Factors affecting viable cell counts of freeze-driedCryptococcus terricolus cells

Freeze-drying ofCryptococcus terricolus cells in distilled water resulted in a survival of only 0.1% of the cells. The viability could be increased to 16% by the use of a dextran-sucrose-sodium glutamate solution as suspending medium.For freeze-dried material with both low and high survival rates, and for five as well as for ten days old cultures, malt extract solution was the superior reconstitution medium. Less, but still distinct, protective action was found with a synthetic glucose-urea-salt solution.The viable cell counts of cells freeze-dried in dextran-sucrose-sodium glutamate solution were independent of the medium used for plating. When distilled water was used as a medium for freeze-drying, two to four times higher counts were obtained with malt extract agar than with synthetic glucose-urea-salt agar.Of the twenty different media tried for freeze-drying, sucrose solution gave the best protection. The viability was greatly influenced by the concentration used, maximum values being obtained when more than 10% of sucrose was added. The survival rate increased with the age of the cells until the fifth day, but was independent of the concentration of cells in the suspension. Under optimum conditions a survival rate of more than 80% was reached.     

Cytological studies on the planarian neoblast

Summary The paper is a study of the cytology of the regeneration cells (neoblasts) in Planaria vitta.The morphology of the living cells has first been examined to provide a reference for an investigation of the fixed neoblasts as studied by ordinary cytological, cytochemical and electron microscopical technics.A rather selective staining method has been devised based on the strong basophilic properties of the scanty cytoplasm. The morphology of the fixed neoblasts and their distribution in the intact animal have been described, using this method.The marked cytoplasmic basophilia was found to be exclusively due to ribonucleic acid, and not to desoxyribonucleic acid or acid mucopolysaccharides.The cytoplasm contains moderate to considerable amounts of basic proteins. Tyrosine, cysteine/cystin, arginine, lysine and perhaps histidine were present, while tryptophan could not be demonstrated.No enzymes could be demonstrated apart perhaps from cytochrome oxidase.The mitochondria are small and inconspicuous and more or less evenly distributed throughout the cytoplasm. A Golgi apparatus could not be demonstrated.The electron microscopic picture is very characteristic, because of the high electron density of the cytoplasm. This density is the result of the presence of a great number of ribonucleoprotein granules. Most of the granules are free and only a minor part bound to the membranes of the endoplasmatic reticulum. The interesting features of the cell membrane are discussed in relation to the structure of the parenchyma.The cytochemical properties of the neoblast (RNA and sulfhydryl-groupcontaining protein) and the fine structure as revealed in the electron microscope characterize the neoblast as a morphogenetically active cell.     

Light-Dark Transients in Levels of Intermediate Compounds during Photosynthesis in Air-Adapted Chlorella

The labeling of intermediate compounds and photosynthetic cofactors during photosynthesis and periods of darkness by Chlorella pyrenoidosa in the presence of ³²P-labeled phosphate and ¹⁴CO₂ have heen investigated. Algae adapted to photosynthesis in air were used, and the level of carbon dioxide was maintained at approximately 0.04 % and at constant specific radioactivity during the course of the experiments. The transient changes which occur in the levels of labeled fructose-1,6-diphosphate and in sedoheptulose-1,7-diphosphate, and in the corresponding monophosphates when the light is turned off suggest a light activation of the diphosphatase enzymes which decays after about 2 minutes of darkness. It is suggested that a light-dark switch in enzymic activities permits photosynthesis and glycolysis to occur in light and dark respectively with the same enzymic apparatus. The greatly diminished rate of disappearanec of the carboxylation substrate, ribulose-1,5-diphosphate, after about 2 minutes suggests that there is also a light activation of the carboxylation reaction in vivo. Large transient changes in the level of pyrophosphate between light and dark indicate that there may be an unstable cofactor which decomposes to give pyrophosphate during or alter killing of the algal cells. The possibility that this cofactor is involved in an activation of Carbon dioxide for the carboxylation reaction in vivo is suggested. Light-dark transient changes in labeling of other compounds of the photosynthetic carbon reduction cycle and related compounds were determined, and possible significance of these changes is discussed.(PDF DAMAGED)     

