Intracellular and Extracellular Antifreeze Protein Significantly Improves Mammalian Cell Cryopreservation

Cell cryopreservation is an essential part of the biotechnology, food, and health care industries. There is a need to develop more effective, less toxic cryoprotective agents (CPAs) and methods, especially for mammalian cells. We investigated the impact of an insect antifreeze protein from Anatolica polita (ApAFP752) on mammalian cell cryopreservation using the human embryonic kidney cell line HEK 293T. An enhanced green fluorescent protein (EGFP)-tagged antifreeze protein, EGFP–ApAFP752, was transfected into the cells and the GFP was used to determine the efficiency of transfection. AFP was assessed for its cryoprotective effects intra- and extracellularly and both simultaneously at different concentrations with and without dimethyl sulfoxide (DMSO) at different concentrations. Comparisons were made to DMSO or medium alone. Cells were cryopreserved at −196 °C for ≥4 weeks. Upon thawing, cellular viability was determined using trypan blue, cellular damage was assessed by lactate dehydrogenase (LDH) assay, and cellular metabolism was measured using a metabolic activity assay (MTS). The use of this AFP significantly improved cryopreserved cell survival when used with DMSO intracellularly. Extracellular AFP also significantly improved cell survival when included in the DMSO freezing medium. Intra- and extracellular AFP used together demonstrated the most significantly increased cryoprotection compared to DMSO alone. These findings present a potential method to improve the viability of cryopreserved mammalian cells

Pterins of the cyanobacterium Aphanizomenon flos-aquae

The cyanobacterium Alphanizomenon flos-aquae contains various glycosides of a pterin that had previously been tentatively identified as 6-(threo-1,2-dihydroxypropyl)-pterin. The pterin is now identified as 6-(l-threo-1,2-dihydroxypropyl)pterin(6-l-threo-biopterin) by NMR and mass spectrometry, thinchromatographic comparisons with authentic samples and by optical rotation. The 6-l-threo-biopterin glycoside-1 from toxic strain NH-1 gave rise to xylose and glucose on hydrolysis, whereas its glycoside-2 from Cambridge Collection and nontoxic NH-1 strains gave mannose and glucose. This pterin may be a useful marker for certain species of cyanobacteria

Studies on Aphantoxin from Aphanizomenon Flos-Aquae in New Hampshire

Toxic Cyanobacteria (blue-green algae) bloom in eutrophic, freshwater lakes and ponds in New England and have caused environmental, health, legal and recreational problems over the past 15 years. Although several species have been implicated with animal kills and water fouling, a common offender was Aphanizomenon flos-aquae. Representative strains of A. flos-aquae bloom in New Hampshire intermittently, in both toxic (aphantoxins) and non-toxic forms. Research has focused on methods of: a) toxin accumulation from natural blooms and laboratory cultivation, b) toxin assay, using the mouse bioassay and a modified fluorometric technique developed for paralytic shellfish poisons, c) toxin characterization and purification, using solvent separation and molecular weight filters, and d) testing active extracts on nerve and muscle preparations to determine the specific sites and modes of action of aphantoxins. Aphantoxin samples were passed through molecular weight filters (10,000 and 500 daltons), lyophilized and weighed, prior to physiological testing. Microgram quantities of toxin reversibly blocked compound action potentials in amphibian nerves as well as mechanical activity in skeletal muscle. No effect was measured on the transmembrane resting potential or on spontaneous miniature end-place potentials (mepps). Tests on lateral and medial giant s from crayfish gave similar salts. The Na+ dependence of the crayfish preparation was verified. The aphantoxins (4 μg/ml) reversibly blocked intracellular recordings of action potentials with no alteration of the resting potential. Amphibian and crustacean cardiac activity was blocked in diastolic arrest, while bivalve hearts were unaffected at increased dose levels. Aphantoxins may block excitability by affecting ion conductance pathways as do toxins from several marine dinoflagellates and may be useful in basic studies on membrane systems

Uso conjunto de dois modelos cognitivos para a compreensão do processo de escolha do consumidor Joint use of two cognitive models in order to understand consumer choice process

O presente estudo tem por objetivo gerar um melhor entendimento do comportamento do consumidor, baseando-se em dois modelos cognitivos: risco percebido e conjunto de consideração. Neste sentido, teve foco no processo de avaliação e na escolha de alternativas dos consumidores, considerando diferentes percepções de risco em dois contextos de serviços. Uma amostra de 772 respondentes foi consultada através de um questionário estruturado. Os resultados indicam que a quantidade de marcas a serem consideradas como alternativa de consumo está associada apenas a algumas das dimensões do risco percebido. Também foram identificados quais atributos são considerados mais importantes na prestação dos serviços analisados e as estratégias redutoras de risco mais utilizadas pelos consumidores com diferentes níveis de percepção de risco. Conclusões e implicações gerenciais também são discutidas.<br>This study aims to generate a better understanding about consumer behavior based on two cognitive models: risk perception and consideration set. In this sense, it has focus on consumer evaluation and choice processes, considering different risk perception levels in two service settings. A survey with a sample of 772 respondents was performed through a structured questionnaire. Results indicate that the number of brands considered for choice is associated with some risk perception dimensions. It was also identified which attributes are considered the most important in service delivery as well as the most used risk reducing strategies by consumers with varying patterns of risk perception. Conclusions and managerial implications are also discussed