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PubMedDate not supplied

The storage of cells from different tumor lines in a mechanical freezer at -80 degrees C. Comparison to cryopreservation in liquid nitrogen.

This paper demonstrates that it is possible to store cells from different tumor lineages in a mechanical freezer at -80 degrees C. Tumor cells cryopreserved without controlled-rate freezing in medium containing 10% dimethyl sulfoxide (DMSO) and 10% Bovine Calf Serum (BCS) for 6 months displayed similar post-thawing viability as cells cryopreserved in liquid nitrogen at -196 degrees C. We recommend this method of cryopreservation as a convenient and less expensive alternative to liquid nitrogen storage. Further studies concerning the maximum storage time at -80 degrees C and studies comparing the optimal composition of the storage medium will further define the limitations and potential of this method.

Animals↗
PubMedDate not supplied

Comparative evaluation of fresh and washed human sperm cryopreserved in vapor and liquid phases of liquid nitrogen.

Infectious organisms such as hepatitis B were recently shown to survive in liquid nitrogen. To prevent cross-contamination of semen samples via liquid nitrogen, studies were undertaken to evaluate human sperm survival in the vapor phase of liquid nitrogen at -189 degrees C. The study was conducted in 2 separate experiments. In the first experiment, a total of 30 unwashed, fresh semen samples (15 normozoospermic and 15 oligozoospermic) were evaluated for motility, vitality, and morphology after freeze-thaw survival in vaporous (-189 degrees C) and liquid nitrogen (-196 degrees C; control) phases. Similar evaluations were carried out in a second experiment on 27 samples (15 normozoospermic and 12 oligozoospermic) that were previously washed by the swim-up method. Motile sperm recovery rates were significantly different between liquid and vapor phases (unwashed, normozoospermic: 42.76% +/- 3.23% vs 45.52% +/- 4.44%, P < .05; washed, normozoospermic: 34.44% +/- 4.41% vs 37.58% +/- 3.90%, P < .05; unwashed, oligozoospermic: 16.53% +/- 3.34% vs 18.25% +/- 4.36%, P < .05; washed, oligozoospermic: 10.32% +/- 2.54% vs 12.25% +/- 2.81%, P < .05). Recovery rates for motility were much higher for unwashed samples compared with washed semen samples. In all experiments the recovery of normal and live forms showed no significant differences between the vapor and liquid nitrogen storage phases (P > .05). The results demonstrate that both washed and unwashed human sperm survive satisfactorily with good recovery in the vapor of liquid nitrogen and can be recommended for future storage in medically assisted conception programs.

Adult↗
PubMed2002

The role of the megagametophyte in maintaining loblolly pine (Pinus taeda L.) seedling arginase gene expression in vitro.

Following loblolly pine (Pinus taeda L.) seed germination, storage-protein breakdown in the megagametophyte and in the seedling results in a large increase in the seedling's free amino acid pool. A substantial portion of both the storage proteins and the amino acid pool is arginine, a very efficient nitrogen-storage compound. Free arginine is hydrolyzed in the seedling by the enzyme arginase (EC 3.5.3.1), which is under strong developmental control. At present, regulation of arginase in conifers is not well understood. Here we report the utilization of an in vitro culture system to address the separate impacts of the seedling and megagametophyte tissues on arginase enzyme activity, protein levels and patterns of gene expression. We also describe the generation of an anti-arginase antibody prepared from a histidine-tagged loblolly pine arginase fusion protein expressed in Escherichia coli. Our results indicate that arginase gene expression in the seedling is initiated by the seedling itself and then maintained or up-regulated by the megagametophyte. The contribution of storage-protein breakdown and the free amino acid pool, particularly arginine, in this regulation is also addressed.

Arginase↗
PubMed1986

Measurement of fibronectin in human body fluids.

Two facts must be taken into consideration when quantifying fibronectin in biological samples: the sensitivity of the assay method and the appropriate handling of samples. A three-antibody, non-competitive ELISA was used in the present study. This procedure offers a simple, inexpensive and very sensitive technique for evaluating fibronectin: reproducible standard curves in the range 5-100 micrograms/l, 3% variability in the assay, and a detection limit of 0.5 ng/well that allows accurate measurement of fibronectin in segmental bronchoalveolar lavage and in ascites liquid samples. Typical dilution ranged from 1/10000 for plasma to 1/100 for bronchoalveolar lavages. For long-term storage of samples two procedures are recommended. First, a rapid freezing with liquid nitrogen, storage at -20 degrees C and thawing once at 37 degrees C avoiding refreezing. Losses of immunoreactive fibronectin after 35 days storage were 15, 15 and 22% in plasma, bronchoalveolar lavages and ascites fluid, respectively. Alternatively, samples can be stored as ammonium sulphate precipitates. This allows the easy availability of the samples when assays have to be repeated, and avoids fractionation in vials and freeze-thawing cycles. Using this storage procedure, losses are, however, slightly higher (22, 25, 25%, respectively, after 35 days).