Mechanism of reaction of a suggested superoxide-dismutase mimic, Fe(II)-N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

The interaction of a recently developed intracellular superoxide dismutase analogue, Fe(II)-N,N,N',N'-tetrakis(2- pyridylmethyl)ethylenediamine (Fe(II)-TPEN), with reactive oxygen species was investigated under in vitro conditions. The complex catalyzed the dismutation of enzyme- or radiolysis-generated superoxide with the production of H2O2; under steady-state conditions the equilibrium was strongly shifted toward Fe(III)-TPEN. Fe(II)-TPEN reacted with H2O2 to generate hydroxyl radicals in a Fenton reaction. The oxidized Fe(III)-TPEN was readily reduced by ascorbate or glutathione. Given the capacity to produce hydroxyl radicals and the reaction with cellular reductants it seems unlikely that Fe-TPEN may find widespread use as an intracellular superoxide dismutase substitute.     

Growth of asparagus in a commercial peat mix containing vesicular-arbuscular mycorrhizal (VAM) fungi and the effects of applied phosphorus

Commercially prepared, peat-based mycorrhizal inocula were studied for growth effects on asparagus grown under greenhouse and field (fumigated) conditions. The fungi tested were Glomus clarum (GC), G. intraradix (GI), G. monosporum (GM), G. versifomre (GVR) and G. vesiculiferum (GVS). GI significantly increased plant dry weight in the greenhouse and the field. Survival of mycorrhizal tissue-cultured transplants after 14 months in the field was increased by twofold over the control. In a second experiment asparagus was grown from seed in the greenhouse in peat inoculated with a G. fasciculatum-like fungus (GF), GI and GVR with applied P levels of 0, 50, 100 and 150 ppm and harvested after 13 and 17 weeks. Total dry weights of GI and GVR plants were significantly increased over those of the control and GF. Dry weight in this second experiment was positively correlated with root colonization. Root colonization at week 13 was slightly reduced with increasing levels of applied P, but not at week 17. The data suggest that the increased growth of mycorrhizal plants was not related to an increase in tissue P concentration, since there was no growth response to applied P and tissue P concentration in the mycorrhizal plants was lower than in the non-mycorrhizal plants.     

Cerebral Polyamine Metabolism in Reversible Hypoglycemia of Rat: Relationship to Energy Metabolites and Calcium

Thirty minutes of insulin-induced reversible hypoglycemic coma (defined in terms of cessation of EEG activity) was produced in anesthetized rats. At the end of the hypoglycemic coma or after recovery for 3, 24, or 72 h induced by glucose infusion, the animals were reanesthetized and their brains frozen in situ. Two control groups were used: untreated controls without prior manipulations, and insulin controls, which received injections of insulin followed by glucose infusion to maintain blood glucose within the physiological range. The brains of these latter animals were frozen 3, 24, or 72 h after glucose infusion. Tissue samples from the cortex, striatum, hippocampus, and thalamus were taken to measure ornithine decarboxylase (ODC) activity, and putrescine and spermidine levels, as well as phosphocreatine (PCr), ATP, glucose, and lactate content. In addition, 20-microns thick coronal sections taken from the striatum and dorsal hippocampus were used for histological evaluation of cell damage and also stained for calcium. Insulin in the absence of hypoglycemia produced a significant increase in ODC activity and putrescine level but had no effect on the profiles of energy metabolites or spermidine. During hypoglycemic coma, brain PCr, ATP, glucose, and lactate levels were sharply reduced, as expected. Energy metabolites normalized after 3 h of recovery. In the striatum, significant secondary decreases in PCr and ATP contents and rises in glucose and lactate levels were observed after 24 h of recovery. ODC activity, and putrescine and spermidine levels were unchanged during hypoglycemic coma. After 3 h of recovery, ODC activity increased markedly throughout the brain, except in the striatum. After 24 h of recovery, ODC activity decreased and approached control values 2 days later. Putrescine levels increased significantly throughout the brain after reversible hypoglycemic coma, the highest values observed after 24 h of recovery (p less than or equal to 0.001, compared with controls). After 72 h of recovery, putrescine levels decreased, but still significantly exceeded control values. Reversible hypoglycemic coma did not produce significant changes in regional spermidine levels except in the striatum, where an approximately 30% increase was observed after 3 and 72 h of recovery (p less than or equal to 0.01 and p less than or equal to 0.05, respectively). Twenty-four hours after hypoglycemic coma, intense calcium staining was apparent in layer III of the cerebral cortex, the lateral striatum, and the crest of the dentate gyrus. After 72 h of recovery, the intense calcium staining included also cortical layer II, the septal nuclei, the subiculum, and the hippocampal CA1-subfield.(ABSTRACT TRUNCATED AT 400 WORDS)     

