Cold Induction Research Articles

Importance and Determinants of Induction of Cold-Induced DEAD RNA Helicase in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Thermococcus kodakarensis, which grows optimally at 85°C, expresses cold stress-inducible DEAD box RNA helicase (Tk-deaD) when shifted to 60°C. A DDA1 deletion (ΔTk-deaD) mutant exhibited decreased cell growth, and cells underwent lysis at 60°C in nutrient broth in the absence of elemental sulfur. In contrast, cells in medium containing elemental sulfur at 60°C did not undergo lysis, suggesting that Tk-deaD is necessary for cell growth in sulfur-free medium. To identify the element responsible for the cold response, a pTKR expression probe plasmid was constructed using thermostable catalase from Pyrobaculum calidifontis as a reporter. The plasmid pTKRD, which contained the transcription factor B recognition element, TATA region, and Shine-Dalgarno (SD) region, including the initiation codon of the Tk-deaD gene, exhibited cold inducibility. We also constructed a series of deletion and chimeric constructs with the glutamate dehydrogenase (gdh) promoter, whose expression is constitutive independent of culture temperatures and catalase expression. Reporter assay experiments indicated that the regulatory element is located in the region between the SD region and the initiation codon (ATG). Nucleotide sequences in the upstream regions of Tk-deaD and gdh were compared and revealed a five-adenosine (AAAAA) sequence between SD and ATG of Tk-deaD that was not present in gdh. Replacement of the repeated adenosine sequence with other sequences revealed that the AAAAA sequence is important for cold induction. This sequence-specific mechanism is unique and is one that has not been identified in other known cold-inducible genes.

Roles of CAMTA transcription factors and salicylic acid in configuring the low‐temperature transcriptome and freezing tolerance of Arabidopsis

Previous studies in Arabidopsis thaliana established roles for CALMODULIN BINDING TRANSCRIPTION ACTIVATOR3 (CAMTA3) in the rapid cold induction of CRT/DRE BINDING FACTOR (CBF) genes CBF1 and CBF2, and the repression of salicylic acid (SA) biosynthesis at warm temperature. Here we show that CAMTA1 and CAMTA2 work in concert with CAMTA3 at low temperature (4°C) to induce peak transcript levels of CBF1, CBF2 and CBF3 at 2h, contribute to up-regulation of approximately 15% of the genes induced at 24h, most of which fall outside the CBF pathway, and increase plant freezing tolerance. In addition, CAMTA1, CAMTA2 and CAMTA3 function together to inhibit SA biosynthesis at warm temperature (22°C). However, SA levels increase in Arabidopsis plants that are exposed to low temperature for more than 1week. We show that this chilling-induced SA biosynthesis proceeds through the isochorismate synthase (ICS) pathway, with cold induction of ICS1 (which encodes ICS), and two genes encoding transcription factors that positively regulate ICS1 - CBP60g and SARD1 -, paralleling SA accumulation. The three CAMTA proteins effectively repress the accumulation of ICS1, CBP60g and SARD1 transcripts at warm temperature but not at low temperature. This impairment of CAMTA function may involve post-transcriptional regulation, as CAMTA transcript levels did not decrease at low temperature. Salicylic acid biosynthesis at low temperature did not contribute to freezing tolerance, but had a major role in configuring the transcriptome, including the induction of 'defense response' genes, suggesting the possible existence of a pre-emptive defense strategy programmed by prolonged chilling temperatures.

Comparison of long-term up-regulated genes during induction of freezing tolerance by cold and ABA in bromegrass cell cultures revealed by microarray analyses

