Ocean acidification bends the mermaid's wineglass.

Ocean acidification lowers the saturation state of calcium carbonate, decreasing net calcification and compromising the skeletons of organisms such as corals, molluscs and algae. These calcified structures can protect organisms from predation and improve access to light, nutrients and dispersive currents. While some species (such as urchins, corals and mussels) survive with decreased calcification, they can suffer from inferior mechanical performance. Here, we used cantilever beam theory to test the hypothesis that decreased calcification would impair the mechanical performance of the green alga Acetabularia acetabulum along a CO2 gradient created by volcanic seeps off Vulcano, Italy. Calcification and mechanical properties declined as calcium carbonate saturation fell; algae at 2283 µatm CO2 were 32% less calcified, 40% less stiff and 40% droopier. Moreover, calcification was not a linear proxy for mechanical performance; stem stiffness decreased exponentially with reduced calcification. Although calcifying organisms can tolerate high CO2 conditions, even subtle changes in calcification can cause dramatic changes in skeletal performance, which may in turn affect key biotic and abiotic interactions.

Spectroscopic and biochemical analysis of regions of the cell wall of the unicellular 'mannan weed', Acetabularia acetabulum.

Although the Dasycladalean alga Acetabularia acetabulum has long been known to contain mannan-rich walls, it is not known to what extent wall composition varies as a function of the elaborate cellular differentiation of this cell, nor has it been determined what other polysaccharides accompany the mannans. Cell walls were prepared from rhizoids, stalks, hairs, hair scars, apical septa, gametophores and gametangia, subjected to nuclear magnetic resonance and Fourier transform infrared spectroscopy, and analyzed for monosaccharide composition and linkage, although material limitations prevented some cell regions from being analyzed by some of the methods. In diplophase, walls contain a para-crystalline mannan, with other polysaccharides accounting for 10-20% of the wall mass; in haplophase, gametangia have a cellulosic wall, with mannans and other polymers representing about a quarter of the mass. In the walls of the diplophase, the mannan appears less crystalline than typical of cellulose. The walls of both diploid and haploid phases contain little if any xyloglucan or pectic polysaccharides, but appear to contain small amounts of a homorhamnan, galactomannans and glucogalactomannans, and branched xylans. These ancillary polysaccharides are approximately as abundant in the cellulose-rich gametangia as in the mannan-rich diplophase. In the diplophase, different regions of the cell differ modestly but reproducibly in the composition of the cell wall. These results suggest unique cell wall architecture for the mannan-rich cell walls of the Dasycladales.

H+ -pumping rhodopsin from the marine alga Acetabularia.

An opsin-encoding cDNA was cloned from the marine alga Acetabularia acetabulum. The cDNA was expressed in Xenopus oocytes into functional Acetabularia rhodopsin (AR) mediating H+ carried outward photocurrents of up to 1.2 microA with an action spectrum maximum at 518 nm (AR518). AR is the first ion-pumping rhodopsin found in a plant organism. Steady-state photocurrents of AR are always positive and rise sigmoidally from negative to positive transmembrane voltages. Numerous kinetic details (amplitudes and time constants), including voltage-dependent recovery of the dark state after light-off, are documented with respect to their sensitivities to light, internal and external pH, and the transmembrane voltage. The results are analyzed by enzyme kinetic formalisms using a simplified version of the known photocycle of bacteriorhodopsin (BR). Blue-light causes a shunt of the photocycle under H+ reuptake from the extracellular side. Similarities and differences of AR with BR are pointed out. This detailed electrophysiological characterization highlights voltage dependencies in catalytic membrane processes of this eukaryotic, H+ -pumping rhodopsin and of microbial-type rhodopsins in general.

New models for circadian systems in microorganisms.

Microorganisms provide important model systems for studying circadian rhythms, and they are overturning established ideas about the molecular mechanisms of rhythmicity. The transcription/translation feedback model that has been accepted as the basis of circadian clock mechanisms in eukaryotes does not account for old data from the alga Acetabularia demonstrating that transcription is not required for rhythmicity. Moreover, new results showing in vitro rhythmicity of KaiC protein phosphorylation in the cyanobacterium Synechococcus, and rhythmicity in strains of the fungus Neurospora carrying clock gene null mutations, require new ways of looking at circadian systems.

Development and organization of the central vacuole of Acetabularia acetabulum.

