Arabidopsis Cor15am is a chloroplast stromal protein that has cryoprotective activity and forms oligomers

Many plants acquire increased freezing tolerance when they are exposed to nonfreezing temperatures of a certain duration. This process is known as cold acclimation and allows plants to protect themselves from freezing injury. A wide variety of polypeptides are induced during cold acclimation, among which is one encoded by COR15A in Arabidopsis (Arabidopsis thaliana). Previous studies showed that the COR15A gene encodes a small, plastid-targeted polypeptide that is processed to a mature form called Cor15am. In this study, we examined the biochemical properties and activities of Cor15am in more detail. We provide evidence that Cor15am localizes almost exclusively to the chloroplast stroma. In addition, the cold-regulated accumulation of Cor15am is affected by chloroplast functionality. Both gel-filtration chromatography and protein cross-linking reveal that Cor15am forms oligomers in the stroma of chloroplasts. Although Cor15am accumulates in response to low temperature, cold acclimation is not a prerequisite for oligomerization of Cor15am. Structural analysis suggests that Cor15am is composed of both ordered and random structures, and can stay soluble with small structural change after boiling and freeze-thaw treatments. Recombinant Cor15am exhibits in vitro cryoprotection of a freeze-labile enzyme, l-lactate dehydrogenase. Furthermore, Cor15am is capable of associating with l-lactate dehydrogenase in vitro and with potential stromal substrates in vivo. On the basis of these results, we propose that Arabidopsis Cor15am is a cryoprotective protein that forms oligomers in the chloroplast stroma, and that direct association of Cor15am with its substrates is part of its cryoprotective mechanism.     

Simple and rapid analytical method for detection of amino acids in blood using blood spot on filter paper, fast-GC/MS and isotope dilution technique

A simple and rapid method for quantitative analysis of amino acids, including valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) and phenylalanine (Phe), in whole blood has been developed using GC/MS. In this method, whole blood was collected using a filter paper technique, and a 1/8 in. blood spot punch was used for sample preparation. Amino acids were extracted from the sample, and the extracts were purified using cation-exchange resins. The isotope dilution method using ²H₈-Val, ²H₃-Leu, ²H₃-Met and ²H₅-Phe as internal standards was applied. Following propyl chloroformate derivatization, the derivatives were analyzed using fast-GC/MS. The extraction recoveries using these techniques ranged from 69.8% to 87.9%, and analysis time for each sample was approximately 26 min. Calibration curves at concentrations from 0.0 to 1666.7 μmol/l for Val, Leu, Ile and Phe and from 0.0 to 333.3 μmol/l for Met showed good linearity with regression coefficients = 1. The method detection limits for Val, Leu, Ile, Met and Phe were 24.2, 16.7, 8.7, 1.5 and 12.9 μmol/l, respectively. This method was applied to blood spot samples obtained from patients with phenylketonuria (PKU), maple syrup urine disease (MSUD), hypermethionine and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and the analysis results showed that the concentrations of amino acids that characterize these diseases were increased. These results indicate that this method provides a simple and rapid procedure for precise determination of amino acids in whole blood.     

多育赛多孢菌性鼻窦炎1例——致病菌的鉴定和体外抗真菌药物敏感性研究

目的报告国内首例多育赛多孢菌致鼻窦炎,并探讨致病菌的鉴定及其对抗真菌药物体外敏感性。方法取患者左侧上颌窦分泌物进行真菌培养和形态学鉴定,分离菌株β-球蛋白、rDNAITS序列分析确切鉴定,对分离菌进行7种抗真菌药体外药敏试验。结果根据菌株的形态学特点和基因序列结果鉴定为多育赛多孢菌。体外药敏试验显示该菌对7种抗真菌药物耐药。结论多育赛多孢菌所致的真菌病较少见,其确切鉴定靠形态学特征和基因分析。该菌株对多种抗真菌药物耐药。     

Protection against CCl4-induced injury in liver by adenovirally introduced thioredoxin gene

Antioxidation therapy is a promising strategy for treating or preventing oxidative stress-related liver diseases. The human thioredoxin (TRX) gene was inserted into an adenovirus vector (Adv-TRX), which was administered to mice. The mice were treated with 1 ml/kg CCl4 48 h after the infection. Blood samples were taken and the liver was excised 24 h after the CCl4 treatment. Serum ammonia, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were determined, and liver sections were stained with hematoxylin and eosin. RT-PCR analysis showed that the introduced TRX gene was expressed only in the liver. Adv-TRX decreased the serum ammonia, AST, and ALT levels. Hematoxylin-eosin staining indicated that the CCl4-induced injury was significantly prevented by the Adv-TRX infection. The gene delivery of TRX, which plays a central role in intracellular redox control, was shown to be effective in protecting the liver against oxidative stress-induced injury.     