Enzyme-Linked Immunosorbent Assay↗
PubMed1997

Type 1 ribosome-inactivating proteins are the most abundant proteins in iris (Iris hollandica var. Professor Blaauw) bulbs: characterization and molecular cloning.

The most abundant protein of Iris bulbs has been identified as a type 1 ribosome-inactivating protein (RIP). Analysis of the purified proteins and molecular cloning of the corresponding cDNAs demonstrated that this type 1 RIP is a mixture of three isoforms that exhibit a high degree of sequence identity and have similar, though not identical, ribosome-inactivating and polynucleotide:adenosine glycosidase activities. The accumulation of large quantities of type 1 RIP in a vegetative storage organ suggests that this presumed defence-related protein also plays a role in the nitrogen-storage metabolism of the bulb.

Amino Acid Sequence↗
PubMed1966

Long-term preservation of fungus cultures with liquid nitrogen refrigeration.

A preservation technique was tested on 162 strains of culturally fastidious fungi sensitive to lyophilization, representing five classes. The results indicated that liquid nitrogen storage of frozen specimens may be used as an alternative to lyophilization for long-term preservation of stock cultures of fungi. The fungus was frozen in 10% (v/v) glycerol-water menstruum in heat-sealed ampoules. The cooling from ambient temperatures to -35 C was controlled at a rate of approximately 1 C per minute. Further cooling to the storage temperature of -165 to -196 C was uncontrolled and took place at an accelerated rate. Frozen ampoules were thawed in a water bath at 38 to 40 C. Viable and unmutated cultures were developed from reactivated specimens after storage for as long as 5 years.

Freezing↗
PubMed1991

Methyl jasmonate treatment eliminates cell-specific expression of vegetative storage protein genes in soybean leaves.

Soybean (Glycine max) plants accumulate a vacuolar glycoprotein in the parenchymal cells of leaves, petioles, stems, seed pods, and germinating cotyledons that acts in temporary nitrogen storage during vegetative growth. In situ immunolocalization of this vegetative storage protein (VSP) revealed that it accumulates in those parenchymal cells in close proximity to existing and developing vasculature, as well as in epidermal and cortical cells. The protein was more prevalent in younger, nitrogen-importing tissues before pod and seed development. Removal of actively growing seed pods greatly enhanced VSP accumulation, primarily in bundle sheath and paraveinal mesophyll cells. In situ hybridization of a VSP RNA probe to mRNA in leaf sections demonstrated that cell-specific mRNA accumulation corresponded with the pattern of protein localization. Treatment of leaf explants with 50 micromolar methyl jasmonate resulted in accumulation of VSP mRNA and protein in all cell types.

Journal Article↗
PubMed2006

PII-regulated arginine synthesis controls accumulation of cyanophycin in Synechocystis sp. strain PCC 6803.

Cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is a nitrogen storage polymer found in most cyanobacteria and some heterotrophic bacteria. The cyanobacterium Synechocystis sp. strain PCC 6803 accumulates cyanophycin following a transition from nitrogen-limited to nitrogen-excess conditions. Here we show that the accumulation of cyanophycin depends on the activation of the key enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase, by signal transduction protein PII.

Arginine↗
PubMed1981

Pretransplantation treatment of human teeth in tissue culture combined with storage in liquid nitrogen. A scanning electron microscopic study.

A combined method of tissue culture and storage in liquid nitrogen is described, which is suitable for covering the root surface of transplantable teeth with a dense cell layer: after an initial period of 10 days in tissue culture the teeth are stored in liquid nitrogen. 10 days before a transplantation the teeth are thawed and cultured again for 10 days. By this pretransplantation treatment a densely grown periodontal cell layer as well as an unlimited storage time are achieved.

Culture Techniques↗
PubMed2004

Nitrogen- and storage-affected carbohydrate partitioning in high-light-adapted Pelargonium cuttings in relation to survival and adventitious root formation under low light.

