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5 páginas -- PAGS nros. 293-297, Changes have been reported in the pattern of gene expression in Arabidopsis on exposure to microgravity. Plant cell growth and proliferation are functions that are potentially affected by such changes in gene expression. In the present investigation, the cell proliferation rate, the regulation of cell cycle progression and the rate of ribosome biogenesis (this latter taken to estimate cell growth) have been studied using morphometric markers or parameters evaluated by light and electron microscopy in real microgravity on the International Space Station (ISS) and in ground-based simulated microgravity, using the Random Positioning Machine and the Magnetic Levitation Instrument. Results showed enhanced cell proliferation but depleted cell growth in both real and simulated microgravity, indicating that the two processes are uncoupled, unlike the situation under normal gravity on Earth in which they are strictly co-ordinated events. It is concluded that microgravity is an important stress condition for plant cells compared to normal ground gravity conditions

The proteolytic activites of Artemia salina immediately after hatching are found masked in a precursor of high molecular weight (∼ 100000). The molecular weight of this precursor decreases slightly as development proceeds. However, its kinetic and chromatographic properties vary greatly. Shortly after hatching, the activities are highly inhibited, can be activated severalfold by the chaotropic agent potassium iodide and are not proportional to the amount of enzyme-containing extract added. Later after hatching, its kinetics become normal. From these observations we have concluded that (a) the majority, if not all, of the proteinase activities found later in development are already present immediately after hatching, although in an inhibited state that is gradually activated; (b) the proteinases are not found free in the cytoplasm but in a complex which may allow regulation of their activity., This work has been supported by grants of the J. March Foundation and Fondo Nacional para la Investigación Científica R. G. is a fellow of the Consejo Superior de Investigaciones Científicas.

We explored the use of microwave technology in fixation with the objective of achieving quicker fixation regimes, lower concentrations of toxic and volatile reagents, and enhanced antigen detection. We used a modified domestic microwave oven (900 W) and a low-power (5 W) microwave bench. The work was done on plant materials. The oven was supplemented with a cooling device, a stirring system, and a record of the sample temperature and the time of effective irradiation. The sample, immersed in a fixative solution of 1% paraformaldehyde (PFA) in PBS, was irradiated for only 10 minutes. The sample temperature did not exceed 37 degrees C. In these mild conditions, the quality of the (ultra)structural preservation of the samples, morphometrically assessed, was at the same level as obtained with the same fixative, using conventional methods. On the contrary, samples fixed in the same conditions without irradiation showed a poor structural preservation. The antigenic preservation of the irradiated samples was excellent, since the labeling levels of two nucleolar proteins, detected by immunogold, were three times higher than in conventionally fixed samples. In the so-called microwave bench, the pathway of microwaves is guided, so that low-power microwaves directly hit the sample and there is no dispersion of energy. Temperature of fixative did not increase after microwave irradiation. Fixation in the bench with either 4% PFA, or 1% PFA, for 20 minutes resulted in structural preservation of samples similar in quality as obtained with conventional fixation and in a similar or better level of antigen preservation. Therefore, controlling temperature and effective irradiation is crucial in order to obtain optimal structural and antigen preservation with microwave-enhanced fixation. The dramatic differences observed between microwave-irradiated samples and samples fixed in the same conditions without irradiation, strongly support the existence of specific effects of microwaves on fixation, independent from the mere heating of the samples.

Few data are available on enzyme activity in amphibian plasma or erythrocytes. We measured the activity of several blood enzymes in the urodele amphibian Pleurodeles waltl reared under standard laboratory conditions. In subsequent experiments, we will estimate and compare the physiological and biochemical conditions of P. waltl when reared under extreme temperature or microgravity conditions. The enzymes selected were glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, superoxide dismutase, catalase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. In fresh plasma samples, enzyme activity in females was higher than in males, except for aspartate and alanine aminotransferases, which were equivalent in females and males. Glutamate dehydrogenase activity was higher in males than in females. In female erythrocytes, the activity of all enzymes was higher than in male erythrocytes. We have also studied the storage conditions of samples and observed that for most enzymes, the activity in freshly isolated plasma and erythrocyte preparations decreased after storage at −18 or +4°C.

