Viability of dopaminergic neurones is influenced by serum and astroglial cells in vitro

Transplantation of embryonic nigral grafts into the striatum of Parkinson's disease patients is not optimal, mainly due to low survival of grafted neurones. Current strategies focus on enhancing neuronal survival by transplanting enriched neuronal cell populations. There is growing evidence for the importance of astroglia in neuronal survival.To characterise the effects of glial cells on dopaminergic neurones, 5-fluoro-2′-deoxyuridine was added to embryonic rat ventral mesencephalic cultures in the presence or absence of serum. The survival and morphology of glial fibrillary acidic protein immunopositive astroglia and tyrosine hydroxylase immunopositive dopaminergic neurones was examined. In serum-containing medium, astroglial cells predominated and 5-fluoro-2′-deoxyuridine had no significant effect on either astroglia or dopaminergic neurone survival. In serum-free medium, astroglial growth was attenuated and numbers were significantly lower in 5-fluoro-2′-deoxyuridine treated compared with untreated cultures. There was no significant difference in the numbers of dopaminergic neurones between 5-fluoro-2′-deoxyuridine treated and untreated cultures. However, by the eighth day in vitro, there were differences in the morphology of these neurones between treated and untreated cultures. This study shows that the use of 5-fluoro-2′-deoxyuridine and serum-free medium can produce a neurone-enriched culture. However, the dopaminergic neurone population present in these cultures appeared to be morphologically dissimilar to those found in control cultures as neurites were retracted and the cell somas of these cells appeared enlarged. These results provide information on the effects of astrocytes on dopaminergic neurones in ventral mesencephalic cultures and thus have implications for transplantation in Parkinson's disease.     

Sorbitol and other carbohydrates in dormant apple shoots as influenced by controlled temperatures

The influence of controlled temperatures on levels of sorbitol and other carbohydrates was determined to provide further information on dormancy of apple trees (Malus sylvestris Mill.). For 3 years, 2-year-old “Red Delicious” apple shoots were collected from mature trees in an orchard at intervals during the autumn and winter, and shoots were stored for 6 hr to 1 week at temperatures from 18 to ?60 °C. Sorbitol and other carbohydrates were estimated in the sap or wood by gas chromatography.Controlled temperatures had a marked influence on the carbohydrate content of excised 2-year-old apple shoots. Levels of sorbitol in the sap were maximum at ?0.6 °C. The increase was greatest at the earliest sampling before complete hardening had occurred in each of the 3 years tested. Total sorbitol in the wood was less influenced by storage at various controlled temperatures than sorbitol in the sap. Levels of fructose, glucose, and sucrose in the wood were higher at temperatures below ?0.6 °C than at warmer temperatures. Levels of starch were usually inversely related to soluble sugars.     

Flowering of Watsonia laccata as influenced by corm storage and forcing temperatures

The genus Watsonia, belonging to the family Iridaceae, is comprised of about 50 species including W. laccata (Jacquin) Ker Gawler that flowers from September to November following low temperature and winter rainfall. Therefore, we hypothesized that flowering would be favored by forcing at low greenhouse temperatures. Using clonal W. laccata corms, four experiments were designed to investigate the effect of temperatures during corm storage, forcing, and their interaction on growth and flowering. Corm formation is favored by growing plants at 18°–20°/15°–17 °C and 21°–23°/18°–20 °C, day/night temperatures. Flowering was earliest with corms produced at 24°–26°/18°–20 °C and forced at 18°–20/15°–17 °C, and was significantly delayed when forced at 27°–29°/24°–26 °C. Flowering was, however, favored by 2 or 4 weeks of high temperatures (27°–29°/24°–26 °C) prior to forcing at low temperatures (18°–20°/15°–17 °C). The number of florets was not significantly affected by corm storage, forcing temperatures, or their interaction, although forcing at high temperatures tends to reduce the floret number. Burn symptom at the tips of leaves was frequently observed, and further studies are required to understand the cause of the tip burn and how to correct the symptom.     

