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Abstracts: CryoLetters 31 (5), 2010

 

 

Volume 31, No. 5 September/October 2010

ISSN 0143-2044

 

 


Seasonal changes in antifreeze protein gene transcription and water content of beetle Microdera punctipennis (coleoptera: tenebrionidae) from gurbantonggut desert in central asia
F. Hou, J. Ma, X. Liu, Y.Wang, XN. Liu and FC. Zhang

359-370

 

 


Importance of a three-stage cooling regime and induced ice nucleation during cryopreservation on colony-forming potential and differentiation in mesenchymal stem/progenitor cells from human fetal liver
Nataliya G. Skorobogatova, Alexander N. Novikov, Barry J. Fuller, and Alexander Yu. Petrenko

371-379

 

 


Influence of cryopreservation on the cytosine methylation state of potato genomic dna
Anja Kaczmarczyk, Andreas Houben, E.R. Joachim Keller and Michael F. Mette

380-391

 

 


Mitochondrial metabolites in tissues as indicators of metabolic alterations during hibernation
Nadezhda I. Fedotcheva, Elena G. Litvinova, Svetlana V. Kamzolova, Igor G. Morgunov and Zarif G. Amerkhanov

392-400

 

 


Stress tolerance and transcriptional response in mouse embryos treated with high hydrostatic pressure to enhance cryotolerance
Istvan Bock, Eszter Losonczi, Solomon Mamo, Zsuzsanna Polgar, Andrea Harnos, Andras Dinnyes and Csaba Pribenszky

401-412

 

 


Polyamine concentration, transglutaminase activity and changes in protein synthesis during cryopreservation of shoot tips of apple variety annurca
Cinzia Forni, Roberto Braglia, Simone Beninati, Alessandro Lentini, Maurizio Ronci, Andrea Urbani, Bruno Provenzano, Andrea Frattarelli, Claudio Tabolacci and Carmine Damiano

413-425

 

 


Cryopreservation of protocorm-like bodies of the hybrid orchid Bratonia (Miltonia flavescens × Brassia longissima)
Elena Popova, Nikolai Bukhov, Alexandr Popov and Haeng-Hoon Kim

426-437

 

 

 

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CryoLetters 31 (5), 359-370 (2010)
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SEASONAL CHANGES IN ANTIFREEZE PROTEIN GENE TRANSCRIPTION AND WATER CONTENT OF BEETLE Microdera punctipennis (COLEOPTERA: TENEBRIONIDAE) FROM GURBANTONGGUT DESERT IN CENTRAL ASIA

F. Hou, J. Ma*, X. Liu, Y.Wang, XN. Liu and FC. Zhang

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China
*Corresponding author  e-mail:
majibrge@yahoo.cn

Abstract

Desert beetle Microdera punctipennis (Coleoptera: Tenebriondae) is a special species in Gurbantonggut Desert in Central Asia. To investigate the possible strategy it employs for cold survival, seasonal changes in supercooling point (SCP), body water content, haemolymph osmolality and antifreeze protein gene (Mpafp) expression were measured over 13 months. Our results show SCPs in M. punctipennis adults changed from -8.0° in summer to -18.7° in winter. During winter, adults endured modest water loss; total water decreased from 65.36% in summer to 55.88% in winter. Mpafp mRNAs level increased by 13.1 fold from summer to early winter, and haemolymph osmolality increased accordingly from 550mOsm to 1486mOsm. Correlation coefficient of Mpafp mRNAs level and SCP indicates that Mpafp mRNA explained 65.28% of the variation in SCPs. The correlation between Mpafp mRNA level and total water reflected an indirect influence of antifreeze protein on water content via reducing SCP.

Keywords: desert beetle; Microdera punctipennis; antifreeze protein; supercooling point; water content; real-time quantitative PCR.

