A procedure for the rapid freezing of whole embryos

Summary A procedure for the rapid freezing of whole chick embryos for histochemical treatment is described. The problems of deformation during preparation for quenching and orientation for sectioning have been largely overcome by placing embryos inside lengths of chicken trachea. The subsequenct disorientation of tissues that follows cracking and shattering due to the rapid freezing of whole embryos is avoided. The method permitted a more precise identification of the position and time of appearance of formaldehyde-induced fluorescence and myosin antibody immunofluorescence in serially sectioned embryos.We gratefully thank Dr Ute Gröschel-Stewart for preparing sections for immunofluorescence.     

A procedure for the rapid freezing of whole embryos.

A procedure for the rapid freezing of whole chick embryos for histochemical treatment is described. The problems of deformation during preparation for quenching and orientation for sectioning have been largely overcome by placing embryos inside lengths of chicken trachea. The subsequent disorientation of tissues that follows cracking and shattering due to the rapid freezing of whole embryos is avoided. The method permitted a more precise identification of the position and time of appearance of formaldehyde-induced fluorescence and myosin antibody immunofluorescence in serially sectioned embryos.     

Preservation of hematopoietic stem cells by slow freezing and elimination of the heat of fusion]

Previous experiments, in dogs with fresh bone marrow or bone marrow frozen with a modified freezing system have demonstrated a 100% recovery of frozen stem cells, stored for periods up to 5 months. Five patients, three with drug resistant acute leukemia and two with metastic carcinomas, have been treated with a high dose combination chemotherapy regimen (TACC) followed by reinfusion of marrow cryopreserved with the same modified freezing system. Following the reinfusion of marrow, autologous engraftment was demonstrated on bone marrow aspiration between days 5 and 10.     

Cryo-bioorganic chemistry: freezing effect on stereoselection of l- and dl-leucine cooligomerization in aqueous solution

The chirality of l-/dl-leucine (50–50%) cooligomerization was investigated in liquid and frozen aqueous solutions. Cooligomerization was carried out by carbonyldiimidazole activation without initiator at an ambient (+22°C) and frozen (−18°C) temperature, respectively. The separated samples obtained after different time intervals of treatment were completely hydrolyzed (HCl) and the diastereomeric l- and d-leucine derivates of Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-l-alanine amide) were then traced and evaluated by RP-HPLC analysis. After 9 days of oligomerization, the l-Leu content was slightly enhanced in the liquid (57%) and somewhat more enhanced in the frozen (64%) samples. After 17 days, however, the l-Leu content had decreased in the liquid (53%) and frozen (56%) conditions. These l-enantiomer amplifications indicate that an l-antipode is preferentially incorporated into the α-helical turn of the oligomer in the earlier stage of cooligomerization, while, later, the d-antipode is also incorporated. The role of ice in the improved stereoselection is discussed. This is the first recorded example of the effect of freezing on stereoselection. Received 27 October 2000; revised 11 December 2000; accepted 4 January 2000     

Adrenergic nerves and mast cells after skin freezing. A hypothesis based on fluorescence microscope observations in the rat

Summary After freezing and thawing of rat skin, degranulation and disappearance of mast cell fluorescence became apparent in the skin up to 1 h after thawing. Gradual disappearance of catecholamines from the adrenergic nerves of the injured area occurred during the 1st 24 h. Both mast cells and adrenergic nerves may play a role in tissue destruction after freezing injury.Supported by grants from the Research Foundation of Oy. Orion, the Paolo Foundation and the Finish Medical Foundation.     

Variation in the number and size of myelinated nerve fibers regenerated after repeated localized freezing of the sciatic nerve of rats]

The number and size of myelinated fibres have been determined in the nerve to medial head of the gastrocnemius muscle of Rats in which the sciatic nerve had been frozen locally 1 to 5 times at monthly intervals. The contralateral nerve was used as a control. When the measurements were made one month after the last freezing, the number of fibres increased progressively until the 3rd freezing, reaching about 220% of the normal value. No higher values were observed after 4 or 5 freezings. The mean diameter of fibres decreased with the number of operations. When the measurements were made 3, 6, 12 or 18 months after the 3rd freezing, the number of fibres decreased by about 30% between the 1st and the 3rd month and then stabilized. The mean diameter of fibres increased progressively. However, at the 18th month, the size of the myelinated fibres had not reached the normal value.     

La conservation de la vie par le froid

Summary It has been found that, by addition of a certain amount of glycerol, it is possible to store living animal tissues in the frozen state over very long periods of time. The cooling velocity, the temperature of storage and the rate of thawing are important factors. Under the optimal conditions, experiments done on the heart of the chick embryo have shown that complete recovery of the normal physiological activities can be achieved after freezing in liquid nitrogen.     