A suspension culture of bromegrass (Bromus inermis Leyss cv. Manchar) provides a unique system for studying cold hardiness mechanism, where freezing tolerance can be independently induced by two different stimuli, cold and exogenous abscisic acid (ABA). Freezing tolerance was induced in bromegrass cells by exposure to low temperatures for 7 days (LT50: −8.1 °C) and also by incubation with ABA for 7 days at 25 °C (LT50: −12.3 °C) compared to the control cells grown without ABA at 25 °C (LT50: −3.9 °C). To characterize freezing tolerance mechanisms involved in both systems, bromegrass genes up-regulated after 7 days of low temperature or ABA treatment were analyzed using rice cDNA microarrays. In total, 300 and 479 clones were identified as cold- and ABA-inducible genes, respectively. Among them, 149 clones were induced both by cold and ABA treatments. Several genes were newly found to be cold-inducible from our microarray results. The increased expression of 8 selected genes in the microarray results was confirmed by Northern blot analyses. Northern blot analyses of three of these genes during 13 days of exposure to 4 °C confirmed a gradually increased and long-term up-regulated expression during cold acclimation. In agreement with physiological and protein studies, these two cold hardiness induction systems exhibited fairly different transcriptome profiles: cold stress was characterized by triggering numerous genes involved in protein degradation/synthesis and RNA maintenance in addition to cold stress-related genes whilst ABA treatment was characterized by inducing numerous genes involved in seed formation and functional genes related to biotic and abiotic stresses.

파이로그린공정 희토류폐기물 유리화 타당성 연구

파이로그린공정의 염폐기물처리과정에서 발생되는 주요 산화물 형태의 폐기물에는 희토류폐기물이 있으며 주요 구성 핵종은 Y, La, Ce, Pr, Nd, Sm, Eu, Gd 등 8종이다. 최종적인 희토류폐기물의 형태는 산화물 형태로 발생된다. 본 연구에서는 붕규산 유리계 내에서 희토류 산화물의 유리화 타당성을 평가 하기 위하여 6종의 유리조성을 개발하였다. 희토류 8핵종 혼합에 대한 solubility는 <TEX>$1,200^{\circ}C$</TEX>에서 25wt% 미만, <TEX>$1,300^{\circ}C$</TEX>에서 30wt% 미만 waste loading으로 온도 상승에 따라 증가하는 것으로 나타났으며 liquidus temperature는 균질한 유리가 형성된 20wt% waste loading에서 <TEX>$950^{\circ}C$</TEX> 이하로 평가되었다. 희토류 산화물의 유리매질 내 solubility 이상에서는 희토류-oxide-silicate 결정이 생성된 유리세라믹을 이차상으로 형성하였으며 20~25wt% waste loading의 표면균질성이 양호한 유리는 용융온도 <TEX>$1,200{\sim}1,300^{\circ}C$</TEX> 범위에서 점도 100 poise 이하, 전기전도도 1 S/cm 이상으로 유도가열식 저온용융로설비에서의 운전 용이성이 매우 양호한 것으로 평가되었다. 개발된 유리조성에 대한 기타 물리 화학적 특성 평가를 위한 실험들이 향후 수행될 예정이다. The rare earth oxide wastes consisting of major 8 nuclides Y, La, Ce, Pr, Nd, Sm, Eu and Gd, are generated during the salt waste treatment of PyroGreen process. The final form of the rare earth is generated as the oxide state. In this study, six candidate glasses were developed to evaluate the feasibility for vitrifying the rare earth oxide wastes within the borosilicate glass system. The solubilities of the mixture of the rare earth oxide waste showed less than 25wt% at <TEX>$1,200^{\circ}C$</TEX>, less than 30wt% at <TEX>$1,300^{\circ}C$</TEX>, respectively. It means that solubility is increased with the temperature increment. The liquidus temperature of the homogeneous glass with 20wt% waste loading was determined as less than <TEX>$950^{\circ}C$</TEX>. In more than solubility of rare earth oxides glass, formation of rare earth-oxide-silicate crystal in glass-ceramic occurred as the secondary phase. As their viscosity at melting temperature <TEX>$1,200{\sim}1,300^{\circ}C$</TEX> was less than 100 poise, electrical conductivity was higher than 1 S/cm, 20~25wt% waste loading glasses with good surface homogeneity are judged to have good operability in cold crucible induction melter. Other physicochemical properties of the developed glasses are going to be experimented in the future.

