Preservation of tissue mucins by freeze-drying and vapour fixation

Summary Histochemical studies previously undertaken showed that tissue mucins (glycoproteins) of the rat submaxillary salivary gland and other organs are preserved very satisfactorily by formaldehyde vapour treatment applied after freeze-drying of the tissue.It was thought desirable to confirm these histochemical findings by quantitative chemical data. This was performed by studying the effect of formaldehyde vapour treatment on the solubility of proteins in the freeze-dried rat submaxillary gland.Large quantities of protein (about 30 to 60 per cent of the dry weight) could be removed by aqueous extraction from the freeze-dried control samples, which had not received any formaldehyde vapour treatment, but very little protein (about 0.5 to 4 per cent of the dry weight) could be extracted from those samples which had been exposed to this vapour at 50°C for 3 hours.Each of the experiments performed confirmed this overall picture, but there were differences in the amount of protein extracted among the control samples, as well as among the formaldehyde vapour treated ones; it has been suggested that these differences were due to variations in the proportion and/or type of protein present, most probably caused by fluctuations in the content of secretory mucins.In part fulfilment for the Doctorate of Philosophy, University of London.     

Proceedings: Preservation of bacteriophages by freezing and freeze-drying

Losses in titer determined before and after freezing and freeze-drying of bacteriophages were matched with the following known characters of the phages: particle morphology, size, chloroform sensitivity, nucleic acid content, and “osmotic” sensitivity. A high percentage of morphological type A phages are unusually sensitive to freezing and freeze-drying; type B phages are less sensitive. Large phages do not freeze-dry as well as small ones. Osmotically sensitive phages are not preserved as successfully as osmotically resistant ones.     

Changes in the cell membrane of Lactobacillus bulgaricus during storage following freeze-drying

Summary The mechanism of inactivation of freeze-dried Lactobacillus bulgaricus during storage in maltodextrin under controlled humidity was investigated. Evidence is presented supporting the hypothesis that membrane damage occurs during storage. A study on the lipid composition of the cells by gas chromatography showed a decrease in the unsaturated and saturated fatty acid content of the cell. Further evidence indicating membrane damage includes a decrease in membrane bound proton-translocating ATPase activity.     

Survival rate of microbes after freeze-drying and long-term storage

The survival rates of 10 species of microorganisms were investigated after freeze-drying and preserving in a vacuum at 5 degrees C. The survival rates varied with species. The survival rates immediately after freeze-drying were different among yeast, gram-positive bacteria, and gram-negative bacteria, and the change in the 10-year survival rate was species-specific. The survival rate of yeast, Saccharomyces cerevisiae, was about 10% immediately after drying, and the rate did not decrease significantly during the 10-year storage period. Survival rates after the drying of gram-positive bacteria, i.e., Brevibacterium flavum, B. lactofermentum, Corynebacterium acetoacidophilum, C. gultamicum, and Streptococcus mutans, were around 80%. The survival rate of Brevibacterium and Corynebacterium did not decrease greatly during the storage period, whereas the rate of S. mutans decreased to about 20% after 10 years. Survival rates after the drying of gram-negative bacteria, i.e., Escherichia coli, Pseudomonas putida, Serratia marcescens, and Alcaligenes faecalis, were around 50%. The survival rate decreased for the first 5 years and then stabilized to around 10% thereafter.     

Preservation of integrity of the inner mitochondrial membrane after freeze-thawing and freeze-drying

The results of this paper illustrate that trehalose partially preserves inner mitochondrial membrane integrity after freeze-thawing and freeze-drying with subsequent rehydration in water. The 2,4-dinitrophenol stimulation of ATPase activity was used as a criterion for membrane integrity. The results show that ATPase activity of lyophilized-rehydrated mitochondria was stimulated up to two to three times.     

Preservation of functional integrity during long term storage of a biological membrane

Sarcoplasmic reticulum vesicles freeze-dried in the presence of trehalose retain most of their original biological activity for short periods. When the dry vesicles are stored for longer periods in air, Ca2+-transport becomes uncoupled from ATPase activity within a few days. However, when they are stored under vacuum, ATPase activity, Ca2+ transport, and coupling between Ca2+ transport and ATP utilization are maintained essentially intact for at least 110 days.     

Preservation of functional integrity during long term storage of a biological membrane

Sarcoplasmic reticulum vesicles freeze-dried in the presence of trehalose retain most of their original biological activity for short periods. When the dry vesicles are stored for longer periods in air, Ca²⁺-transport becomes uncoupled from ATPase activity within a few days. However, when they are stored under vacuum, ATPase activity, Ca²⁺ transport, and coupling between Ca²⁺ transport and ATP utilization are maintained essentially intact for at least 110 days.     

Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying

Sugars inhibit protein unfolding during the drying step of lyophilization by replacing hydrogen bonds to the protein lost upon removal of water. In many cases, polymers fail to inhibit dehydration-induced damage to proteins because steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been shown to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into subunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically distinguishable hybrid tetramers during reversible dissociation. We examined hybridization and recovery of catalytic activity during freeze-thawing and freeze-drying in the presence of polymers (dextran, Ficoll, and polyethylene glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80, Brij 35, hydroxy-propyl beta-cyclodextrin). The surfactants did not protect LDH during freeze-thawing or freeze-drying. Rather, in the presence of Brij 35, enhanced damage was seen during both freeze-thawing and freeze-drying, and the presence of Tween 80 exacerbated loss of active protein during freeze-drying. Polymers and sugars prevented dissociation of LDH during the freezing step of lyophilization, resulting in greater recovery of enzyme activity after lyophilization and rehydration. This beneficial effect was observed even in systems that do not form glassy solids during freezing and drying. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dissociated monomers. Polymers inhibit freezing-induced dissociation thermodynamically because they are preferentially excluded from the surface of proteins, which increases the free energy of dissociation and denaturation.     

Proceedings: Preservation of marine and fresh water algae by means of freezing and freeze-drying

Several species of marine and fresh water algae have been isolated from various habitats. Recently they were examined for their viability after freezing and freeze-drying procedures.The addition of suspending agents to algal cultures has resulted in greater viability for most of the green algae, but has shown little effect on the blue-green algae.It is considered that the preservation of algae by means of freezing and freeze-drying procedures are of great benefit as they offer the possibility of long-term preservation of viable collections, especially for patent depository for industrial applications.     

Preservation of mitochondrial structure and function after Bid- or Bax-mediated cytochrome c release

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1-mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.     

The function of the suspending medium during the freeze-drying preservation of Escherichia coli

The role of added solutes in the freeze-drying preservation of bacteria is examined. Escherichia coli were washed, suspended in solutions of selected hydroxy-substituted compounds of various molecular weights, and frozen at rates of the order of one degree C per minute. The frozen materials were freeze-dried and rehydrated in several different ways. Freeze-drying survival was correlated with the development and persistence of an amorphous solute matrix and the desorption of residual water. Protection from potentially harmful effects of freeze-drying was attributed to the dispersion, by the aforementioned amorphous matrix, of metabolites released from the cells during the preparation, and freezing of the bacterial suspensions.     

Significance of freeze-drying in long term storage of date palm pollen

The authors attempt to determine the optimal conditions for maintaining long term viability of date palm pollen using a special method of freeze-drying. They have undertaken a systematic study of the viability over time of a sample of the same pollen stored and conserved under different conditions (freeze-drying, temperature, gaseous atmosphere and storage time).

The results are thoroughly analysed by means of different approaches of multiparametric modelisation. These numerical treatments permit us to:

Les auteurs ont déterminé, par une méthode particulière de lyophilisation, les conditions optimales permettant le maintien de la viabilité du pollen du palmier dattier (Phoenix dactylifera) à long terme. Une étude systématique a été réalisée sur des lots provenant d'un même échantillon de pollen conservé et stocké selon différentes conditions: (lyophilisation, température, atmosphère gazeuse et en fonction du temps de stockage).

Ils ont cherché à analyser de manière approfondie les résultats obtenus au moyen de plusieurs approches de modélisation multiparamétrique.

Ces traitements numériques ont permis:

1. demonstrate that such a phenomenon submits to the usual laws and can be modelled

2. propose a general approach for the study of the ageing of pollen with time and with respect to different conservation and storage conditions.

1. de démontrer qu'un tel phénomène obéit globalement à des lois statistiques simples, et qu'il est de ce fait modélisable

2. de proposer une approche plus générale du vieillissement des grains de pollen en fonction du temps et des conditions de conservation et de stockage.     

Preservation of liver mitochondria function in hemorrhage-resistant rats during severe long-term hypotension

In contrast to the reported data evidencing early impairment of the liver mitochondrial function in the Wiggers model of hemorrhagic shock at the arterial blood pressure 30-40 mm Hg, lasting not over 6 h, a group of hemorrhage-resistant rats was discovered. In these rats, the lifetime was about 20 h, with the blood pressure being the same as indicated above. Rectal temperature decreased to 24-25 degrees C during shock. No substantial disorders were recorded in oxidative phosphorylation and ATPase activity of the mitochondria isolated from the liver in the irreversible stage of shock (70% blood return) or in the terminal state of animals. It is assumed that hypothermia plays the protective role. The conclusion is made that the damage to the mitochondria is not indispensable factor of the development of irreversible shock.