 

 

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CryoLetters 31 (5), 371-379 (2010)
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IMPORTANCE OF A THREE-STAGE COOLING REGIME AND INDUCED ICE NUCLEATION DURING CRYOPRESERVATION ON COLONY-FORMING POTENTIAL AND DIFFERENTIATION IN MESENCHYMAL STEM/PROGENITOR CELLS FROM HUMAN FETAL LIVER

Nataliya G. Skorobogatova1, Alexander N. Novikov1,
Barry J. Fuller2, and Alexander Yu. Petrenko1*

1Department of Biochemistry of Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, 23 Pereyaslavskaya Str., 61015 Kharkov, Ukraine.
2 University Department of Surgery, Royal Free and University College Medical School, London, UK.
* Corresponding author email:
alexander_petrenko@cryo.org.ua   Tel: 380-57-3734135

Abstract

Colony-forming activity, as well as osteogenic and adipogenic capacities of primary human fetal liver (HFL) mesenchymal stem or progenitor cells (MSCs) were compared before and after cryopreservation using a standard three-step cooling protocol (Cryo3-S) or the same protocol with induced ice nucleation (Cryo3-IIN) and 5% and 10% w/v dimethyl sulphoxide (Me2SO). Cell viability, using the Cryo3-S protocol with 5% and 10% Me2SO, was about 60-70% as assessed by the trypan blue staining method, but the ability to undergo growth in culture and form colonies was completely lost. Cryopreservation using Cryo3-IIN resulted in conservation of colony-forming MSCs. Colony-forming efficiency (CFE) of the cell samples cryopreserved with Cryo3-IIN and 5% Me2SO was on average 0.4 ± 0.1 colonies per 105 cells, whereas with 10% Me2SO 1.6 ± 0.7 colonies were obtained. HFL MSCs recovered after cryopreservation in the both groups demonstrated capacity to be expanded and induced into either osteogenic or adipogenic differentiation.

Keywords: cryopreservation, mesenchymal stem/progenitor cells; human fetal liver; induced ice nucleation; human fetal liver; dimethyl sulphoxide.

 

 

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CryoLetters 31 (5), 380-391 (2010)
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INFLUENCE OF CRYOPRESERVATION ON THE CYTOSINE METHYLATION STATE OF POTATO GENOMIC DNA

Anja Kaczmarczyk*1, Andreas Houben, E.R. Joachim Keller
and Michael F. Mette

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany.
1 present address: Curtin University of Technology, Western Australian Biomedical Research Institute, School of Biomedical Sciences, GPO Box U1987, Perth, WA 6845 Australia
*Corresponding author  e-mail:
A.Kaczmarczyk@curtin.edu.au

Abstract

Shoot tips of Solanum tuberosum 'Désirée' were successfully cryopreserved by the DMSO droplet method and stored for almost 7 years, while control material was maintained in vitro for the same period of time. To analyse potential epigenetic changes, the DNA methylation status was assayed by methylation-sensitive amplified polymorphism (MSAP) analysis using restriction endonucleases MspI and HpaII. An amount of 93.6% of the analysed MSAP signals were stable among all cryopreserved and in vitro maintained samples tested, indicating extensive stability of DNA methylation. Only 0.9% of MSAP signals showed results that differed between the two treatments and at the same time matched for all three biological replications within each treatment. These can be seen as indicating directed effects of the two treatments on the DNA methylation. Cryopreserved samples displayed in comparison to in vitro stored samples consistent hypomethylation for 0.6% (3 of 469) of MSAP signals (Table 4, pattern 4) and consistent hypermethylation for 0.2% (1 of 469), respectively. For 5.6% of all MSAP signals, inconsistent results were observed among the three biological replications at least for one of the two treatments. These were interpreted as resulting from stochastic DNA methylation changes in individual samples. As results for two biological replications were identical and different from the result for the third biological replication, the direction of methylation change could be determined in those cases. Cases of stochastic loss of CG methylation in cryopreserved samples were most frequent among them, adding up to 3.4% of MSAP signals. Stochastic loss of CG methylation was also found in material maintained in vitro, only for 0.6% of all MSAP signals. In conclusion, methylation changes occurred in long-term cryopreservation of potato, in a random rather than directed fashion. Hence, cryopreservation and long-term in vitro maintenance both induce limited changes of DNA methylation status. The order of magnitude of methylation changes observed was consistent with other studies, where similar rates of DNA methylation changes have been found.