Glasses for immobilization of low- and intermediate-level radioactive waste

Reprocessing of spent nuclear fuel (SNF) for recovery of fissionable elements is a precondition of long-term development of nuclear energetics. Solution of this problem is hindered by the production of a great amount of liquid waste; 99% of its volume is low- and intermediate-level radioactive waste (LILW). The volume of high-level radioactive waste (HLW), which is characterized by high heat release, does not exceed a fraction of a percent. Solubility of glasses at an elevated temperature makes them unfit for immobilization of HLW, the insulation of which is ensured only by mineral-like matrices. At the same time, glasses are a perfect matrix for LILW, which are distinguished by low heat release. The solubility of borosilicate glass at a low temperature is so low that even a glass with relatively low resistance enables them to retain safety of under-ground LILW depositories without additional engineering barriers. The optimal technology of liquid confinement is their concentration and immobilization in borosilicate glasses, which are disposed in shallow-seated geological repositories. The vitrification of 1 m3 liquid LILW with a salt concentration of ∼300 kg/m3 leaves behind only 0.2 m3 waste, that is, 4–6 times less than by bitumen impregnation and 10 times less than by cementation. Environmental and economic advantages of LILW vitrification result from (1) low solubility of the vitrified LILW in natural water; (2) significant reduction of LILW volume; (3) possibility to dispose the vitrified waste without additional engineering barriers under shallow conditions and in diverse geological media; (4) the strength of glass makes its transportation and storage possible; and finally (5) reliable longterm safety of repositories. When the composition of the glass matrix for LILW is being chosen, attention should be paid to the factors that ensure high technological and economic efficiency of vitrification. The study of vitrified LILW from the Kursk nuclear power plant with high-power channel reactors (HPCR; equivalent Russian acronym, RBMK) and the Kalinin nuclear power plant with pressurized water reactors (PWR; equivalent Russian acronym VVER) after their 14-yr storage in the shallow-seated repository at the MosNPO Radon testing ground has confirmed the safety of repositories ensured by confinement properties of borosilicate matrix. The most efficient vitrification technology is based on cold crucible induction melting. If the content of a chemical element in waste exceeds its solubility in glass, a crystalline phase is formed in the course of vitrification, so that the glass ceramics become a matrix for such waste. Vitrified waste with high Fe; Na and Al; Na, Fe, and Al; Na and B is characterized. The composition of frit and its proportion to waste depends on waste composition. This procedure requires careful laboratory testing.

Cold-Induced Activities of Cytochromes P450 1A1 and 1A2 in Rat Liver: Putative Role of Endogenous Compounds in Induction Mechanism

Adaptation to cold includes adaptive changes at the organism and molecular levels. One of the interesting facts is induction of cytochromes P450 subfamily 1A (CYP1A) in the liver of rats, inducible enzymes participating in biotransformation of procarcinogenic xenobiotics, under the effect of moderate cold exposure. Cold activation of CYP1A can be mediated by adaptive changes and the resultant redistribution or intensification of the synthesis of mediator compounds. This hypothesis is verified in the present study. The role of bilirubin, tocopherol, and corticosterone as mediators of cold induction of CYP1A in the rat liver was evaluated. The results indicate that these compounds can be involved in cold induction of CYP1A, but none of them is the only mediator in this process.

The metabolic fate of citric acid as affected by cold storage in blood oranges

Citrus fruit are natural source of several phytochemicals useful in the maintenance of a satisfactory status of the human health. As citric acid is one of the main determinant of citrus fruit quality, researches focusing the elucidation of citrate metabolism have been constantly carried out, often leading to a deeper understanding of the biochemical and physiological changes occurring during fruit development and ripening. Lower interest has been directed to the knowledge of the metabolic fate of citrate during fruit storage, although the temperature controlled conservation is a spread and, in many cases, mandatory practise. In this work, we evaluated the expression of the main enzymes involved in the citrate metabolism, such citrate synthase, citrate lyase and both the cytosolic and mitochondrial isoforms of aconitase, in blood oranges subjected to cold storage (4 °C × 15 days). Total acidity (TA) and total soluble solid (TSS) were also determined to correlate such fruit quality parameters with gene expression. The results suggest that cold storage influences both the analytical parameters and gene expression, in particular, a strong induction of the transcription of all the selected gene was observed simultaneously with the sudden reduction in acidity. These findings suggest that aconitase gene products localized in the cytosolic compartment are strongly implicated in the consumption of the citrate released from vacuoles and meanwhile mitochondrial aconitase is involved in the catabolism of organelle-localized citrate. The expression of citrate lyase turned out to be also induced during cold storage of blood oranges. The alternative breakdown of citrate through the citrate lyase, which is ruled out during fruit development and ripening, might be a pathway exclusively activated in response to cold storage and therefore correlated with the sharp cold induction of the flavonoid biosynthesis which is supplied by the citrate lyase reaction products.

