Freeze tolerance in Aporrectodea caliginosa and other earthworms from Finland

Earthworms that live in subarctic and cold temperate areas must deal with frost even though winter temperatures in the soil are often more moderate than air temperatures. Most lumbricid earthworms can survive temperatures down to the melting point of their body fluids but only few species are freeze tolerant, i.e. tolerate internal ice formation. In the present study, earthworms from Finland were tested for freeze tolerance, and the glycogen reserves and glucose mobilization (as a cryoprotectant) was investigated. Freeze tolerance was observed in Aporrectodea caliginosa, Dendrobaena octaedra, and Dendrodrilus rubidus, but not in Lumbricus rubellus. A. caliginosa tolerated freezing at -5 degrees C with about 40% survival. Some individuals of D. octaedra tolerated freezing even at -20 degrees C. Glycogen storage was largest in D. octaedra where up to 13% of dry weight consisted of this carbohydrate, whereas the other species had only 3-4% glycogen of tissue dry weight. Also glucose accumulation was largest in D. octaedra which was the most freeze-tolerant species, but occurred in all four species upon freezing. It is discussed that freeze tolerance may be a more common phenomenon in earthworms than previously thought.     

Freeze tolerance and accumulation of cryoprotectants in the enchytraeid Enchytraeus albidus (Oligochaeta) from Greenland and Europe

The freeze tolerance and accumulation of cryoprotectants was investigated in three geographically different populations of the enchytraeid Enchytraeus albidus (Oligochaeta). E. albidus is widely distributed from the high Arctic to temperate Western Europe. Our results show that E. albidus is freeze tolerant, with freeze tolerance varying extensively between Greenlandic and European populations. Two populations from sub Arctic (Nuuk) and high Arctic Greenland (Zackenberg) survived freezing at −15 °C, whereas only 30% of a German population survived this temperature. When frozen, E. albidus responded by catabolising glycogen to glucose, which likely acted as a cryoprotectant. The average glucose concentrations were similar in the three populations when worms were frozen at −2 °C, approximately 50 μg glucose mg⁻¹ tissue dry weight (DW). At −14 °C the glucose concentrations increased to between 110 and 170 μg mg⁻¹ DW in worms from Greenland. The average glycogen content of worms from Zackenberg and Nuuk were about 300 μg mg⁻¹ DW, but only 230 μg mg⁻¹ DW in worms from Germany showing that not all glycogen was catabolised during the experiment. Nuclear magnetic resonance spectrometry (NMR) was used to screen for other putative cryoprotectants. Proline, glutamine and alanine were up regulated in frozen worms at −2 °C but only in relatively small concentrations suggesting that they were of little significance for freeze survival. The present study confirms earlier reports that freeze tolerant enchytraeids, like other freeze tolerant oligochaete earthworms, accumulate high concentrations of glucose as a primary cryoprotectant.     

The Plasma Membrane of Myxosporidian Valve Cells: Freeze Fracture Data

Freeze fracturing of Myxosporidian spores reveals the occurrence of a continuous layer of transmembrane particles all over the surface area of the valve cells which form the spore envelope. These particles are densely packed all over the P face membrane. Due to their polygonal outline, their diameter (6-7 nm) and their central core, they resemble the particles forming the connections of gap junctions which metabolically couple the neighboring cells in animal tissues. In the present report, the role of the transmembrane particles is still hypothetical. However, they might represent a membrane structural specialization of the spores which are submitted to osmotic variations of the fluid external medium. Furthermore similar transmembrane particles are observed at the level of the septate junction which seals the valve cells. In this occurrence, they are arranged in a series of 40 double rows parallel to the suture of the spore envelope. These findings support the view that Myxosporidia are Metazoa and raise the problem of their origin.     

