Effect of cooling rate,cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.)

Reflexed saltmarsh-grass suspension cultures produced by seed callus were frozen to the liquid nitrogen temperature. Cooling rates, cryoprotectants and holding times were taken as a function of transfer temperatures. The highest survival of cells (45%) was found at a freezing rate of 1°C min^-1, without cryoprotectant treatments. The cryoprotectants (proline, dimethyl sulphoxide, glycerol), used at different concentrations and transfer temperatures, increased the survival rate. The maximum value was 78% at 12.5% (w/v) of proline with –30°C transfer temperature. Considerable improvement of viability (from 0% to 95%) among the 12.5 and 15.0% (v/v) dimethyl sulphoxide cryopreserved cells was achieved by holding them at – 20°C for 10–30 min before plunging into the liquid nitrogen. A 20 min holding time at 15.0% (v/v) glycerol level and – 30°C transfer temperature significantly enhanced the viability of the explants from 42% to 92%. Plants were successfully regenerated from cells cryopreserved with proline (w/v) and dimethyl sulfoxide (v/v) levels of 12.5 and 15.0%, respectively.     

The dynamics of sparse random networks

Recurrent neural networks with full symmetric connectivity have been extensively studied as associative memories and pattern recognition devices. However, there is considerable evidence that sparse, asymmetrically connected, mainly excitatory networks with broadly directed inhibition are more consistent with biological reality. In this paper, we use the technique of return maps to study the dynamics of random networks with sparse, asymmetric connectivity and nonspecific inhibition. These networks show three qualitatively different kinds of behavior: fixed points, cycles of low period, and extremely long cycles verging on aperiodicity. Using statistical arguments, we relate these behaviors to network parameters and present empirical evidence for the accuracy of this statistical model. The model, in turn, leads to methods for controlling the level of activity in networks. Studying random, untrained networks provides an understanding of the intrinsic dynamics of these systems. Such dynamics could provide a substrate for the much more complex behavior shown when synaptic modification is allowed.     

Studies on dehydro-l-ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

l-threo-2,3-Hexodiulosono-1,4-lactone 2-(arylhydrazones) (2) were prepared by condensation of dehydro-l-ascorbic acid with various arylhydrazines. Reaction of 2 with hydroxylamine gave the 2-(arylhydrazone) 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl-l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones (4). On treatment of 4 with liquid ammonia, 2-aryl-4-(l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxamides (5) were obtained. Acetylation of 5 with acetic anhydride-pyridine gave the triacetates, and vigorous acetylation with boiling acetic anhydride gave the tetraacetyl derivatives. Periodate oxidation of 5 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxamides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamides, characterized as the monoacetates and diacetates. Controlled reaction of 2 with sodium hydroxide, followed by neutralization, gave 3-(l-threo-glycerol-l-yl)-4,5-isoxazolinedione 4-(arylhydrazones), characterized by their triacetates. Reaction of 2 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy-l-threo-2,3-hexodiulosono-1,4-lactone 2-(arylhydrazones); these were converted into 4-(2-O-acetyl-3-bromo-3-deoxy-l-threo-glycerol-l-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones on treatment with acetic anhydride-pyridine.     

Effect of the organophosphorous insecticide, chlorpyrifos (Dursban), on growth, fecundity and mortality of Biomphalaria alexandrina and on the production of Schistosoma mansoni cercariae in the snail.

Exposure of Biomphalaria alexandrina to sublethal concentrations (0.125, 0.25 and 0.05 ppm) of the organophosphorous insecticide, chlorpyrifos (Dursban), induced a reduction in egg production and egg hatchability. Exposure of Schistosoma mansoni miracidia to the insecticide (60 min, 0.50 ppm) prior to infection of B. alexandrina did not affect the subsequent production of cercariae. However, exposure of S. mansoni-infected snails to the insecticide until day 55, from day 20 to day 62 and from day 35 to 62 following infection resulted in blockage of cercarial shedding. Cercarial shedding commenced in some snails when the treatment stopped. Exposure to the insecticide in concentrations of 0.125 and 0.25 ppm during the first 20 days following infection did not affect the subsequent production of cercariae, but exposure to 0.5 ppm during the first 20 days affected markedly the production of cercariae due to a high snail mortality. The findings indicate that the cercaria is the target stage for the activity of chlorpyrifos on the intramolluscan larval development. It is suggested that S. mansoni cercarial production in B. alexandrina may be a useful system for monitoring the effect of low concentrations of pesticides on the aquatic environment, and that the ability by chemical means to interrupt the cercarial production might be a useful tool in further analyses of important aspects of the snail/parasite relationship.     

Nucleotide requirements for the bone marrow heme oxygenase system

Rat bone marrow microsomal heme oxygenase activity has been studied and optimal condition for the measurement of this activity are described. The activity of bone marrow heme oxygenase was linear with time and protein concentration as measured under these assay conditions. Bilirubin formation by the heme oxygenase complex system was observed with either a NADPH generating system or with NADH as electron donor. The enzyme activity for heme degradation supported by NADH proceeds at a comparable rate to that observed with NADPH as reducing equivalent. It thus appears that this oxidation reaction must be more complex than simply involving NADPH as the sole election donor as has been previously proposed.     

Oxygen supply to bacterial suspensions of high cell densities by hydrogen peroxide

The supply of heterotrophically growing suspensions of Alcaligenes eutrophus PHB^?4 with oxygen formed by the continuous addition of H₂O₂ in the presence of bovine liver catalase was found to be restricted to well-defined conditions. The catalase-H₂O₂ system proved to be suitable during the growth at low cell densities equivalent to 2 g dry weight/liter. When under these conditions the oxygen concentration was held constant at 1.8 mg O₂/liter, the cells grew for 6–8 hr at a rate almost identical to that observed with conventional aeration. However, aeration with H₂O₂ for longer durations (10–20 hr) and at higher cell densities (5?20 g dry weight/liter) led invariably to cell damage and retardation of growth. The impairment of growth observed during the oxygen supply by the catalase?H₂O₂ system was traced back to the formation of gradually increasing steady-state concentrations of H₂O₂ in the medium. Possible sites of cell damage by H₂O₂ such as membrane function, excretion and function of siderophores, and synthesis of cell polymers have been studied, and the cytotoxic mechanism of low concentrations of H₂O₂ was discussed.