Cold-induced modulation of CbICE53 gene activates endogenous genes to enhance acclimation in transgenic tobacco

Inducers of C-repeat binding factor (CBF) expression [inducer of CBF expression 1 (ICE1)-like], being MYC-type transcription factors, play an important role in plant tolerance to low temperatures and other abiotic environmental stresses. These ICEs are the key activators binding to C-repeat/dehydration-responsive elements, triggering the response of CBF signaling and other cold-related genes under cold stress. Our previous report documented a novel ICE transcription factor from Capsella bursa-pastoris, named CbICE53 (GenBank accession no. AY506804), which is responsive to cold, salt stress, and exogenous IAA, GA3, ABA, MeJA, and SA. In the present study, subcellular localization analyses reveal that CbICE53 is specifically localized to the nucleus. Similarly to AtICE1, CbICE53 exhibited slightly increased transcript level under cold induction. Compared with wild-type plants, transformants overexpressing CbICE53 showed increased tolerance to both chilling and freezing temperature according to the index of electrolyte leakage, relative water content and glucose content, and survival rate assay. The expressions of endogenous cold-responsive genes in transgenic tobacco (NtDREB1, NtDREB3, NtERD10a, and NtERD10b) were obviously upregulated only in cold condition. These results suggest that CbICE53 increases plant tolerance to low temperature via activating downstream cold-responsive pathways, which can be considered as a potential candidate for transgenic engineering in plant breeding for cold-tolerant crops.

Cold Shock Genes cspA and cspB from Caulobacter crescentus Are Posttranscriptionally Regulated and Important for Cold Adaptation

Cold shock proteins (CSPs) are nucleic acid binding chaperones, first described as being induced to solve the problem of mRNA stabilization after temperature downshift. Caulobacter crescentus has four CSPs: CspA and CspB, which are cold induced, and CspC and CspD, which are induced only in stationary phase. In this work we have determined that the synthesis of both CspA and CspB reaches the maximum levels early in the acclimation phase. The deletion of cspA causes a decrease in growth at low temperature, whereas the strain with a deletion of cspB has a very subtle and transient cold-related growth phenotype. The cspA cspB double mutant has a slightly more severe phenotype than that of the cspA mutant, suggesting that although CspA may be more important to cold adaptation than CspB, both proteins have a role in this process. Gene expression analyses were carried out using cspA and cspB regulatory fusions to the lacZ reporter gene and showed that both genes are regulated at the transcriptional and posttranscriptional levels. Deletion mapping of the long 5'-untranslated region (5'-UTR) of each gene identified a common region important for cold induction, probably via translation enhancement. In contrast to what was reported for other bacteria, these cold shock genes have no regulatory regions downstream from ATG that are important for cold induction. This work shows that the importance of CspA and CspB to C. crescentus cold adaptation, mechanisms of regulation, and pattern of expression during the acclimation phase apparently differs in many aspects from what has been described so far for other bacteria.