Keywords: DNA methylation, DMSO droplet method, genetic stability, Solanum tuberosum

 

 

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CryoLetters 31 (5), 392-400 (2010)
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MITOCHONDRIAL METABOLITES IN TISSUES AS INDICATORS OF METABOLIC ALTERATIONS DURING HIBERNATION

Nadezhda I. Fedotcheva1*, Elena G. Litvinova1, Svetlana V. Kamzolova2,
Igor G. Morgunov2 and Zarif G. Amerkhanov 3

 

1 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia
2 Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia
3 Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia
* Corresponding author e-mail:
nfedotcheva@mail.ru

Abstract

The decrease in metabolism is one of mechanisms for hibernating animals to resist hypoxia and oxidative stress. Assuming that the inhibition of mitochondria; respiration in torpor and its activation upon arousal are accompanied by changes in the content of mitochondrial substrates, we estimated the levels of endogenous metabolites of the tricarboxylic acid (TCA) cycle in the liver, brown adipose tissue, and the brain of the arctic ground squirrels as possible indicators of mitochondrial processes. The level of lactate in the same tissues and serum was determined as marker of hypoxia. It was found that the isocitrate (ISC) concentration in all tissues was one order of magnitude higher than that of alpha-ketoglutarate (KGL), while succinate was not detected in any of tissues, indicating the inhibition at the initial stages of the TCA cycle. During the torpor, the concentrations of ISC, KGL and lactate predominantly decreased in tissues. Serum lactate decreased five-fold in torpor and was restored in a temperature-dependent manner with a long period of persistence of stable concentration in the range of body temperature between 12 and 27°C upon arousal. The data obtained indicate the development of metabolic depression rather than hypoxia in these tissues.

Keywords: hibernation; isocitrate; -ketoglutarate; lactate; hypoxia

 

 

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CryoLetters 31 (5), 401-412 (2010)
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STRESS TOLERANCE AND TRANSCRIPTIONAL RESPONSE IN MOUSE EMBRYOS TREATED WITH HIGH HYDROSTATIC PRESSURE TO ENHANCE CRYOTOLERANCE

Istvan Bock1,2*, Eszter Losonczi3, Solomon Mamo4,
Zsuzsanna Polgar5, Andrea Harnos6, Andras Dinnyes1,2
and Csaba Pribenszky7

 

1Molecular Animal Biotechnology Laboratory, Institute for Basic Animal Sciences, Faculty of Agricultural and Environmental Sciences, Szent István University, Páter K. u. 1. H-2103 Gödöllő, Hungary.
2BioTalentum Ltd., Aulich Lajos u. 26. H-2100 Gödöllő, Hungary.
3ARTechnic Co. Csapó u. 13. H-4024 Debrecen, Hungary.
4University College of Dublin, Lyons Research Farm, Newcastle Co. Dublin, Ireland.
5Faculty of Natural Sciences, Constantine the Philosopher University, Tr. Andreja Hlinku 1, SK-949 74 Nitra, Slovakia.
6Szent István University, Faculty of Veterinary Science, Department of Biomathematics and Informatics, István u. 2. H-1078 Budapest, Hungary.
7Szent István University, Faculty of Veterinary Science, Department of Animal Breeding and Genetics, István u. 2. H-1078 Budapest, Hungary.
I. Bock and E. Losonczi contributed equally to the manuscript.
*Corresponding author   e-mail:
istvan.bock@biotalentum.hu

Abstract

   Sublethal high hydrostatic pressure (HHP) treatment of cells was reported to enhance stress tolerance and to increase post-thawing survival after cryopreservation in mouse, swine and cattle. The goal of this study was to define if HHP stress tolerance depends on the embryos' stage of development and culture conditions, to describe long term in vivo effects and transcriptional alterations of selected stress related genes. Studies showed that impacts greater than 60 MPa caused blastomere and membrane injuries to the two-cell stage embryos, while even 80 MPa was well tolerated by blastocysts. HHP treatment caused significant upregulation of Azin1, Sod2 and Gadd45g genes, detected by RT-qPCR. The transfer of HHP treated blastocysts revealed normal in vivo development and reproductive function in a two generation study. The cell type and the embryos' development stage shall be taken into account when optimizing sublethal HHP stress treatment protocol of different cells.