Standard viable preservation methods for biological samples using low temperatures have been investigated concerning their storage capabilities under higher temperature levels than usual. For a representative set of organism classes (plants, mammalian cells, arthropods and aquatic invertebrates), the minimum appropriate storage conditions have been identified by screening storage temperatures at -196 degrees, -80 degrees, -20 degrees, +4 degrees, +20 degrees/25 degrees C for periods from 2 days to 4 weeks. For storage below 0 degree C, as a typical cryopreservative, dimethylsulfoxide (DMSO) was used. For some samples, the addition of trehalose (as cryopreservative) and the use of a nitrogen atmosphere were investigated. After storage, the material was tested for vitality. The findings demonstrated that acceptable preservation can be achieved under higher storage temperatures than are typically applied. Small, dense cultured plant cells survive for 21 d when moderately cooled (+4 degrees to -20 degrees C); addition of trehalose enhances viability at -20 degrees C. For mammalian cells, the results show that human lymphocytes can be preserved for 3 d at 25 degrees C, 7 d at 4 degrees C and 28 d at -80 degrees C. Friend leukaemia virus transformed cells can be stored for 3 d at 25 degrees C, 14 d at 4 degrees C and 28 d at -80 degrees C. Hybridoma cells can be kept 7 d at 4 degrees C and 28 d at -20 degrees C or -80 degrees C. Model arthropod systems are well preserved for 2 weeks if maintained at lower temperatures that vary depending on the species and/or stage of development; e.g., 12 degrees C for Drosophila imagoes and 4-6 degrees C for Artemia nauplii. For aquatic invertebrates such as sea urchins, embryonic and larval stages can be preserved for several weeks at +6 degrees C, whereas sperm and eggs can best be stored at + 4 degrees C for up to 5 d at maximum. These results enhance the range of feasible space experiments with biological systems. Moreover, for typical terrestrial preservation methods, considerable modification potential is identified.

The well-known technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects, our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoy's solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue in the microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.

10 páginas, 7 figuras, 1 tabla -- PAGS nros. 1244-1253, Seeds of Arabidopsis thaliana were sent to the International Space Station in the “Cervantes Mission” (Spanish Soyuz Mission). Seed germination was initiated in flight by supplying culture medium. Seedlings were grown for 4 days at 22 °C, and growth was stopped by the addition of paraformaldehyde fixative. Once back on the ground, samples were immediately processed for microscopy. A ground control experiment was simultaneously replicated. Glutaraldehyde-fixed root cells from seedlings grown in the Biorack on board of the Space Shuttle (STS-84 Mission) in similar conditions were also ultrastructurally examined. The length of seedlings grown at 1g was conspicuously shorter than parallel samples grown under microgravity. We examined the morphology of the root meristematic cells, with a focus on their nucleoli in the cortex and stele. In general, root cortical cells proliferate at a higher rate and their nucleoli are more active than those of stele cells. While the stele showed longer cells with larger nucleoli in the flight samples, cortical cells from space-grown seedlings were shorter, more numerous and more densely packed than ground controls. However, nucleoli were smaller and less active in fast proliferating flight cells than in the ground controls. This reduced level of ribosome synthesis in the flight samples is probably the result of an accelerated cell cycle. An altered rate of cell proliferation may be detrimental for the plant and could be the reason for the reported smaller size of older space-grown seedlings. Finally, two-dimensional protein electrophoresis showed noticeable differences between space samples and ground controls, This work has been supported by the Spanish “Programa Nacional de Investigación Espacial” Grants Nos. ESP2002-12062-E, ESP2002-12686-E, and ESP2003-09475-C02-02

Taking advantage of the fact than segmentation inArtemia is largely a postembryonic process making it more susceptible to environmental influences, heat treatments ofArtemia newly-hatched nauplii were shown to induce a severe inhibition of mesodermal structures, without apparently affecting the corresponding ectodermal and endodermal derivaives. This inhibition was reversible and with enough time the missing mesodermal structures developed. These results indicate that ectoderm and endoderm development can proceed without neccessarily a concomitant mesodermal differentiation, which in turn can be largely uncoupled from that of the rest of the germ layer derivatives.

Fluorescamine is a useful reagent in monitoring protein-DNA interactions only if a convenient method of separating the complex from free protein is available. Sedimentation of the complex provides such a method at least in the case of histone-like proteins capable of extensive interaction with DNA. This approach is therefore complementary to the filter binding assay. When the interaction of protein and DNA is compared by both methods, a clear-cut distinction between two steps is obtained: (i) a nucleation step that can be measured by the filter binding assay: and (ii) the cooperative growth of the complex that can only be measured by the sedimentation assay. The method is also useful to detect small amounts of protease contamination in DNA preparations.