Photoperiodic regulation of the C-repeat binding factor (CBF) cold acclimation pathway and freezing tolerance inArabidopsis thaliana

The CBF (C-repeat binding factor) pathway has a major role in plant cold acclimation, the process whereby certain plants increase in freezing tolerance in response to low nonfreezing temperatures. In Arabidopsis thaliana, the pathway is characterized by rapid cold induction of CBF1, CBF2, and CBF3, which encode transcriptional activators, followed by induction of CBF-targeted genes that impart freezing tolerance. At warm temperatures, CBF transcript levels are low, but oscillate due to circadian regulation with peak expression occurring at 8 h after dawn (zeitgeber time 8; ZT8). Here, we establish that the CBF pathway is also regulated by photoperiod at warm temperatures. At ZT8, CBF transcript levels in short-day (SD; 8-h photoperiod) plants were three- to fivefold higher than in long-day plants (LD; 16-h photoperiod). Moreover, the freezing tolerance of SD plants was greater than that of LD plants. Genetic analysis indicated that phytochrome B (PHYB) and two phytochrome-interacting factors, PIF4 and PIF7, act to down-regulate the CBF pathway and freezing tolerance under LD conditions. Down-regulation of the CBF pathway in LD plants correlated with higher PIF4 and PIF7 transcript levels and greater stability of the PIF4 and PIF7 proteins under LD conditions. Our results indicate that during the warm LD growing season, the CBF pathway is actively repressed by PHYB, PIF4, and PIF7, thus mitigating allocation of energy and nutrient resources toward unneeded frost protection. This repression is relieved by shortening day length resulting in up-regulation of the CBF pathway and increased freezing tolerance in preparation for coming cold temperatures.

Corrosion study of a highly durable electrolyzer based on cold crucible technique for pyrochemical reprocessing of spent nuclear oxide fuel

The application of the cold crucible technique to a pyrochemical electrolyzer used in the oxide-electrowinning method, which is a method for the pyrochemical reprocessing of spent nuclear oxide fuel, is proposed as a means for improving corrosion resistance. The electrolyzer suffers from a severe corrosion environment consisting of molten salt and corrosive gas. In this study, corrosion tests for several metals in molten 2CsCl–NaCl at 923K with purging chlorine gas were conducted under controlled material temperature conditions. The results revealed that the corrosion rates of several materials were significantly decreased by the material cooling effect. In particular, Hastelloy C-22 showed excellent corrosion resistance with a corrosion rate of just under 0.01mm/y in both molten salt and vapor phases by controlling the material surface at 473K. Finally, an engineering-scale crucible composed of Hastelloy C-22 was manufactured to demonstrate the basic function of the cold crucible. The cold crucible induction melting system with the new concept Hastelloy crucible showed good compatibility with respect to its heating and cooling performances.

A simplified mathematical model of glass melt convection in a cold crucible induction melter

Cold crucible induction melting is emerging as a promising technology for immobilizing nuclear waste in glass matrices. Since the transport properties such as viscosity and electrical conductivity of molten glass exhibit strong temperature dependencies, performance of the cold crucible induction melter is highly sensitive to the thermal field prevailing in the molten glass pool. A simplified mathematical model was developed to numerically investigate the impact of molten glass properties such as viscosity, thermal conductivity and electrical conductivity on the performance of a cold crucible induction melter meant for high level radioactive waste vitrification. The present investigation of the thermal convection using the simplified model confirms the findings of previous studies. Numerical simulation of thermal convection shows that low electrical conductivity and high viscosity existing in the cooler parts of the molten glass bath can lead to poor electromagnetic induction and localized heating in the present melter-inductor configuration. The stable thermal stratification due to bottom cooling leads to a relatively stagnant fluid layer in the lower part of the glass melt. These features of the thermal convection can limit the heat transfer and mixing in the glass melt which in turn can affect both the melting capacity and product homogeneity adversely. Mechanical stirring using a water-cooled stirrer can overcome these limitations. The present study confirms that mechanical stirring of the glass melt can enhance the electromagnetic induction through thermal homogenization. Mechanical mixing eliminates the relatively stagnant fluid layer observed in thermal convection. Distribution of induced power, temperature and velocity predicted by the simplified model exhibit matching characteristics of the results obtained by other investigators. The simplified model reduces the computational load substantially as it eliminates complex electromagnetic computations.

