADP-Glc pyrophosphorylase1 produced a large number of RCBs, while starch-excess mutants starch-excess1 and maltose-excess1 produced fewer RCBs. In nitrogen-limited plants, as leaf carbohydrates were accumulated, RCB production was suppressed. We propose that there exists a close relationship between the degradation of chloroplast proteins via RCBs and leaf carbon but not nitrogen status in autophagy. We also found that the appearance of non-RCB-type autophagic bodies was not suppressed in the light and somewhat responded to nitrogen in excised leaves, unlike RCBs. These results imply that the degradation of chloroplast proteins via RCBs is specifically controlled in autophagy.

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.

Chloroplasts are the characteristic organelle of photoautoprophs. To acquire carbohydrates, the majority of leaf nitrogen is distributed to chloroplasts as photosynthetic proteins. During age-related senescence or under starvation conditions, chloroplasts become a major source of carbon and nitrogen for recycling. While bulk degradation of the cytosol and organelles must occur by autophagy in plants, the role of autophagy in chloroplast degradation is still unclear. Our recent results confirm the role of autophagy in both partial and whole chloroplast degradation, at least during promoted senescence of individually-darkened leaves.

To assess the effect of human interferon-alpha (IFNα) on shipping fever of Thoroughbred racehorses subjected to long-distance transportation, an IFNα preparation was orally administered to 48 horses three times (once daily, 3 successive days) before transportation (IFNα group). In the control group (25 horses), maltose was administered in the same way. These treatments induced no abnormal findings in Thoroughbred racehorses before transportation. Immediately after transportation, significant increases in rectal temperature were observed in both treatment groups, whereas the rectal temperature of the IFNα group tended to be lower than that of the control group. Although WBC, Fbg, and SAA immediately after transportation were significantly increased due to transportation in both groups, the extent of the increases in the IFNα group was significantly smaller than in the control group. Long-distance transportation had a relatively profound impact on Thoroughbred racehorses, which was mitigated by IFNα treatment.

DNA is known to be aggregated by metal ions including Mn(2+) ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn(2+) ions ([Mn]/[P] = 46.3) at 70 degrees C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol-chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), as transition element metal ions, were effective as to the degradation into dNMP. Mg(2+), Ca(2+), Sr(2+), and Ba(2+), as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl(-), CH(3)OO(-), and NO(3)(-) were found to increase the degradation rate. Sixty mug of the 120 mug of the starting DNA in 450 mul was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn(2+) ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH(-) ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5'-NMP, based on the H(1)NMR spectra. This prosess should prove to be a new process for the production of 5'-dNMP in addtion to the exonuclease.

DNA is well known to be aggregated by metal ions including Mn ions, however, analysis of the aggregation process from a chemical aspect, which means identification of the product yielded during the process, has not been performed yet. On determination of what kinds of degraded materials were in the supernatant obtained on centrifugation of a DNA mixture aggregated under the conditions of 10 mM Mn ions ([Mn]/[P]=46.3) at 70 degrees C for 1 h, dAMP, dCMP, dGMP, and TMP produced through self-cleavage of DNA were found in the water-soluble part. These mononucleotides were purified by HPLC using TSKgel ODS-80Ts, and identified by LC-TOF/MS. The self-cleavage was effectively occurred under the conditions of more than 5 mM Mn ions, a reaction temperature of more than 70 degrees C, a reaction time of more than 30 min, and the use of DNA with a molecular weight of more than 140 bp. The self-cleavage was affected by the molecular size of the DNA.

Objective. The information derived from central venous catheters is underused. We developed an EKG-R synchronization and averaging system to obtained distinct CVP waveforms and analyzed components of these. Methods. Twenty-five paralyzed surgical patients undergoing CVP monitoring under mechanical ventilation were studied. CVP and EKG signals were analyzed employing our system, the mean CVP and CVP at end-diastole during expiration were compared, and CVP waveform components were measured using this system. Results. CVP waveforms were clearly visualized in all patients. They showed the a peak to be 1.8+/- 0.7 mmHg, which was the highest of three peaks, and the x trough to be lower than the y trough (-1.6+/- 0.7mmHgand-0.9+/- 0.5mmHg, respectively), with a mean pulse pressure of 3.4mm Hg.The difference between the mean CVP and CVP at end-diastole during expiration was 0.58+/- 0.81 mmHg. Conclusions. The mean CVP can be used as an index of right ventricular preload in patients under mechanical ventilation with regular sinus rhythm. Our newly developed system is useful for clinical monitoring and for education in circulatory physiology.

Objective—To determine the effects of immersion in warm springwater (38° to 40°C) on autonomic nervous activity in horses. Animals—10 male Thoroughbreds. Procedure—Electrocardiograms were recorded from horses for 15 minutes during a warm springwater bath after being recorded for 15 minutes during stall rest. Variations in heart rate (HR) were evaluated from the power spectrum in terms of low frequency (LF, 0.01 to 0.07 Hz) power and high frequency (HF, 0.07 to 0.6 Hz) power as indices of autonomic nervous activity. Results—Mean (±SE) HR during stall rest and immersion in warm springwater was 31.1 ± 1.7 and 30.3 ± 1.0 beat/min, respectively. No significant difference was found between the HR recorded during stall rest and that recorded during immersion in warm springwater. The HF power significantly increased from 1,361 ± 466 milliseconds2 during stall rest to 2,344 ± 720 milliseconds2 during immersion in warm springwater. The LF power during stall rest and immersion in warm springwater was 3,847 ± 663 and 5,120 ± 1,094 milliseconds2, respectively, and were not significantly different from each other. Similarly, the LF:HF ratio did not change during immersion in warm springwater. The frequency of second-degree atrioventricular block, which was observed in 2 horses, increased during immersion in warm springwater, compared with during stall rest. Conclusions and Clinical Relevance—Increases in HF power indicates that the parasympathetic nervous activity in horses increases during immersion in warm springwater. Thus, immersion in warm springwater may provide a means of relaxation for horses. ( Am J Vet Res 2003;64:1482–1485)

The ZNF219 gene is a member of the Kruppel-like zinc finger gene family that is involved in a diverse range of biological processes. The ZNF219 gene encodes a 77-kDa nuclear protein containing nine sets of C 2 H 2 zinc finger structures. By using a random oligonucleotide selection assay and the electromobility gel shift assay, we have revealed that the ZNF219 protein recognizes two copies of CCCCCA. The DNA binding core element is CCCCC. 3' flanking A residues enhance binding of the ZNF219 protein. Use of the various truncated ZNF219 constructs demonstrated that zinc finger 1 to 3 or zinc finger 5 and 6 domains are sufficient to allow specific DNA binding. Both domains independently recognized the same consensus sequence, CCCCCA. Proteins expressed from human cDNA clones KIAA0390 and KIAA0222, which have partial similarities to ZNF219, also showed specific binding to the same core DNA sequence. Potential ZNF219 binding sites were found in the HMGN1 promoter. To examine the function of ZNF219 in the modulation of transcription, we constructed Gal4 DNA binding domain (DBD)/ZNF219 fusion proteins and demonstrated that ZNF219 functioned as a transcriptional repressor for the HMGN1 promoter. Experiments with the truncated ZNF219 constructs suggest that the proline-rich sequence (226-272 a.a., proline content 49%) was responsible for part of the observed repression. These findings provide us with an important start point in our understanding of the functional role of ZNF219 in vivo.