Eine neue Methode zum schnellen Einfrieren von Geweben

Summary A new unit for the rapid freezing of tissues in liquid nitrogen is described.     

Schnelles Einfrieren von Geweben und Flüssigkeiten

Summary A new unit for rapid freezing of tissues and liquids in liquid nitrogen is described.     

Cryo-bioorganic chemistry: molecular interactions at low temperature

Freezing of aqueous or organic solutions plays a pivotal role in enhancement of rate and/or yield of biomolecular reactions. The smooth conditions of the frozen state at low temperature can also suppress racemization and side-product formation of the reactions. Molecular interactions in liquid undercooled solutions, on the other hand, offer the possibility to study enzyme activity mechanisms in vitro and a chance for survival of organisms in vivo. This review illustrates the differences between frozen and liquid conditions on several small and large biomolecules, together with the synthetic use of freezing. In relation to the freezing effect on enzyme activity, a peculiar phenomenon is discussed: 'cryo-oscillations' are temporal motions of trypsin activity in frozen solution in the presence of Mn2+ ion. The molecular basis of cold adaptation is also discussed, which points to mechanisms evolved by organisms living at subzero temperatures. The factors involved in the freezing effect are shown; i.e. the role of freeze-concentration and frozen solvent surface is demonstrated and elucidated using several examples.     

The Ehrenfest Classification of Phase Transitions: Introduction and Evolution

The first classification of general types of transition between phases of matter, introduced by Paul Ehrenfest in 1933, lies at a crossroads in the thermodynamical study of critical phenomena. It arose following the discovery in 1932 of a suprising new phase transition in liquid helium, the “lambda transition,” when W. H. Keesom and coworkers in Leiden, Holland observed a λhaped “jump” discontinuity in the curve giving the temperature dependence of the specific heat of helium at a critical value. This apparent jump led Ehrenfest to introduce a classification of phase transitions on the basis of jumps in derivatives of the free energy function. This classification was immediately applied by A.J. Rutgers to the study of the transition from the normal to superconducting state in metals. Eduard Justi and Max von Laue soon questioned the possibility of its class of “second-order phase transitions” -- of which the “lambda transition was believed to be the arche type -- but C.J. Gorter and H.B.G. Casimir used an “order parameter to demonstrate their existence in superconductors. As a crossroads of study, the Ehrenfest classification was forced to undergo a slow, adaptive evolution during subsequent decades. During the 1940s the classification was increasingly used in discussions of liquid-gas, order-disorder, paramagnetic-ferromagnetic and normal-super-conducting phase transitions. Already in 1944 however, Lars Onsagers solution of the Ising model for two-dimensional magnets was seen to possess a derivative with a logarithmic divergence rather than a jump as the critical point was approached. In the 1950s, experiments further revealed the lambda transition in helium to exhibit similar behavior. Rather than being a prime example of an Ehrenfest phase transition, the lambda transition was seen to lie outside the Ehrenfest classification. The Ehrenfest scheme was then extended to include such singularities, most notably by A. Brain Pippard in 1957, with widespread acceptance. During the 1960s these logarithmic infinities were the focus of the investigation of “scaling” by Leo Kadanoff, B. Widom and others. By the 1970s, a radically simplified binary classification of phase transitions into “first-order” and “continuous” transitions was increasingly adopted.     

Zur Methodik der Gefrierschnitt-Mikroautoradiographie

Summary A microautoradiographic technique for frozen sections is described. The method makes it possible to follow the distribution of inhaled radon in the organism with high degree of accuracy. After inhaling radon, the mice were killed and stored in liquid air (freezing method ofRajewsky ⁹). The organ sections (5µ thick) were prepared by means of a freezing microtome in the Kryostat. A thin fixing and protecting layer of chromic alum gelatine was placed between the tissue-sections and the photographic emulsion. The exposure took place in an atmosphere of nitrogen.     

Un développement nouveau de la lyophilisation: La cryodessiccation des systèmes non aqueux

Summary A new development is made to enlarge the field of lyophilization using non-aqueous media. Unstable compounds are preserved by freezing and drying their solutions in organic solvents. The same technique applied to chemicals dissolved in liquid ammonia enables the preparation of stable concentrated free radicals, due to the very low temperature of sublimation of frozen NH₃. Finally, experiments are made demonstrating the possibilities of high velocity, low temperature sublimation of solutions in solid CO₂ at normal atmospheric pressure.      

Measuring cellulose decomposition using Benchkote-paper,for the estimation of soil pollution with copper

Summary Cellulose decomposition in paddy soil polluted by copper was determined by the use of polyethylene-backed absorbent paper (Benchkote) as a source of cellulose. It was shown that the paper is a suitable material for measuring the rate of cellulose decomposition. Correlations were found between copper content in the soil, the growth of rice plants and the cellulose decomposition rate.Acknowledgment. Gratitude is extend to Dr M.H. Martin, Department of Botany, University of Bristol, England, for suggesting the use of Benchkote paper.     