Triploid Penaeus monodon: Sex ratio and growth rate

This study seeks to determine the best method for preventing the second body (PBII) extrusion as a means to induce triploidy in the black tiger shrimp Penaeus monodon. Chemical (cytochalasine-B and 6-dimethylaminopurine) and temperature (heat and cold) shocks were applied to newly fertilized eggs. Cold shock that was administered at 8°C, for 10min at 8min post-spawning, was the best method for PBII triploidy induction, as evidenced by the highest percentage of forming three pronuclei in syngamy. Therefore, cold shock induction was employed to further explore the advantage of triploid over diploid shrimp. After hatching of the cold-shocked eggs, the larvae were allowed to reach juvenile and adult levels of development. The number and amount of chromosomes were determined in juveniles and adult stages, using Fluorescence Activating Cell Sorting methods, by which the shrimp were divided into diploid and triploid groups. At day 150 in culture, the average body weight of the triploid females (35.2±3.2g) and triploid males (31.5±3.5g) was significantly higher (P<0.0001) than that of their diploid counterparts (24.5±0.5g for females and 23.1±3.8g for males), having a ratio of 2 females:1 male for triploid shrimp, and 2 females:3 males for diploid shrimp. These results reveal the advantages of growing triploid over diploid P. monodon, and its feasibility for commercial production.

The effect of tepid amino acid-enriched induction cardioplegia on the outcome of infants undergoing cardiac surgery

Despite promising experimental results, no information has been published on the clinical effects of amino acid-enriched induction cardioplegic solution on outcome in children undergoing cardiac surgery. This is a retrospective study of 185 consecutive patients younger than 12 months with one of the following defects undergoing open heart surgery: atrioventricular septal defect, transposition of the great arteries, tetralogy of Fallot or ventricular septal defect. Patients were divided into two groups according to the following myocardial protection approaches: tepid substrate-enriched induction cardioplegia followed by cold blood cardioplegia (n=113) or only cold blood induction cardioplegia (n=72). Patient allocation was determined by the anesthesiologist in charge of cardiopulmonary bypass (CPB). The primary outcome measure was postoperative myocardial injury assessed by troponin T level and inotrope score. Demographic data were similar for both groups. Cardioplegic induction had no overall effect for inotrope score (16.3 ± 9.2 vs.17.9 ± 10.0, p=0.276) or lactate release (1.8 ± 1.3 vs. 1.6 ± 0.8, p=0.110) on arrival to the paediatric intensive care unit. On the first postoperative day, there were no significant differences between the cardioplegia groups for inotrope score (13.7 ± 8.7 vs.14.3 ± 9.1, p=0.657), troponin T (2.4 ± 1.6 vs. 2.8 ± 2.7 μg/L, p=0.267), lactate (1.5 ± 2.0 vs. 1.5 ± 0.8, p=0.972), or any of the other clinical outcome measures. Compared to cold cardioplegia alone, the administration of tepid induction cardioplegia had no effect on the clinical outcome of infants who underwent cardiac surgery.

Arabidopsis Type-III Phosphatidylinositol 4-Kinases β1 and β2 are Upstream of the Phospholipase C Pathway Triggered by Cold Exposure

Phosphatidylinositol-4-phosphate (PtdIns4P) is the most abundant phosphoinositide in plants and the precursor of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)]. This lipid is the substrate of phosphoinositide-dependent phospholipase C (PI-PLC) that produces diacylglycerol (DAG) which can be phosphorylated to phosphatidic acid (PtdOH). In plants, it has been suggested that PtdIns4P may also be a direct substrate of PI-PLC. Whether PtdIns4P is the precursor of PtdIns(4,5)P(2) or a substrate of PI-PLC, its production by phosphatidylinositol-4-kinases (PI4Ks) is the first step in generating the phosphoinositides hydrolyzed by PI-PLC. PI4Ks can be divided into type-II and type-III. In plants, the identity of the PI4K upstream of PI-PLC is unknown. In Arabidopsis, cold triggers PI-PLC activation, resulting in PtdOH production which is paralleled by decreases in PtdIns4P and PtdIns(4,5)P(2). In suspension cells, both the PtdIns4P decrease and the PtdOH increase in response to cold were impaired by 30 μM wortmannin, a type-III PI4K inhibitor. Type-III PI4Ks include AtPI4KIIIα1, β1 and β2 isoforms. In this work we show that PtdOH resulting from the PI-PLC pathway is significantly lowered in a pi4kIIIβ1β2 double mutant exposed to cold stress. Such a decrease was not detected in single pi4kIIIβ1 and pi4kIIIβ2 mutants, indicating that AtPI4KIIIβ1 and AtPI4KIIIβ2 can both act upstream of the PI-PLC. Although several short-term to long-term responses to cold were unchanged in pi4kIIIβ1β2, cold induction of several genes was impaired in the double mutant and its germination was hypersensitive to chilling. We also provide evidence that de novo synthesis of PtdIns4P by PI4Ks occurs in parallel to PI-PLC activation.