Brain Tryptophan Hydroxylation in the Portacaval Shunted Rat: A Hypothesis for the Regulation of Serotonin Turnover In Vivo

Regional and whole-brain tryptophan-hydroxylating activity and serotonin turnover were investigated in portacaval shunted (PCS) rats using an in vivo decarboxylase inhibition assay. To saturate tryptophan hydroxylation with amino acid substrate, rats were administered a high dose of tryptophan 1 h prior to analysis of brain tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid. The analysis revealed, as expected, higher brain concentrations of tryptophan and 5-hydroxyindoles and increased serotonin synthesis rate in PCS rats as compared with shamoperated controls. Saturating levels of brain tryptophan were achieved in both PCS and sham animals after exogenous tryptophan administration. The tryptophan load resulted in increased brain serotonin turnover in all regions and in whole brain compared with rats that did not receive a tryptophan load. Tryptophan-loaded PCS rats showed increased brain serotonin turnover compared with tryptophan-loaded sham rats. Regionally, this supranormal tryptophan-hydroxylating activity was most pronounced in the mesencephalon-pons followed by the cortex. It is concluded that, at least in the PCS rat, brain tryptophan hydroxylation is an inducible process. Since it is known that brain tissue from PCS rats undergoes a redox shift toward a reduced state and that the essential cofactor tetrahydrobiopterin is active in tryptophan hydroxylation only when present in its reduced form, it is hypothesized that this is the reason for the supranormal tryptophan-hydroxylating activity displayed by the PCS rats. The hypothesis further suggests that alterations in tetrahydrobiopterin availability may serve as a mechanism by which brain tryptophan hydroxylation, and therefore serotonin turnover, can be regulated with high sensitivity in vivo.     

Growth and survival of Pseudomonas cepacia DBO1(pRO101) in soil amended with 2,4-dichlorophenoxyacetic acid

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading pseudomonad, Pseudomonas cepacia DBO1(pRO101), was inoculated at approximately 10⁷ CFU/g into sterile and non-sterile soil amended with 0, 5 or 500 ppm 2,4-D and the survival of the strain was studied for a period of 44 days. In general, the strain survived best in sterile soil. When the sterile soil was amended with 2,4-D, the strain survived at a significantly higher level than in non-amended sterile soil. In non-sterile soil either non-amended or amended with 5 ppm 2,4-D the strain died out, whereas with 500 ppm 2,4-D the strain only declined one order of magnitude through the 44 days.The influence of 0,0.06, 12 and 600 ppm 2,4-D on short-term (48 h) survival of P. cepacia DBO1(pRO101) inoculated to a level of 6×10⁴, 6×10⁶ or 1×10⁸ CFU/g soil was studied in non-sterile soil. Both inoculum level and 2,4-D concentration were found to have a positive influence on numbers of P. cepacia DBO1(pRO101). At 600 ppm 2,4-D growth was significant irrespective of the inoculation level, and at 12 ppm growth was stimulated at the two lowest inocula levels. P. cepacia DBO1(pRO101) was able to survive for 15 months in sterile buffers kept at room temperature. During this starvation, cells shrunk to about one third the volume of exponentially growing cells.Abbreviations AODC acridine orange direct count - CFU colony forming units - PTYG-Agar peptone, tryptone, yeast & glucose agar - TET tetracycline - LB Luria Bertani medium     