Fluorescamine, a reagent capable of reacting with a wide variety of nucleophiles, only gives fluorescent products when reacted with primary amines. The results of a reexamination of the principal parameters affecting the reaction are presented with emphasis on practical aspects. In particular, the potential competition of other nucleophiles (secondary amines and thiols) and the effect of the pH on the reaction and on the fluorescence of the product are presented. Polyalcohols, due to their reversible interaction with fluorescamine, affect the velocity without interfering with its extent. The initial hydrolysis product of fluorescamine under mild alkaline conditions has been isolated and characterized. Although unreactive with most amines, it is shown that at pHs below 7 it will react with amines that show abnormal low pH-dependent reactivity with fluorescamine. Histidine is the only amino-acid that under these conditions will react giving a fluorescent product. Among other amino groups containing molecules tested, only polyamines, histamine, and N-amino terminal-free peptides have been found to give this type of reaction. The factors affecting the limit of sensitivity of the reaction are also studied. The very low, but significant, fluorescence of the blanks sets the actual limit of sensitivity of the reaction. A micromethod that reduces the amount of fluorescamine in the assay under conditions in which the reaction is quantitative is described. A similar micromethod can be used with the O-phtalaldehyde reaction. The particular application of fluorescamine in the assay of proteins is compared to other methods. Alkaline and acid hydrolysis are shown to decrease the variability of the reaction shown by different proteins, while increasing the sensitivity. The different practical alternatives in the assay of proteins with fluorescamine and O-phtalaldehyde are discussed.

Mini M13 phages, about one-half as long and with one-half the DNA of wild-type filaments, contain a full amount of the gene 3 protein. This finding supports earlier evidence for an adsorption function of the gene 3 protein and its location at the end(s) rather than along the length of the filament. An unusual property of the extracted protein is its aggregation even under strongly denaturing and reducing conditions.

Several molecules of a protein specified by gene 3 of M13 comprise a minor fraction of the phage coat and have been assigned a role in adsorption to the bacterial cell. We find that the gene-3 protein molecules of the virion are fully conserved in phage that have attached irreversibly to the host cell, and they form a complex with the phage DNA when it has been converted to a duplex replicative form. In cells infected at a restrictive temperature with a thermosensitive mutant in gene 3, there is no conversion of the phage DNA to the replicative form. Both the adsorbed phage and the complex of replicative form DNA with the gene-3 protein were isolated with the inner membrane fraction of the cell. We suggest that the gene-3 protein may perform an essential function in the synthesis of replicative form by linking the phage DNA to a cellular replicative system in or on the inner cell membrane.

As described in this article, visualization and/or photography under uv light of 4-chloro-1-naphthol-developed, peroxidase-marked immunoblots allows an increase in sensitivity of more than 100 times over the apparent staining results observable under normal visible white light. This increase in sensitivity can be obtained with the minimal additional requirement of an uv lamp, with the actual chloronaphthol staining procedure remaining unaltered and thereby allowing the monitoring of specific reactions with much smaller quantities of antigen or antibodies. Substantial shortening of the procedure is another advantage, making it possible to complete in 20 min or even less a procedure usually requiring 3 to 6 h. The phenomenon depends on the uv absorption and the fluorescence quenching properties of the products of the peroxidase reaction. The absorption spectra of the membranes with or without peroxidase products indicate that an intermediate in the peroxidase reaction is responsible for the absorption under uv light. This intermediate accumulates under conditions where the final product absorbing in the visible light has not begun to be produced, thus explaining the large increase in sensitivity. The behaviors of three types of membranes, nitrocellulose, nylon, and Immobilon (PVDF), are compared. Due to its lower uv absorption, PVDF gives by far the best results, followed by nitrocellulose.

Interaction between the filamentous phage M13 and the Escherichia coli host progresses through stages of binding, eclipse, and penetration, defined as follows: binding of phage to cell receptor may be reversible or irreversible, eclipse of phage makes it noninfective and susceptible to DNase, and penetration entails uptake of phage components in a manner tightly coupled to replication. Binding (or adsorption) was specific for F+ cells, was relatively slow, did not follow simple bimolecular kinetics, and attained a value of about one phage per cell. It was blocked by low temperature, cyanide, or starvation and was unaffected by rifampicin. Binding capacity of phages was removed by subtilisin treatment which also released the gene 3 (adsorption) protein from the viral particle. Rifampicin which blocks replication prevents penetration of an irreversibly attached and altered (“eclipsed”) phage. Thus, decapsidation and penetration of the phage are coupled to replication of the viral DNA. Binding or eclipse triggered an alteration of the gene 3 protein to a lower molecular-weight form. The coat proteins (gene 3 and gene 8) were taken up by the cells to the same extent as the DNA. Cells disrupted by lysozyme-detergent treatment, as judged by release of β-galactosidase, retained their capacity to bind and eclipse the phage particle; binding to disrupted cells remained cyanide sensitive. After this treatment F− cells were as active in the binding of M13 as F+ cells. Such preparations from both F+ and F− cells had the capacity of replicating the DNA in intact phage particles to the duplex replicative form. In a discussion of these findings, current models for the key role of the F pilus (of F+ cells) in phage adsorption are discussed and a larger role for the phage adsorption protein is proposed.