Proteomic and biochemical analysis of maize anthers after cold pretreatment and induction of androgenesis reveals an important role of anti-oxidative enzymes

In stress conditions, microspores and young pollen grains can be switched from their normal pollen development toward an embryogenic pathway via a process called androgenesis. Androgenic embryos can produce completely homozygous, haploid or double-haploid plants. This study aimed to investigate changes in the abundance of protein species during cold pretreatment and subsequent cultivation of maize anthers on induction media using gel-based proteomics. Proteins upregulated on the third day of anther induction were identified and discussed here. Simultaneous microscopic observations revealed that the first division occurred in microspores within this period. Using 2-D electrophoresis combined with MALDI TOF/TOF MS/MS analysis 19 unique proteins were identified and classified into 8 functional groups. Proteins closely associated with metabolism, protein synthesis and cell structure were the most abundant ones. Importantly, ascorbate peroxidase, an enzyme decomposing hydrogen peroxide, was also upregulated. Isozyme analysis of peroxidases validated the proteomic data and showed increased peroxidase activities during androgenic induction. Further, the isozyme pattern of SOD revealed increased activity of the MnSOD, which could provide hydrogen peroxide as a substrate for in vivo peroxidase reactions (including ascorbate peroxidase). Together, these data reveal the role of enzymes controlling oxidative stress during induction of maize androgenesis.

English

Thaumatin-like proteins (TLPs) constitutes a homogeneous family, members of which are produced by plants in response to different kinds of stress. NP24 protein is one of such salt-induced protein from tomato&nbsp;(Solanum lycopersicum) and it belongs to TLPs family. NP24 is a 24 kDa (207 amino acid) antimicrobial TLP found in tomato fruits. One isoform (NP24I) of NP24 was discovered in the outer pericarp of tomato fruit and is reported to play a possible role in ripening of the fruit in addition to its antimicrobial activity. In this study, the total RNA was isolated successfully from the outer pericarp of ripe (red) tomato fruit. cDNA was prepared and the gene coding for NP24I protein was amplified using conventional polymerase chain reaction (PCR). The gene was then cloned into Mach1&trade;- T1&reg;&nbsp;Escherichia colicells, then subcloned into the over-expression vector pET-28a (+) using&nbsp;BL21expression bacteria.&nbsp;Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) analysis showed that the gene was over-expressed in&nbsp;E. coli&nbsp;as inclusion bodies. Optimization of the recombinant NP24I protein solubility was achieved by cold induction through decreasing both expression temperature andIsopropyl-beta-thio galactopyranoside (IPTG) concentration. The recombinant NP24I protein was purified using Ni-NTA resin, and then the antimicrobial activity of the purified recombinant NP24I protein was tested. The aims of this work were to study the cloning and expression of NP24 protein from local tomato cultivar in a prokaryotic system and to test the activity of the recombinant NP24, as well as to prove the activity of native protein on the bacterial as well as fungal growth. &nbsp; Key words:&nbsp;Pathogenesis-related proteins (PR), low molecular weight thaumatin-like protein (TLPs), cloning,&nbsp;Escherichia coli, cold induction, antimicrobial activity, tomato&nbsp;(Solanum lycopersicum).