Ultrastructure of the Vegetative Cells of Nostoc flagelliforme Prepared with High Pressure Freezing and Freeze Substitution Technique

The ultrastructure of the vegetative cells of Nostoc fiagelliforme Born. et Flah. was investigated with high pressure freezing and freeze substitution technique and compared with the results obtained by using conventional preparation methods. During the processes of chemical fixation, dehydration and embedding, the cell structures might be more artificially modified than that obtained from high pressure freezing and freeze substitution. With the present method, the sheath of N. fiageUiforme could be well-penetrated and no extra big space could exist between the cell and the sheath. The cell protoplasm rarely shrinked. Some fine structures of cell inclusions and unit membranes became visualized. Many bacteria were harbored in the sheath. In addition, the presence of big vacuoles in the cell of N. fiageUiforme as well as the presence of bacteria in the sheath shown in the present preparation for cyanobacteria has not been described so far in the literature.     

Ultrastructure of the Floral Nectary of Arabidopsis thaliana L. Prepared from High Pressure Freezing and Freeze Substitution

High pressure freezing and freeze substitution techniques were used to study the floral nectary of Arabidopsis thaliana L. focused on its ultrastmcture of mature nectary at pre-secretory, early secretory and heavy secretory stages. Questions of whether or not the transported vesicles fuse with the plasma]emma, and whether or not the "densely-stained cells" were comparable to companion cells during the secretory process were especially emphasized. The flowers of A. thaliana contained a pair of rather large lateral nectaries and 2 to 4 median ones. The median nectaries were situated at the bases of the two long stamens or between them while each of the. lateral ones was situated nearby each short stamen and between two petals. Before and during the secretion changes in the size of vacuoles and starch grains in the chloroplasts, the number of dictyosomes and endoplasmic mticulum and the distribution of mitechondria occurred before and during the stage of secretion. It appears that transported vesicles from the dictyosomes were transferred directly from one nectarfferous cell to another or to the exterior. They did not fuse with the plasmalemma when they left the cells. A great number of special "densely-stained cells" were different from the companion cells nearby the sieve elements. It was suggested that a tunnel be formed by these "densely-stained cells" connecting the stomata at the top of nectary leading the out-flow of nectar.     

春季-冻融交替时期9种落叶及半常绿小灌木抗寒性综合评价

在春季—冻融交替时期,以北京地区迎春、金叶女贞、小花溲疏等9种落叶及半常绿小灌木一年生枝条为试材,通过人工模拟降温,测定其在4 ~ -20℃低温胁迫下的6个生理响应指标,并对各指标进行相关性分析。结果表明,相对电导率、半致死温度、游离-脯氨酸含量和相对含水量这4个生理指标可作为9种小灌木抗寒性的评价指标。利用主成分分析、隶属函数法进行抗寒性综合评价,发现9种小灌木的抗寒性强弱顺序为：小花溲疏＞ 紫叶小檗＞ 太平花＞ 红王子锦带＞ 迎春＞ 连翘＞ 金叶女贞＞ 棣棠＞ 金山绣线菊。通过聚类分析可将9种小灌木分为3类,小花溲疏和紫叶小檗归为一类,其抗寒性相对较好;太平花、红王子锦带、迎春和连翘聚成一组,抗寒性一般;金叶女贞、棣棠和金山绣线菊聚在一起,抗寒性相对较差。     

春季—冻融交替时期9种落叶及半常绿小灌木抗寒性综合评价

在春季—冻融交替时期,以北京地区迎春、金叶女贞、小花溲疏等9种落叶及半常绿小灌木一年生枝条为试材,通过人工模拟降温,测定其在4~-20℃低温胁迫下的6个生理响应指标,并对各指标进行相关性分析。结果表明,相对电导率、半致死温度、游离-脯氨酸含量和相对含水量这4个生理指标可作为9种小灌木抗寒性的评价指标。利用主成分分析、隶属函数法进行抗寒性综合评价,发现9种小灌木的抗寒性强弱顺序为:小花溲疏紫叶小檗太平花红王子锦带迎春连翘金叶女贞棣棠金山绣线菊。通过聚类分析可将9种小灌木分为3类,小花溲疏和紫叶小檗归为一类,其抗寒性相对较好;太平花、红王子锦带、迎春和连翘聚成一组,抗寒性一般;金叶女贞、棣棠和金山绣线菊聚在一起,抗寒性相对较差。     