ATP levels in algal cells as influenced by environmental conditions

The cellular content of adenosine triphosphate (ATP) relativeto cell size and cellular organic carbon has been investigatedin 30 different algal cultures representing 7 phyla. Duringexponential growth in batch culture, cellular contents of ATPremained at fairly uniform levels in all these unicellular algaeand averaged 0.35% of the cellular organic carbon content. Duringextreme nitrogen or phosphorus deficiency the cellular levelsof ATP decreased to 20–50% of that found in exponentially-growingcells, but these percentages may be low due to detrital carbonin the senescent cultures. The steady state levels of ATP in cells were similar in lightor in dark, although ATP concentrations fluctuated for a fewminutes upon any sudden change in light conditions. When thelight was turned on there was a rapid increase in ATP levels,followed by a slow decrease; when the light was turned off,there was a rapid fall in cellular ATP levels, which then rosewithin a few minutes to achieve the steady state concentration.The cellular concentrations of ATP in these algae and in othermicrobial groups are discussed relative to studies where ATPdeterminations are used to estimate microbial biomass. (Received June 2, 1970; )     

Synergistic induction of antigen-specific CTL by fusions of TLR-stimulated dendritic cells and heat-stressed tumor cells

Dendritic cell (DC)/tumor cell fusion cells (FCs) can induce potent CTL responses. The therapeutic efficacy of a vaccine requires the improved immunogenicity of both DCs and tumor cells. The DCs stimulated with the TLR agonist penicillin-killed Streptococcus pyogenes (OK-432; OK-DCs) showed higher expression levels of MHC class I and II, CD80, CD86, CD83, IL-12, and heat shock proteins (HSPs) than did immature DCs. Moreover, heat-treated autologous tumor cells displayed a characteristic phenotype with increased expression of HSPs, carcinoembryonic Ag (CEA), MUC1, and MHC class I (HLA-A2 and/or A24). In this study, we have created four types of FC preparation by alternating fusion cell partners: 1) immature DCs fused with unheated tumor cells; 2) immature DCs fused with heat-treated tumor cells; 3) OK-DCs fused with unheated tumor cells; and 4) OK-DCs fused with heat-treated tumor cells. Although OK-DCs fused with unheated tumor cells efficiently enhanced CTL induction, OK-DCs fused with heat-treated tumor cells were most active, as demonstrated by: 1) up-regulation of multiple HSPs, MHC class I and II, CEA, CD80, CD86, CD83, and IL-12; 2) activation of CD4+ and CD8+ T cells able to produce IFN- gamma at higher levels; 3) efficient induction of CTL activity specific for CEA or MUC1 or both against autologous tumor; and 4) superior abilities to induce CD107+ IFN-gamma+ CD8+ T cells and CD154+ IFN-gamma+ CD4+ T cells. These results strongly suggest that synergism between OK-DCs and heat-treated tumor cells enhances the immunogenicity of FCs and provides a promising means of inducing therapeutic antitumor immunity.     

Structure of thylakoids in cells of Rhodopseudomonas viridis as influenced by growth conditions

A vessel was constructed for growth of photosynthetic bacteria at defined light intensity, temperature and partial pressure of oxygen.Under growth conditions at light intensities larger than 1,000 lx, the particles exposed by freeze-fracturing of thylakoids are unordered.Under growth conditions at light intensities lower than 30 lx, the particles seen are hexagonally arranged. If the oxygen partial pressure is increased from 0 to 30 mm Hg while keeping the light intensity at 30 lx, the particles seen in the thylakoids are found to be unregularly arranged.The protein pattern of thylakoids isolated from bacteria grown either at 2,000 lx or at 30 lx revealed a constant ratio of reaction centre polypeptide to either of the membrane polypeptides of 8 kdalton apparent Mr and 12 kdalton apparent Mr.Dedicated to Prof. Dr. G. Drews on occasion of his 60th birthday     

Killing and mutation induction in quiescent V79 cells as influenced by nalidixic acid

Involvement of topoisomerase II in the repair of damage by N-methyl-N'-nitro-N-nitrosoguanidine, UV light and gamma-rays has been studied using quiescent V79 cells. In each case, the presence of nalidixic acid, the inhibitor of topoisomerase II, decreased the survival through suppression of potentially lethal damage repair. There was also an increase in the yield of mutants because of such suppression. The observations were in contrast with those made with exponential cells in the following aspects: (a) density-inhibited cells showed a positive response to suppression of topoisomerase II activity after treatment with UV light and gamma-rays and (b) for MNNG exposure, mutational yield increased instead of decreasing as in exponential cells. The results showed that topoisomerase II played an important part in the repair of damage of density-inhibited cells.     