Genetic analysis reveals a complex regulatory network modulating CBF gene expression and Arabidopsis response to abiotic stress

Arabidopsis CBF genes (CBF1–CBF3) encode transcription factors having a major role in cold acclimation, the adaptive process whereby certain plants increase their freezing tolerance in response to low non-freezing temperatures. Under these conditions, the CBF genes are induced and their corresponding proteins stimulate the expression of target genes configuring low-temperature transcriptome and conditioning Arabidopsis freezing tolerance. CBF2 seems to be the most determinant of the CBFs since it also regulates CBF1 and CBF3 expression. Despite the relevance of CBF genes in cold acclimation, little is known about the molecular components that control their expression. To uncover factors acting upstream of CBF2, mutagenized Arabidopsis containing the luciferase reporter gene under the control of the CBF2 promoter were screened for plants with de-regulated CBF2 expression. Here, the identification and characterization of five of these mutants, named acex (altered CBF2 expression), is presented. Three mutants show increased levels of cold-induced CBF2 transcripts compared with wild-type plants, the other two exhibiting reduced levels. Some mutants are also affected in cold induction of CBF1 and CBF3. Furthermore, the mutants characterized display unique phenotypes for tolerance to abiotic stresses, including freezing, dehydration, and high salt. These results demonstrate that cold induction of CBF2 is subjected to both positive and negative regulation through different signal transduction pathways, some of them also mediating the expression of other CBF genes as well as Arabidopsis responses to abiotic stresses.

Derivation, characterization and expansion of fetal chondrocytes on different microcarriers

Fetal chondrocytes (FCs) have recently been identified as an alternative cell source for cartilage tissue engineering applications because of their partially chondrogenically differentiated phenotype and developmental plasticity. In this study, chondrocytes derived from fetal bovine cartilage were characterized and then cultured on commercially available Cytodex-1 and Biosilon microcarriers and thermosensitive poly(hydroxyethylmethacrylate)-poly(N-isopropylacrylamide) (PHEMA-PNIPAAm) beads produced by us. Growth kinetics of FCs were estimated by means of specific growth rate and metabolic activity assay. Cell detachment from thermosensitive microcarriers was induced by cold treatment at 4°C for 20min or enzymatic treatment was applied for the detachment of cells from Cytodex-1 and Biosilon. Although attachment efficiency and proliferation of FCs on PHEMA-PNIPAAm beads were lower than that of commercial Cytodex-1 and Biosilon microcarriers, these beads also supported growth of FCs. Detached cells from thermosensitive beads by cold induction exhibited a normal proliferative activity. Our results indicated that Cytodex-1 microcarrier was the most suitable material for the production of FCs in high capacity, however, 'thermosensitive microcarrier model' could be considered as an attractive solution to the process scale up for cartilage tissue engineering by improving surface characteristics of PHEMA-PNIPAAm beads.

Age associated low mitochondrial biogenesis may be explained by lack of response of PGC-1α to exercise training

Low mitochondriogenesis is critical to explain loss of muscle function in aging and in the development of frailty. The aim of this work was to explain the mechanism by which mitochondriogenesis is decreased in aging and to determine to which extent it may be prevented by exercise training. We used aged rats and compared them with peroxisome proliferator-activated receptor-γ coactivator-1α deleted mice (PGC-1α KO). PGC-1α KO mice showed a significant decrease in the mitochondriogenic pathway in muscle. In aged rats, we found a loss of exercise-induced expression of PGC-1α, nuclear respiratory factor-1 (NRF-1), and of cytochrome C. Thus muscle mitochondriogenesis, which is activated by exercise training in young animals, is not in aged or PGC-1α KO ones. Other stimuli to increase PGC-1α synthesis apart from exercise training, namely cold induction or thyroid hormone treatment, were effective in young rats but not in aged ones. To sum up, the low mitochondrial biogenesis associated with aging may be due to the lack of response of PGC-1α to different stimuli. Aged rats behave as PGC-1α KO mice. Results reported here highlight the role of PGC-1α in the loss of mitochondriogenesis associated with aging and point to this important transcriptional coactivator as a target for pharmacological interventions to prevent age-associated sarcopenia.