Keywords: High hydrostatic pressure; Gene expression; Morphology; Stress tolerance; Mouse embryo

 

 

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CryoLetters 31 (5), 413-425 (2010)
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POLYAMINE CONCENTRATION, TRANSGLUTAMINASE ACTIVITY AND CHANGES IN PROTEIN SYNTHESIS DURING CRYOPRESERVATION OF SHOOT TIPS OF APPLE VARIETY ANNURCA

Cinzia Forni*1, Roberto Braglia1, Simone Beninati1,
Alessandro Lentini1, Maurizio Ronci2, Andrea Urbani2,3,
Bruno Provenzano1, Andrea Frattarelli4, Claudio Tabolacci1
and Carmine Damiano4

1Department of Biology, University of Rome "Tor Vergata", Via della Ricerca Scientifica, 1 00133 Rome (Italy).
2Laboratory of Proteomics and Metabolomics, S. Lucia Foundation- IRCCS, Via del Fosso di Fiorano, 00143 Rome (Italy).
3Department of Internal Medicine and Department of Laboratory Medicine, University of Rome "Tor Vergata", Via Montpellier, 1 00133 Rome, (Italy).
4Agricultural Research Council. - Fruit Trees Centre (CRA-FRU), Via Fioranello, 52 00134 Rome (Italy).
*Corresponding author  email:
forni@uniroma2.it

Abstract

Changes in metabolism and protein expression were analysed during cryopreservation of the ancient apple variety Annurca. Our experiments concerned transglutaminase activity, polyamine levels and protein expression associated with shoot tip dehydration. Cryopreserved shoot tips displayed 72% regrowth after treatment in liquid medium with 0.75 M sucrose for 1 day followed by dehydration to 19% moisture content (fresh weight basis). After dehydration, the concentration of polyamines putrescine and spermidine decreased compared with untreated controls, while spermine concentration remained unaffected. Transglutaminase activity was slightly reduced in treated samples, while post-thaw regrowth enzyme activity approached control values. We also detected significant changes in protein expression profiles and identified six proteins related with stress response or involved in the slowing down of the cell cycle. The relationship between biochemical parameters, protein synthesis and cryotolerance is discussed.

Keywords: Annurca (Malus x domestica Borkh.), biodiversity, conservation, encapsulation-dehydration, metabolic changes, proteomics.

 

 

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CryoLetters 31 (5), 426-437 (2010)
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CRYOPRESERVATION OF PROTOCORM-LIKE BODIES OF THE HYBRID ORCHID Bratonia (Miltonia flavescens × Brassia longissima)

Elena Popova1*, Nikolai Bukhov2, Alexandr Popov2
and Haeng-Hoon Kim1

1National Agrobiodiversity Center, National Academy of Agricultural Science, RDA, Suwon 441-707, Korea.
2Institute of Plant Physiology of Russian Academy of Sciences, Moscow, Russia
*Corresponding author  e-mail:
elena_aygol@hotmail.com

Abstract

In this study, cryopreservation of Bratonia (Miltonia flavescens (Lindl.) Lindl. × Brassia longissima (Reichb.) Nash), a hybrid tropical orchid, was achieved using protocorm-like bodies (PLBs) multiplied in vitro. Cryopreservation was performed using a vitrification protocol including pretreatment of PLBs with a loading solution (LS, 2.0 M glycerol + 0.4 M sucrose) for 15 min followed by treatment with modified PVS2 vitrification solution (containing PEG instead of ethylene glycol) for 1 h. Increasing benzyladenine (BA) concentration in the recovery medium to 5.0 or 10.0 mg l-1 during the initial 3 weeks after rewarming provided 20.4% post-cryopreservation regrowth. By contrast, preliminary culture of PLBs with abscisic acid (ABA) and high sucrose concentrations (up to 0.3 M) as well as addition of reduced glutathione during the preculture, loading and post-culture steps were not beneficial. Forty to 45 plants were regenerated from each PLB which withstood cryopreservation. No morphological differences were observed between plants regenerated from cryopreserved and untreated PLBs. Investigations into the functional activity of photosystems I and II in PLBs suggest that electron transport was retained in the reaction centers of both photosystems shortly after cryopreservation.

Keywords: Bratonia, orchid, protocorm-like bodies (PLBs), PVS2, vitrification

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