The properties of the reaction of fluorescamine with proteins are the basis for the development of a sensitive, general and simple method for the assay of proteolytic activities. More importantly, the assay measures the initial step(s) of proteolytic attack, making it specially suitable for the examination of the controlling factors that regulate proteolytic degradation and/or the detection of “specific” proteinases. The method allows the simple determination of the general parameters of enzyme action, V and Km, using proteins, i.e. the physiological substrates of the proteinases. The more appropriate proteins to be used as substrates are the N-amino-terminally blocked ones. Many proteins fulfill this requirement. If the particular protein whose degradation has to be studied lacks this modification, three different approaches can be used to study its degradation: (a)the accumulation of N-amino termini in excess over that of the intact substrate; (b)a gel filtration/continuous method and (c) the chemical blockage of its amino groups. The particular advantages of each of these approaches are discussed., This work has been supported by grants from the J. March Foundation and Fondo Nacional para el desarrollo de la Investigación Científica. R. G. and J.V.C. are fellows of the Consejo Superior de Investigaciones Científicas.

A systematic examination of the properties of the clot assay, a highly sensitive and simple method to detect DNase activities, is presented. It is shown that it is both quantitative and specific for monitoring the double-strand cleavage of DNA produced by specific and nonspecific endonucleases. Its simplicity and ease of quantification, which exceeds that of other methods of similar sensitivity, indicates that the clot assay can be advantageous in monitoring the activity of both types of endonucleases, especially if carried out in parallel with other more informative assays on the mechanism of action of the enzymes. As an example of its use, it is shown that pancreatic DNase I is capable, in the absence of external metal addition, of a limited attack on DNA containing bound divalent cations.

The high redox potential of the couple NAD + NADH has limited in the past the application of dehydrogenases in the coupled assay of enzymes. Here the way to obviate this difficulty is presented, by using the chemical analog 3-acetyl-NAD+ and an auxiliary enzyme after the dehydrogenase. A general kinetic scheme of the multienzyme systems is presented in the discussion that helps to explain why this approach succeeds in making these dehydrogenases satisfactory auxiliary enzymes even in the unfavorable direction.

The intracellular location of brain hexokinase and the nature of its association with mitochondria were studied in calf and rat. Three different media were used in order to minimize possible artifacts in the homogenization process. Subcellular fractionation was followed testing, in addition to hexokinase, certain enzymes known as markers for the different cellular fractions. In the conditions used, hexokinase was almost entirely associated with mitochondria in calf, but only partially associated in rat. This is at variance with the rest of the enzymes involved in the glucose 6-phosphate crossroads. The use of a “physiological saline” homogenization medium resulted in a considerable increase in the proportion of soluble hexokinase in rat brain. Repeated washings of mitochondria from calf brain released little hexokinase, but in rat brain a rapid release was observed. Upon submitochondrial fractionation by Sottocasa's method, a substantial amount of hexokinase appeared bound to the inner membrane in the case of calf brain. In rat brain, the evidence found also points to the binding of the greater part of hexokinase to the inner membrane. Brain mitochondria were found to be sensitive to atractyloside, which made it possible to locate mitochondrial bound hexokinase as external to the ATP barrier, both in calf and rat. These results indicate that mitochondrially bound hexokinase is only active on substrates located in the cytosol., We were in receipt of fellowships from the Spanish Ministry of Education and Science.

Summary The pyruvate kinase of rat adipose tissue is strongly inhibited by physiological concentrations of alanine. This inhibition is counteracted by FDP + in the μmolar range of concentrations. These effects can be observed independently of the homogenization medium employed and in partially purified preparations of the enzyme. Aging of the preparations tends to produce the loss of the inhibitory and activatory effects, pointing to their allosteric nature.