Expression and subcellular localization of antiporter regulating protein OsARP in rice induced by submergence, salt and drought stresses

We examined the expression and subcellular localization of antiporter regulating protein OsARP in a submergence tolerant rice (Oryza sativa L.) cultivar FR13A. In the public databases, this protein was designated as putative Os02g0465900 protein. The cDNA containing the full-length sequence of OsARP gene was present in Gene Bank Accession no. AK071205 and this encoded 216 amino acids which had molecular mass of 25 kD. The OsARP gene was first expressed into E. coli and antibody was produced by using purified recombinant protein. The expression of OsARP protein was detected under submergence, salt and drought stresses. This protein was widely expressed in roots, shoots and leaves of rice under salt stress. To get an insight into the functional role of OsARP protein, subcellular localization was done using cell fractionation of rice leaves. Immuno-blotting of 3-day submergence rice leaves cell fractions detected the presence of OsARP protein in plasma-membrane fraction only. This indicates that OsARP is a membrane bound protein of rice which is expressed under submergence, salt and drought stresses.Key words: Drought, antiporter regulating protein, immuno-localization, rice, salinity, submergence

Identification of a Predictive Biomarker for the Beneficial Effect of Keishibukuryogan, a Kampo (Japanese Traditional) Medicine, on Patients with Climacteric Syndrome

Keishibukuryogan (KBG; Guizhi-Fuling-Wan in Chinese) is one of the Kampo (Japanese traditional) medicines used to treat patients with climacteric syndrome. KBG can be used by patients who cannot undergo hormone replacement therapy due to a history of breast cancer. We evaluated whether cytosine-adenine (CA) repeat polymorphism of the estrogen receptor β gene can be a predictor of the beneficial effect of KBG on climacteric syndrome. We also investigated the relationship between CA repeat polymorphism, the patients’ profiles, and the therapeutic effect. We found that CA was an SS, SL, or LL genotype according to the number of repeats. We studied 39 consecutive patients with climacteric disorders who took KBG for 12 weeks. The diagnosis of climacteric disorders was made on the basis of the Kupperman index. KBG significantly improved the patients’ climacteric symptoms (i.e., vasomotor symptoms in the patients with the LL genotype and melancholia in the patients with the SL genotype). No relationship between the patients’ profiles and CA repeat polymorphism was recognized. CA repeat polymorphism could thus be a potential biomarker to predict the efficacy of KBG in climacteric syndrome, and its use will help to reduce the cost of treating this syndrome by focusing the administration of KBG on those most likely to benefit from it

Effect of Zinc Acetate Dihydrate (NobelzinR) Treatment on Anemia and Taste Disorders in Patients with Chronic Kidney Disease with Hypozincemia

Some patients with chronic kidney disease (CKD) receiving hemodialysis develop erythropoietin-resistant anemia, possibly due to zinc deficiency. The frequency of zinc deficiency in CKD (stages 1-5 and 5D) and CKD improvement via zinc supplementation are not completely verified. Here 500 CKD patients (Stage 1/2, n=100; Stage 3, n=100; Stage 4, n=100, Stage n=5, 100; Stage 5D, n=100) will be recruited to determine the frequency of serum zinc deficiency at each CKD stage. Patients with serum zinc concentrations <80 μg/dL will be treated with zinc acetate dihydrate (NobelzinR) to evaluate its effects on hypozincemia, taste disturbances, and anemia

Negative Priming Under Rapid Serial Visual Presentation

Negative priming (NP) was examined under a new paradigm wherein a target and distractors were temporally separated using rapid serial visual presentation (RSVP). The results from the two experiments revealed that (a) NP was robust under RSVP, such that the responses to a target were slower when the target served as a distractor in a previous trial than when it did not; (b) NP was found regardless of whether the distractors appeared before or after the targets; and (c) NP was stronger when the distractor was more distinctive. These findings are generally similar to those on NP in the spatial search task. The implications for the processes causing NP under RSVP are discussed in the current paper

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Sphingolipid synthesis is involved in autophagy in Saccharomyces cerevisiae

In eukaryotes, autophagy is a conserved protein degradation system that degrades cytoplasmic components by encompassing them with double-membrane structures, called autophagosomes, and delivering them to the lytic compartments of vacuoles/lysosomes. Certain Atg proteins are known to be involved in autophagy, yet the identity and function of lipid molecules involved remain largely unknown. We investigated the involvement of sphingolipids in autophagy using Saccharomyces cerevisiae. Inhibiting synthesis of the simplest complex sphingolipid, inositol phosphorylceramide (IPC), resulted in reduced autophagic activities. Similar results were obtained using myriocin, an inhibitor of the first step in sphingolipid synthesis. Our results indicate that sphingolipids, especially IPC, are required for autophagy. Inhibition of sphingolipid synthesis had no effect on formation of Atg12-Atg5 or Atg8-phosphatidylethanolamine conjugates, on maturation of vacuolar proteases, or on formation of the pre-autophagosomal structure (PAS). These results suggest that sphingolipids are not involved in the cellular signaling that leads to formation of the PAS, but may be involved in the process of autophagosome formation

Unperverted synthesis of complex sphingolipids is essential for cell survival under nitrogen starvation

Changes in membrane dynamics are known to occur in cells faced with starvation. However, the functions of the major lipid components of biological membranes, sphingolipids, during the starvation response remain unclear. In this study, we found that yeast cells lacking genes encoding mannosylinositol phosphorylceramide (MIPC) synthases (csg1 csh1) underwent rapid cell death upon nitrogen starvation, but not upon carbon starvation or carbon and nitrogen starvation. Addition of NaN3 prevented the nitrogen starvation-induced cell death of the csg1 csh1 cells, indicating that energy production is required for this rapid cell death. IPC) species containing phytosphingosine. Removing Ca2+ by treating the cells with a calcium chelator or by changing the medium to a Ca2+-free medium before nitrogen starvation rescued the cells from death. Approximately half of the cells died shortly after collapse of the vacuole, whereas in the other half, morphological changes in the cytoplasm preceded vacuole disruption. Because the vacuole is the major Ca2+ storage organelle, we suggest that the vacuole is involved in the cell death either directly or indirectly. We report here that normal synthesis of complex sphingolipids is important for cell survival in nitrogen-starved medium