Freeze fracture immunocytochemistry of light-harvesting pigment complexes in a cryptophyte

Summary Immunocytochemical techniques using colloidal gold as the marker have been used to examine the location of the two light harvesting pigment-protein complexes in cryptophyte chloroplasts. A comparison of post-embedding thin section labelling and freeze fracture labelling has been carried out onRhodomonas salina using polyclonal antibodies to a chlorophylla/c ₂ light-harvesting complex, phycoerythrin and the -subunit of phycoerythrin. The effect of different fixation procedures on the intensity of labelling and ac curacy of antigen location have been examined and the effectiveness of uranyl acetate and tannic acid in improving both the preservation of thylakoid structure and labelling density of phycoerythrin has been demonstrated. Freeze fracture labelling gives better spatial res olution of the different antigens than post-embedding labelling, as well as better definition of thylakoid membranes. It confirms the location of phycoerythrin in the thylakoid lumen and the location of the chlorophylla/c ₂ LHC in both appressed and unappressed thylakoid membranes.Abbreviations PE phycoerythrin - chl chlorophyll - LHC light-har-vesting complex     

Further Characterization of In Vitro Conditions Appropriate for GABA Determination in Human CSF: Impact of Acid Deproteinization and Freeze/Thaw

Recently established standardized protocols for collection, handling, and storage of CSF for measurement of gamma-aminobutyric acid (GABA) have proven valuable in the characterization of various CNS disorders. In response to two recent reports which may have an impact on certain widely used protocols, we have, using the confirmed ion-exchange/fluorometric procedure, systematically evaluated the effects of deproteinization with various concentrations of sulfosalicylic acid (SSA) ranging from 0 to 10% (100 mg/ml), as well as the effects of freeze/thaw (F/T) on CSF GABA levels. Results of F/T studies documented that levels are stable to freezing and thawing. Acid deproteinization studies revealed the presence of an equilibrium between strictly free GABA, demonstrable only in acid-free CSF, and a very loosely bound form of GABA, fully demonstrable only in CSF deproteinized with concentrations of SSA above 1% (10 mg/ml). The relationship between GABA concentrations in undeproteinized and acid-deproteinized CSF revealed a highly significant (p less than .001) correlation, suggesting that alterations of central GABAergic activity would be reflected by either the level of strictly free GABA or free plus loosely bound GABA. This hypothesis was upheld in studies of patients with Parkinson's disease (PD) and Huntington's disease (HD), two neurologic disorders in which dysfunctions of the GABA system have been implicated. Results indicated that CSF GABA levels are significantly reduced in both PD and HD patients compared with neurologically normal controls, whether the measurement is of free GABA or free plus loosely bound GABA. Thus, we conclude that the level of strictly free GABA is stable to freezing and thawing and can only be accurately determined in nonacidified CSF; however, existing protocols employing deproteinization in 5% SSA yield data that provide an equally good reflection of central GABAergic transmission.     

Behavioural adaptations of Rana temporaria to cold climates

Environmental conditions at the edge of a species’ ecological optimum can exert great ecological or evolutionary pressure at local populations. For ectotherms like amphibians temperature is one of the most important abiotic factors of their environment as it influences directly their metabolism and sets limits to their distribution. Amphibians have evolved three ways to cope with sub-zero temperatures: freeze tolerance, freeze protection, freeze avoidance. The aim of this study was to assess which strategy common frogs at mid and high elevation use to survive and thrive in cold climates. In particular we (1) tested for the presence of physiological freeze protection, (2) evaluated autumnal activity and overwintering behaviour with respect to freeze avoidance and (3) assessed the importance of different high-elevation microhabitats for behavioural thermoregulation. Common frogs did not exhibit any signs of freeze protection when experiencing temperatures around 0 °C. Instead they retreated to open water for protection and overwintering. High elevation common frogs remained active for around the same period of time than their conspecifics at lower elevation. Our results suggest that at mid and high elevation common frogs use freeze avoidance alone to survive temperatures below 0 °C. The availability of warm microhabitats, such as rock or pasture, provides high elevation frogs with the opportunity of behavioural thermoregulation and thus allows them to remain active at temperatures at which common frogs at lower elevation cease activity.     