BACKGROUND AND AIMS: The aim of this study was to determine the role of nitrogen- and storage-affected carbohydrate availability in rooting of pelargonium cuttings, focusing on the environmental conditions of stock plant cultivation at low latitudes, transport of cuttings, and rooting under the low light that prevails during the winter rooting period in Central European greenhouses. METHODS: Carbohydrate partitioning in high-light-adapted cuttings of the cultivar 'Isabell' was studied in relation to survival and adventitious root formation under low light. Effects of a graduated supply of mineral nitrogen to stock plants and of cutting storage were examined. KEY RESULTS: Nitrogen deficiency raised starch levels in excised cuttings, whereas the concentrations of glucose and total sugars in leaves and the basal stem were positively correlated with internal total nitrogen (Nt). Storage reduced starch to trace levels in all leaves, but sugar levels were only reduced in tissues of non-nitrogen deficient cuttings. Sugars accumulated in the leaf lamina of stored cuttings during the rooting period, whereas carbohydrates were simultaneously exhausted in all other cutting parts including the petioles, thereby promoting leaf senescence. The positive correlation between initial Nt and root number disappeared after storage. Irrespectively of storage, higher pre-rooting leaf glucose promoted subsequent sugar accumulation in the basal stem and final root number. The positive relationships between initial sugar levels in the stems with cutting survival and in leaves with root formation under low light were confirmed in a sample survey with 21 cultivars provided from different sources at low latitudes. CONCLUSIONS: The results indicate that adventitious rooting of pelargonium cuttings can be limited by the initial amount of nitrogen reserves. However, this relationship reveals only small plasticity and is superimposed by a predominant effect of carbohydrate availability that depends on the initial leaf sugar levels, when high-light adaptation and low current light conditions impair net carbon assimilation.

Adaptation, Physiological↗
PubMed1982

Growth and body composition during long-term total parenteral nutrition in the rat.

Detailed metabolic data indicating the adequacy of long-term total parenteral nutrition in the rat are not available. In this study an aseptic catheterization technique and a presumed adequate intravenous diet were used to support the nutrition of growing male Fischer 344 rats for a 21-day period. These animals were compared with sham-operated rats after 21 days of ad libitum access to one of two adequate oral diets. Total parenteral nutrition rats demonstrated weight gains that were similar to those in the rats consuming the oral diets. Body composition and nitrogen storage studies indicated fat and protein accumulation in total parenteral nutrition rats that were similar to those in the orally fed animals, but with a tendency toward increased fat and decreased nitrogen retention. These data indicate that apparently normal growth and development can be achieved with total parenteral nutrition in rats by meticulous attention to detail.

Adipose Tissue↗
PubMed1984

The effect of storage in liquid nitrogen on the recovery of human dental plaque bacteria.

Centrifuged deposits of various individual bacteria from type-culture collections and dental plaque samples were immersed in liquid nitrogen for periods of up to 2 years. All the organisms were recovered after storage in liquid nitrogen but Fusobacterium nucleatum (ATCC 10953) and Campylobacter fetus (NCTC 10354) consistently showed a reduction in the number of colony-forming units after freezing. The main effect on bacterial proportions in plaque samples was a reduction in aerobic Gram-negative cocci. Thus, storage in liquid nitrogen may be useful for comparative studies of dental plaque and as a means of storing individual organisms.

Adolescent↗
PubMed1988

The structure and biosynthesis of new tetrahydropyrimidine derivatives in actinomycin D producer Streptomyces parvulus. Use of 13C- and 15N-labeled L-glutamate and 13C and 15N NMR spectroscopy.

Two novel compounds, 2-methyl, 4-carboxy, 5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A] and 2-methyl, 4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B] have been identified in the pool of Streptomyces parvulus by in vivo and in vitro studies. 13C and 15N were introduced into the compounds by feeding S. parvulus with 15N- and 13C-labeled L-glutamate. High resolution 13C and 15N NMR have been applied to elucidate their structure and biosynthesis in S. parvulus. The splitting patterns and coupling constants of adjacent nitrogen-carbon molecular fragments enable us to unravel their molecular structure. Two different glutamate pools are responsible for their biosynthesis, THP(A) carbon skeleton derives from the extracellular L-[13C]glutamate, whereas THP(B) stems from D-fructose via the intracellular glutamate. During cell growth, THP(A) is synthesized and becomes the major constituent of the intracellular pool. It is consumed after THP(B) is accumulated intracellularly. The onset of THP(A) and -(B) synthesis seems correlated to the time of actinomycin D synthesis. Their high cellular concentrations during actinomycin D synthesis suggest that they may function as nitrogen storage. Other possible functions of THP molecules within the cell are discussed.

