CryoLetters 24 (4), 2003

CryoLetters is a bimonthly, international journal for low temperature science and technology

CryoLetters 24, 201-202 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Editorial – CryoLetters enters a New Phase – Electronic publishing



CryoLetters 24, 203-212 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


L.I. Relina* and A.K. Gulevsky

Department of Biochemistry of Cold Adaptation, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, 23 Pereyaslavskaya Street, Kharkov 61015, Ukraine.
*Corresponding author, Email:


A brief history of molecular chaperones is presented in relation to low temperature stresses across a range of species. The recent knowledge of participation of chaperones in cold adaptive mechanisms is reviewed. Possible applications of molecular chaperones and directions of future investigations are discussed.

Keywords: molecular chaperones, cold stress, cold tolerance.



CryoLetters 24, 213-228 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Euglena gracilis AND Haematococcus pluvialis

Roland A. Fleck1#, Erica E. Benson2*, David H. Bremner2 and John G. Day1

1CCAP, CEH Windermere, Far Sawrey, Ambleside, Cumbria, LA22 0LP.
2Conservation and Environmental Chemistry Centre, School of Contemporary Science, University of Abertay Dundee, Bell Street, Dundee, DD1 1HG.
#Current address: National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts. EN6 3QG.
*Corresponding author


Algal culture collections are required to develop robust and broadly applicable cryogenic storage methods for diverse taxonomic groups and understanding differential responses to cryoinjury helps to achieve this end. Antioxidant profiles were constructed for cryopreserved Euglena gracilis (Klebs CCAP 1224/5Z), a freeze sensitive alga, and Haematococcus pluvialis (Flotow CCAP 34/8), which is a highly freeze-tolerant. H. pluvialis had a coordinated antioxidant response with respect to catalase, superoxide dismutase (SOD) and glutathione reductase; it is postulated that this may contribute to freeze tolerance. Formation  (via SOD) and removal (via catalase) of H2O2 were not fully coordinated in freeze-sensitive E. gracilis and this may exacerbate cryoinjury. Increased SOD activity in the absence of catalase thus compromises survival due to the formation of hydroxyl radicals (.OH) from H2O2.   Changes in sulfhydryl group (SH) status for non-Protein bound SH groups were greater in freeze-tolerant H. pluvialis. Therefore, the tolerant organism may have a range of coordinated protection mechanisms that ameliorate the deleterious effects of oxidative stress during cryopreservation.

Keywords: cryopreservation, antioxidants, free radicals, oxidative stress, Fenton chemistry.



CryoLetters 24, 229-244 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

The physico-mathematical theory of human erythrocyte hypotonic hemolysis phenomenon

E.A. Gordienko*, Yu.E. Gordienko, O.I. Gordienko

Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Pereyaslavskaya St. 23, Kharkov, Ukraine, 61015.


A physico-mathematical model of human erythrocyte hypotonic hemolysis has been developed in this work. It quantitatively describes the kinetics of the phenomenon occurring when cells are suspended in an hypertonic aqueous solution of permeable non-electrolyte.

Keywords: human erythrocyte, physico-mathematical model, hypotonic hemolysis, cell swelling.



CryoLetters 24, 245-252 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Katsuhito Nagano*1, Hiroki Kajihara2, Etuko Suzaki3, Masashi Suzuto1, Katsuko Kataoka3, Michiko Yoshii1 and Koichiro Ozawa1

1 Department of Pharmacotherapy, Graduate School of Medical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
2 Center for foundational Arts and Sciences, Hiroshima Prefectual College of Health Sciences
3 Department of Anatomy, Hiroshima University School of Medicine


This study examined whether a hypothermic environment reduces experimentally-induced atrophy of skeletal muscle, as judged by histochemical findings. The hind limbs of hamsters in a hypothermic group were suspended and flexed into plantar positions at the ankle joint, and housed for one week at 8 to 12°C in a temperature-controlled room, while the normothermic group was housed at 23 to 25°C. Hypothermia did not significantly alter the average caloric intake, and the animals from the hypothermic group lost a significant amount of body weight when compared with the normothermic group. The hypothermic group retained more muscle wet-weight and myofibers cross-sectional area in the soleus and gastrocnemius muscles compared with the normothermic group.  Our results indicate that a hypothermic environment inhibits short-term muscle atrophy. This inhibition may be caused by the increased caloric intake combined with a state similar to hibernation in low-temperature environments.