Isolation of an antifreeze peptide from the Antarctic sponge <Emphasis Type="Italic">Homaxinella balfourensis</Emphasis>

Polar plants and animals survive in subzero waters (-2 degrees C) and many of these marine organisms produce antifreeze proteins (AFPs) to better adapt themselves to these conditions. AFPs prevent the growth of ice crystals which disrupt cellular membranes and destroy cells by inhibiting crystallization of water within the organism. The hydrophilic extract of an Antarctic sponge Homaxinella balfourensis exhibited a non-colligative freezing point depression effect on the crystal morphology of water. The extract was purified by repeated reverse phase high-pressure liquid chromatography, then assayed and shown to contain several AFPs. The major peptide was isolated, analyzed using matrix-assisted laser desorption ionization mass spectrometry and the partial structure of the peptide identified through amino acid sequencing. AFPs have potential applications in agriculture, medicine and the food industry.     

Modifications in the type distribution of muscle fibers induced by repeated local freezings the rat sciatic nerve]

In the Rat, after a localized freezing of the sciatic nerve inducing a complete denervation of the medial head of the gastrocnemius muscle, the reinnervation took place within 16-18 days under our experimental conditions. After only one freezing, a limited and stable "type-grouping" of muscle fibres is observed from 30 to 360 days. After 2 to 5 freezing repeated every three weeks, the muscular changes observed one month after the last freezing are much more pronounced than when only one freezing is performed. These changes consist of a progressive increase in the number of type I and type II C fibres. This transformation is not stable: 3 months after the 3rd freezing, the muscle pattern does not differ from that which is noted after a single freezing.     

The volcanoes of Auvergne

It is with good reason that the name Rutherford is closely linked with the early history of the alpha particle. He discovered them, determined their nature, and from 1909 used them to probe the structure of the atom. From 1898 to 1902 Rutherford construed alpha radiation as a type of non-particulate Röntgen radiation. On his theory of the locomotion of radioactive particles Rutherford proposed that alpha radiation consisted of negatively charged particles. During 1902 he confirmed the particulate nature of alpha radiation but discovered that these alpha particles were positively charged. Although Rutherford suspected from 1903 that these alpha particles were related somehow with helium, the proof required six long years of investigation. By mid-1908 it seemed certain that the alpha particle possessed two units of the elementary charge. Since the e/m ratio had already been determined for alpha particles, this evidence enhanced the suspected connection with helium. However, this gain and loss of charge was still construed as an ionization effect. Since as late as 1908 gaseous ionization was assumed to involve the gain or loss of a single unit of charge, Rutherford's alleged case of doubly ionized alpha particles was presumably an exception. Yet helium was known to be an inert gas and thus hardly a likely candidate for such exceptional ionization behaviour. To establish the connection, therefore, Rutherford resorted to a spectroscopic test. He collected spent alpha particles shot into a thin glass tube and gradually observed the spectrum of helium. Rutherford had thus been correct in his assumption, but a proper explanation was possible only after the confirmation of the nuclear structure of the atom.     

James Franck,the ionization potential of helium,and the experimental discovery of metastable states

In 1920, James Franck together with Fritz Reiche and Paul Knipping found strong experimental evidence that the lowest-lying triplet state in helium is metastable—an atom in this state cannot make a spontaneous transition to the ground state. Even though their evidence was entirely experimental, they tied their results almost inextricably to Alfred Landé’s 1919 model of the helium atom, and in the process, misunderstood the new theoretical selection rules of Adalbert Rubinowicz and Niels Bohr. In an additional complication, experiments of the English physicists Frank Horton and Ann Catherine Davies contradicted Franck’s. Although Franck’s result has held up, the reasons for the discrepancies remain unclear.     

Understanding macroscopic quantum phenomena: The history of superfluidity 1941–1955

In this paper we attempt to investigate the historical and methodological aspects of the developments related to superfluid helium, concentrating on the period between 1941 and 1955. During this period, the various developments constituted a series of steps towards redefining and refining the two-fluid concept devised to explain the unexpected macroscopic behaviour of superfluid helium. The idea that superfluids are essentially ‘quantum structures on a macroscopic scale’ functioned as a heuristic principle which guided the theoretical physicists engaged in the above research programme.     

Freezing and transplantation of brain tissue in rats

Summary Neocortical tissue obtained from rat embryos was frozen and stored at –70°C for 6 h prior to transplantation into the cerebellum of neonatal rats. Growth, differentiation, and integration of this tissue within the host brain was comparable to that obtained from freshly dissected and transplanted tissue. It is suggested that freezing to low temperatures does not adversely effect the viability or transplantability of the neural tissue.Supported by N.I.H. research grants Nos NS-08817 and CA-14650.