Mapping QTLs influencing rice floral morphology using recombinant inbred lines derived from a cross between Oryza sativa L. and Oryza rufipogon Griff.

Theoretical and Applied Genetics -     

Genetic resources of primitive upland rice in Laos

Primitive upland cultivars (Oryza sativa L.) were collected in northern Laos. One-hundred-thirty-two cultivars were collected in upland fields at 27 sites. Morphological and physiological traits were recorded. The materials were classified intoindica and japonica types based on isozyme genotypes. We classified 106japonica, 16indica, two intermediate, and eight heterozygous cultivars. Thejaponica cultivars were characterized by glabrous hulls and sticky grains. Only two out of 16indica cultivars were glabrous. The heterozygotes were estimated to be generated by out-crosses betweenjaponica andindica cultivars in upland fields. The intermediate type would be the progeny of such heterozygotes. Higher polymorphism was found at two isozyme loci—Amp1 andEst2—among thejaponica cultivars. Genotypic frequencies differed between populations collected from upland fields along roads and along a branch of the Mekong river. Such differences would be caused by different origins of these two populations. In this report, isozymes were indicated as valuable markers to recognize the cultivars to be of independent stock.     

Software sensing for glucose concentration in industrial antibiotic fedbatch culture using fuzzy neural network

In order to control glucose concentration during fed-batch culture for antibiotic production, we applied so called “software sensor” which estimates unmeasured variable of interest from measured process variables using software. All data for analysis were collected from industrial scale cultures in a pharmaceutical company. First, we constructed an estimation model for glucose feed rate to keep glucose concentration at target value. In actual fed-batch culture, glucose concentration, was kept at relatively high and measured once a day, and the glucose feed rate until the next measurement time was determined by an expert worker based on the actual consumption rate. Fuzzy neural network (FNN) was applied to construct the estimation model. From the simulation results using this model, the average error for glucose concentration was 0.88 g/L. The FNN model was also applied for a special culture to keep glucose concentration at low level. Selecting the optimal input variables, it was possible to simulate the culture with a low glucose concentration from the data sets of relatively high glucose concentration. Next, a simulation model to estimate time course of glucose concentration during one day was constructed using the on-line measurable process variables, since glucose concentration was only measured off-line once a day. Here, the recursive fuzzy neural network (RFNN) was applied for the simulation model. As the result of the simulation, average error of RFNN model was 0.91 g/L and this model was found to be useful to supervise the fed-batch culture.     

The acceleration of reproductive aging in Nrg1flox/flox;Cyp19‐Cre female mice

Irregular menstrual cycles, reduced responses to exogenous hormonal treatments, and altered endocrine profiles (high FSH/high LH/low AMH) are observed in women with increasing age before menopause. In this study, because the granulosa cell‐specific Nrg1 knockout mice (gcNrg1KO) presented ovarian and endocrine phenotypes similar to older women, we sought to understand the mechanisms of ovarian aging and to develop a new strategy for improving fertility in older women prior to menopause. In the ovary of 6‐month‐old gcNrg1KO mice, follicular development was blocked in bilayer secondary follicles and heterogeneous cells accumulated in ovarian stroma. The heterogeneous cells in ovarian stroma were distinguished as two different types: (i) the LH receptor‐positive endocrine cells and (ii) actin‐rich fibrotic cells expressing collagen. Both the endocrine and fibrotic cells disappeared following long‐term treatment with a GnRH antagonist, indicating that the high levels of serum LH induced the survival of both cell types and the abnormal endocrine profile to reduce fertility. Moreover, follicular development to the antral stages was observed with reduced LH and the disappearance of the abnormal stromal cells. Mice treated with the GnRH antagonist regained normal, recurrent estrous cycles and continuously delivered pups for at least for 3 months. We conclude that endocrine and matrix alternations occur within the ovarian stroma with increasing age and that abolishing these alternations resets the cyclical release of LH. Thus, GnRH antagonist treatments might provide a new, noninvasive strategy for improving fertility in a subset of aging women before menopause.