Carbon Isotopes↗
PubMed1974

Hydrogen sulfide production by Pseudomonas putrefaciens in shrimp experimentally packed in nitrogen.

Shrimp refrigerated in a nitrogen atmosphere develop off-odors not typical of normal spoilage. Investigations of this phenomenon showed that hydrogen sulfide developed in the headspace gas, and a large percentage of the microbial population present on the shrimp stored in nitrogen was capable of hydrogen sulfide production, in contrast to the flora on shrimp stored in air. The predominant hydrogen sulfide-producing organism, Pseudomonas putrefaciens, was present in low numbers on fresh shrimp but usually reached high numbers by day 8 of nitrogen storage. Further studies revealed that cysteine and cystine were the probable substrates in shrimp utilized by this organism for hydrogen sulfide production. When shrimp sterilized by irradiation were inoculated with P. putrefaciens and incubated in an atmosphere of nitrogen, hydrogen sulfide and the characteristic off-odors developed.

Air↗
PubMed1998

Mutation of a gene encoding a putative glycoprotease leads to reduced salt tolerance, altered pigmentation, and cyanophycin accumulation in the cyanobacterium Synechocystis sp. strain PCC 6803.

The salt-sensitive mutant 549 of the cyanobacterium Synechocystis sp. strain PCC 6803 was genetically and physiologically characterized. The mutated site and corresponding wild-type site were cloned and partially sequenced. The genetic analysis revealed that during the mutation about 1.8 kb was deleted from the chromosome of mutant 549. This deletion affected four open reading frames: a gcp gene homolog, the psaFJ genes, and an unknown gene. After construction of mutants with single mutations, only the gcp mutant showed a reduction in salt tolerance comparable to that of the initial mutant, indicating that the deletion of this gene was responsible for the salt sensitivity and that the other genes were of minor importance. Besides the reduced salt tolerance, a remarkable change in pigmentation was observed that became more pronounced in salt-stressed cells. The phycobilipigment content decreased, and that of carotenoids increased. Investigations of changes in the ultrastructure revealed an increase in the amount of characteristic inclusion bodies containing the high-molecular-weight nitrogen storage polymer cyanophycin (polyaspartate and arginine). The salt-induced accumulation of cyanophycin was confirmed by chemical estimations. The putative glycoprotease encoded by the gcp gene might be responsible for the degradation of cyanophycin in Synechocystis. Mutation of this gene leads to nitrogen starvation of the cells, accompanied by characteristic changes in pigmentation, ultrastructure, and salt tolerance level.

Amino Acid Sequence↗
PubMed1986

Mouse embryo cryobanking.

The frozen storage of mouse embryos is a practical means of assuring the preservation of scientifically valuable strains of mice for research. Banks are maintained at over 15 institutions throughout the world. At the Jackson Laboratory eight-cell embryos from approximately 500 strains are preserved in liquid nitrogen storage.

Animals↗
PubMed2004

Ecosystem carbon storage in arctic tundra reduced by long-term nutrient fertilization.

Global warming is predicted to be most pronounced at high latitudes, and observational evidence over the past 25 years suggests that this warming is already under way. One-third of the global soil carbon pool is stored in northern latitudes, so there is considerable interest in understanding how the carbon balance of northern ecosystems will respond to climate warming. Observations of controls over plant productivity in tundra and boreal ecosystems have been used to build a conceptual model of response to warming, where warmer soils and increased decomposition of plant litter increase nutrient availability, which, in turn, stimulates plant production and increases ecosystem carbon storage. Here we present the results of a long-term fertilization experiment in Alaskan tundra, in which increased nutrient availability caused a net ecosystem loss of almost 2,000 grams of carbon per square meter over 20 years. We found that annual aboveground plant production doubled during the experiment. Losses of carbon and nitrogen from deep soil layers, however, were substantial and more than offset the increased carbon and nitrogen storage in plant biomass and litter. Our study suggests that projected release of soil nutrients associated with high-latitude warming may further amplify carbon release from soils, causing a net loss of ecosystem carbon and a positive feedback to climate warming.

Arctic Regions↗