Clonal analysis of epiblast fate during germ layer formation in the mouse embryo.

The fate of cells in the epiblast at prestreak and early primitive streak stages has been studied by injecting horseradish peroxidase (HRP) into single cells in situ of 6.7-day mouse embryos and identifying the labelled descendants at midstreak to neural plate stages after one day of culture. Ectoderm was composed of descendants of epiblast progenitors that had been located in the embryonic axis anterior to the primitive streak. Embryonic mesoderm was derived from all areas of the epiblast except the distal tip and the adjacent region anterior to it: the most anterior mesoderm cells originated posteriorly, traversing the primitive streak early; labelled cells in the posterior part of the streak at the neural plate stage were derived from extreme anterior axial and paraxial epiblast progenitors; head process cells were derived from epiblast at or near the anterior end of the primitive streak. Endoderm descendants were most frequently derived from a region that included, but extended beyond, the region producing the head process: descendants of epiblast were present in endoderm by the midstreak stage, as well as at later stages. Yolk sac and amnion mesoderm developed from posterolateral and posterior epiblast. The resulting fate map is essentially the same as those of the chick and urodele and indicates that, despite geometrical differences, topological fate relationships are conserved among these vertebrates. Clonal descendants were not necessarily confined to a single germ layer or to extraembryonic mesoderm, indicating that these lineages are not separated at the beginning of gastrulation. The embryonic axis lengthened up to the neural plate stage by (1) elongation of the primitive streak through progressive incorporation of the expanding lateral and initially more anterior regions of epiblast and, (2) expansion of the region of epiblast immediately cranial to the anterior end of the primitive streak. The population doubling time of labelled cells was 7.5 h; a calculated 43% were in, or had completed, a 4th cell cycle, and no statistically significant regional differences in the number of descendants were found. This clonal analysis also showed that (1) growth in the epiblast was noncoherent and in most regions anisotropic and directed towards the primitive streak and (2) the midline did not act as a barrier to clonal spread, either in the epiblast in the anterior half of the axis or in the primitive streak. These results taken together with the fate map indicate that, while individual cells in the epiblast sheet behave independently with respect to their neighbours, morphogenetic movement during germ layer formation is coordinated in the population as a whole.     

A study of Pseudomonas fluorescens biovars using the Automated Microbiology Identification System (AMBIS)

The AMBIS is a system which can determine the relationships between microbial strains by comparing the profiles produced after their radiolabelled intracellular proteins are subjected to SDS PAGE. This system was used to compare the profiles of strains representing the five biovars of Pseudomonas fluorescens , a species implicated in food spoilage. The three strains of biovar I, three strains of biovar III and two strains of biovar IV segregated into three distinct clusters with correlation coefficients (cc) of 0·85, 0·85 and 0·87 respectively. Although two of the biovar II strains studied clustered together (cc = 0·74) one of the remaining biovar II strains linked (cc = 0·83) with the cluster of biovar IV strains while the other was linked with biovar I and V strains (cc = 0·68). Biovar V strains (three in total) also failed to form a single cluster which was expected since this biovar is known to be heterogeneous. The findings are in substantial agreement with more comprehensive taxonomic studies of this species. AMBIS may be a useful tool in taxonomic studies of micro-organisms.