Viability of deformed cells

Most of the researchers in the field of cryobiology believe that the mechanism of damage during freezing with low cooling rates is chemical and related to the hypertonicity of the extracellular solution. However, there is some evidence to indicate that cells may be destroyed during freezing also by compression between ice crystals. We have developed an experimental procedure to study the effect of cell compression on viability. Using human prostate primary adenoma cancer cells we show that cell viability decreases steeply when cells are compressed to 30% of their original diameter. If uniform expansion of cell membrane is assumed, this corresponds to a 50% increase in the cell membrane surface area. A simple mathematical model shows that the temperature at which the compression effect may cause cell damage is related to the spacing between ice crystals. When the ice crystals are spaced at distances comparable to the cell diameter the model combined with our experimental data predicts compression damage at about -1.8 degrees C. This is consistent with experimental observation on frozen cell destruction in the presence of antifreeze proteins.     

Activities of phosphomonoesterases as influenced by goethite and pyrite

Laboratory experiments were conducted to measure the activity of phosphomonoesterases in various zones of a geological outcrop. The outcrop contained an Entisol and partially weathered carbonaceous shales of Paleozoic age with differentiating zones rich in pyrite and goethite. Goethite is the most commonly occurring iron oxide in soils, and pyrite is often found in reconstructed soils of coal surface mines. The activity of acid phosphatase was significantly elevated in goethite‐rich zones compared to zones where pyrite was present. Alkaline phosphatase activity was reduced significantly in the presence of goethite but was not affected by pyrite. Experiments with pure pyrite and synthetic goethite substantiated the results obtained from the outcrop samples. The enhancing effect of goethite at lower pH values is probably due to chemisorption and removal of mineralized P, which increases the mineralization of organic P. At higher pH values goethite surfaces are negatively charged and repel phosphate anions. The exact mechanism for the inhibitory effect of pyrite on acid phosphatase is not known.     

Viability and DNA maintenance in nonculturable spiral Campylobacter jejuni cells after long-term exposure to low temperatures.

Survival of Campylobacter jejuni at 4 and 20 degrees C was investigated by using cellular integrity, respiratory activity, two-dimensional (2D) protein profile, and intact DNA content as indicators of potential viability of nonculturable cells. Intact DNA content after 116 days, along with cellular integrity and respiring cells, was detected for up to 7 months at 4 degrees C by pulsed-field gel electrophoresis. Most changes in 2D protein profiles involved up- or down-regulation.     

Frankia growth and activity as influenced by water potential

Summary Growth responses of Frankia isolates to decreasing water potential were monitored in systems where potentials were controlled by KCl, NaCl and Polyethylene glycol. The highest potential tested was −2 bar (basal medium). The general pattern emerging was that isolates fromAlnus glutinosa, A. viridis andComptonia peregrina showed declining growth at potentials below −2 to −5 bar. AMyrica gale isolate showed declining growth with decreasing potential. All isolates were more sensitive to decreases in potential in a matric controlled than an osmotic controlled system. They all showed approximately 50 percent growth reduction at −5 to −8 bar, and meagre growth at −16 bar after 35 days. The Comptonia isolate was the most vigorous at low potentials. Nitrogen fixation ability was monitored for two isolates. Highest specific activities were observed between −3 and −5 bar for the Myrica isolate and between −5 and −7.5 bar for theA. glutinosa isolate.     

Viability of cells reconstituted by virus-induced fusion of minicells with anucleate cells

The addition of 1 mM ATP to rabbit peritoneal polymorphonuclear leukocytes suspended in a solution of glycerol causes a decrease in their volume by 4–17% within 3 min. The suspending medium must not only contain glycerol but be of low ionic strength for ATP to be effective. Divalent cations are also required. Ca²⁺ present alone can sustain the volume increase induced by ATP; in the presence of low concentrations of Ca²⁺, Mg²⁺ is also effective but not to the same extent as Ca²⁺. When the cell volume is contracted by the ATP in the presence of Ca²⁺ the addition of EGTA induces a reexpansion of the volume.The organic mercurial, salyrgan prevents the ATP induced reduction in the volume but ouabain has no effect. Guanosine triphosphate (GTP), uridine triphosphate (UTP), and adenosine diphosphate (ADP) can also decrease the volume of the glycerinated leukocytes but to a distinctly lesser extent than ATP. Adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) are without significant effect.The results indicate that the volume decrease caused by exogenous ATP is unlikely to be a passive osmotic or an active, ouabain-sensitive process. The similarities to the interaction of ATP with actomyosin suggest that the volume decrease might be a result of the contraction of the leukocyte actomyosin by ATP.