Freeze Thaw: A Simple Approach for Prediction of Optimal Cryoprotectant for Freeze Drying

The present study evaluates freeze thaw as a simple approach for screening the most appropriate cryoprotectant. Freeze–thaw study is based on the principle that an excipient, which protects nanoparticles during the first step of freezing, is likely to be an effective cryoprotectant. Nanoparticles of rifampicin with high entrapment efficiency were prepared by the emulsion-solvent diffusion method using dioctyl sodium sulfosuccinate (AOT) as complexing agent and Gantrez AN-119 as polymer. Freeze–thaw study was carried out using trehalose and fructose as cryoprotectants. The concentration of cryoprotectant, concentration of nanoparticles in the dispersion, and the freezing temperature were varied during the freeze–thaw study. Cryoprotection increased with increase in cryoprotectant concentration. Further, trehalose was superior to fructose at equivalent concentrations and moreover permitted use of more concentrated nanosuspensions for freeze drying. Freezing temperature did not influence the freeze–thaw study. Freeze-dried nanoparticles revealed good redispersibility with a size increase that correlated well with the freeze–thaw study at 20% w/v trehalose and fructose. Transmission electron microscopy revealed round particles with a size ∼400 nm, which correlated with photon correlation spectroscopic measurements. Differential scanning calorimetry and X-ray diffraction suggested amorphization of rifampicin. Fourier transfer infrared spectroscopy could not confirm interaction of drug with AOT. Nanoparticles exhibited sustained release of rifampicin, which followed diffusion kinetics. Nanoparticles of rifampicin were found to be stable for 12 months. The good correlation between freeze thaw and freeze drying suggests freeze–thaw study as a simple and quick approach for screening optimal cryoprotectant for freeze drying.     

Freeze branding of juvenile salmon

Freeze branding using liquid nitrogen as a coolant was carried out on salmon parr and a sequential histopathological study was carried out for 16 weeks. The initial darkening of the brand area was due to destruction of melanophore control but the later, more diffuse, pattern was related to the invasion of the area of the stratum spongiosum and hypodermis by melanin containing cells such as are commonly found in healing teleost wounds. The traumatic damage was completely resolved well before the end of the experiment, suggesting that freeze branding is a particularly suitable short term batch marking method.     

Freeze Preservation of Cultured Plant Cells

A basic technique for successful freeze preservation and storage at -196°C of cultured plant cells and an assay of percentage survival following the freezing-storage-thawing procedure are described. These techniques have been applied to suspension cultures of carrot (3 cell lines), belladonna and sycamore. Dimethyl sulfoxide (DMSO) and glycerol, when appropriately applied, were the most effective cryoprotectants tested. Although these cryoprotectants were of low toxicity and did not cause alterations in the cytology and growth potential of the recovered cells, the cell lines differed in their sensitivity to the toxicity of these cryoprotectants. Small meristematic cells survived the freezing-thawing procedure better than larger more highly vacuolated cells. Specific differences in survival are in part explained in terms of differences in cell morphology.     

Freeze Fracture of Intact Plant Tissues

A procedure for dissolving and handling replicas of cutinized leaves and other plant tissues is described. This technique yields comparatively large replicas which can include vascular tissue, epidermal cells and the cuticle. Dissolution of the tissue involves the sequential use of alcoholic KOH, sulfuric acid and chromic acid. The use of clean, uncoated grids for transfer of the tissue and replicas results in rapid, easy handling with a minimum of breakage or loss. The procedure is more efficient than previous methods and produces large replicas. This allows tissue orientation and more positive identification of cell types for better correlation of freeze fracture results with thin sections of similar material.