Keywords: muscle atrophy, hind limb unloading, hypothermic environment, muscle wet-weight, myofibers cross-sectional area



CryoLetters 24, 253-259 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


         Tamás Vajdaa, Marianna Mákb and Miklós Hollósia

aDepartment of Organic Chemistry, Eötvös Loránd University, P.O.Box 32, Budapest 112,
  H-1518 (Hungary), Fax +3613722620.
bG. Richter Ltd., Spectroscopic Res. Lab., Mass Spectrometry Group, Budapest


The possibility of stereoselection through the DLalanineNcarboxyanhydride (NCADLAla) oligomerization, and the effect of freezing on it have been investigated. To this end, the chirality of peptides obtained by oligomerization for 1 and 3 days, respectively, in liquid (+22o C) and frozen (18şC) dioxane solutions, was analyzed. These water-soluble samples were fractionated by gel filtration, aliquots of the fractions were completely hydrolyzed and then derivatized with Marfey reagent (1fluoro2,4dinitrophenyl5Lalanine amide). These derivatives were traced and evaluated by RPHPLC analysis. The relatively best effects appeared in a given fraction, where after 1 day of oligomerization the Lalanine enantiomeric excess (ee) was 3.8% in liquid and 8.6% in frozen conditions. After 3 days, however, the ee contents decreased to 2.0% and 4.1%. The mass spectrometric data of the peptides pointed to the formation of open chain and cyclic peptide mixtures, where the residue numbers of 8-11 and 4-5 dominated. The formation of some chiral peptides from a racemic amino acid suggests the possibility of preferential incorporation of the L-enantiomer into the growing chain, beside the achiral statistical succession of residues. Here we provide the first example of the role of freezing in the increased formation of chirality from racemic amino-acid through oligomerization, together with some speculations about the implications of our model in the events of prebiotic chemistry.

Keywords: freezing, oligomerization, enantiomeric excess, prebiotic chirality generation.

CryoLetters 24, 261-268 (2003)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Milagros C. Esteso1, María R. Fernández-Santos1, Ana J. Soler1 and José J. Garde* 1, 2

1Dpto. Ciencia y Tecnología Agroforestal. Campus Universitario, 02071. Albacete, Spain.
2IREC (UCLM-CSIC-JCCM). E-mail address: (J.J. Garde)


The objective of this study was to evaluate the effects of the thawing procedure on red deer spermatozoa distribution in morphologically distinct subpopulations after freezing and thawing. For this purpose, epididymal spermatozoa were thawed using two different thawing protocols (I = 37 şC for 20 s vs. II = 70 şC for 5 s). The spermatozoa, from 10 Iberian deer stags, were diluted at room temperature in a Triladyl® -20% egg yolk medium and frozen in nitrogen vapor. Standard sperm freezability was judged by microscopic assessments of sperm motility (%). The thawing procedure had an effect on sperm motility percentage (P < 0.05), with the best overall recovery rates found with the use of protocol I (76.8 ± 1.8 vs. 70.6 ± 1.8). Moreover, the morphometric dimensions for a minimum of 200 sperm heads were analyzed from each sample by means of the Sperm-Class Analysez® (SCA), and the mean measurements recorded. Deer sperm heads were significantly (P < 0.01) smaller when spermatozoa were thawed using protocol II than when using procedure I (area = 30.02 μm2 vs. 30.32 μm2; width = 4.47 μm vs. 4.51 μm; length = 8.05 μm vs. 8.11 μm), but not for all stags. All sperm head measurements were placed in a statistical database and a multivariate cluster analysis performed. Mean measurements for all parameters of the major clusters for the two different thawing procedures were compared by ANOVA. The mean values for length, width, area, perimeter, shape factor and width/length in the major cluster of sperm head dimensions for thawing protocol I were significantly different from those for protocol II (P < 0.001). In addition, differences were found within all stags for whole morphometric parameters (P < 0.001), with the smallest overall sperm head dimensions found with the use of protocol II. Additionally, the rapid thawing protocol produced a dramatic loss of heterogeneity. Finally, our results showed that the greater the loss of heterogeneity, the greater the degree of sperm cryoinjury.

Keywords: ASMA, cryopreservation, freezing, morphology, red deer, variation


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