* Here we analyzed the shape of the central vacuole of Acetabularia acetabulum by visualizing its development during diplophase (from juvenility through reproduction) and haplophase (from meiosis through mating). * Light microscopy and whole-organism applications of a pH-sensitive dye, neutral red, were used to visualize the anatomy of the central vacuole. We studied connectivity within the thallus by locally applying dye to morphologically distinct regions (rhizoid, stalk, apex, hairs) and observing dye movements. * In vegetative thalli most of the rhizoid, stalk and young hairs stained with dye. In reproductive structures (caps, gametangia) dye also stained the majority of the interiors. When applied to small areas, dye moved at different rates through each region of the thallus (e.g. within the stalk). Dye moved from younger hairs, but not from older hairs, into the stalk. Errors in incorporation of central vacuole into gametangia occurred at <10(-5). * These data indicate that the central vacuole of A. acetabulum is a ramified polar organelle with, potentially, a gel-like sap that actively remodels its morphology during development.

The search for a biosensor as a witness of a human laying on of hands ritual.

CONTEXT: Intentional healing by laying on of hands is a popular complementary therapy. Previous studies of this therapy have been focused on the influence of laying on of hands with focused intention on the patient or on a biological model that took the place of the patient. OBJECTIVE: Exploring the line of thinking that the consciousness-mediated act of healing during a healer-patient ritual changes a consciousness field that could be detected in another living non-human organism that was present only as a witness and was not the object of any directed intention. DESIGN: A comparison of a biosensor's behavior during healer-patient ritual treatments that were alternated by non-healing periods. SETTING AND PARTICIPANTS: An automatic device for measurement of ultra-weak emission of photons from algae was placed at the location of a healer during a series of experiments consisting of 36 healing sessions with human patients. Neither healer nor patients were aware of the type of measurements that took place. MAIN OUTCOME MEASURES: The number and periodicity of photon counts. RESULTS: Primary data analysis showed that the photon count distributions show some remarkable alterations during the ritual of healer-patient sessions. The data further suggest that during healing a shift in cyclical components of photon emission occurs. CONCLUSIONS: The significance of the experiment lies in the possibility to enter the discussion on a quantitative basis with respect to the relevance of the patient-healer relationship in intentional healing.

Oscillations in ultraweak photon emission of Acetabularia acetabulum (L.).

Ultraweak photon emission of dark-incubated A. acetabulum cells were measured with the use of a sensitive electronphotomultiplier of the Hamamatsu 550 type. The photon count series were subjected to Fourier analysis for 2-1020 sec period range. The average level of photon emission in samples containing 50 cells was approximately. 40% above background. Cell cultures were prepared at least 24 hr before the photon emission measurements and kept un-disturbed ("established cultures"). This paper reports results of Fourier analysis of a number of samples of Acetabularia cells. In a single population cells periodicity of light emission was not defined directly from Fourier transformation. A large number of analyses, however, if they are combined and compared with background data, reveal a cell-culture specific frequency pattern. The results suggest the idea that established cell-cultures are characterized by higher intensities of long period (minutes) oscillations occurs, while a relative decrease was observed in the short period (few seconds) range. The long period oscillations were not detected in cell cultures that were prepared within 1 hr before the photon emission measurements. It is concluded that Fourier analysis of ultraweak photon emission, even with relatively low signals, appears to be possible. It may serve as a non-invasive tool for monitoring the physiological state of cells, or for studying the control of intercellular dynamics.

Poly(A)+ RNA during vegetative development of Acetabularia peniculus.

In the juvenile stage, the diploid giant-celled green algae Acetabularia spp. are differentiated into an upright stalk and an irregularly branched rhizoid. Early amputation and grafting experiments as well as biochemical and molecular analyses have shown that mRNA (as poly(A)+ RNA) is continuously supplied from the primary nucleus in the rhizoid and accumulates in the stalk apex. In the present study, localization of poly(A)+ RNA in the juvenile stage of the Acetabularia peniculus was investigated by fluorescent in situ hybridization using oligo(dT) as a probe. The signal was localized in the apical cytoplasm and, in addition, multiple longitudinal striations throughout the stalk and rhizoid cytoplasm. A large portion of the poly(A)+ RNA striations exhibited structural polarity, broadened at one end and gradually thinned toward the other end. Some of the striations in the rhizoid cytoplasm were continuous with a zone of signal in the area of the perinuclear rim. The poly(A)+ RNA striations were associated with thick bands of longitudinal actin bundles which run through the entire length of the stalk. Cytochalasin D caused fragmentation of the actin bundles and irregular distribution of the fluorescent signal. We suggest that the poly(A)+ RNA striations constitute a hitherto unknown form of packaged mRNA that is transported over large distances along the actin cytoskeleton to be stored and expressed in the growing apex.

Delayed luminescence of biological systems arising from correlated many-soliton states.

The kinetics of the delayed luminescence arising from correlated coherent many-soliton states in low-dimensional macromolecular systems, is calculated and shown to be different from the one arising from independent soliton states. The correlation between coherent electron states is essential at relatively high levels of excitation in the presence of very long macromolecules in a system. These conditions can be fulfilled in such biological systems, like algae Acetabularia Acetabulum. The cytoskeleton of this unicellular alga contains macromolecular structures (actin filaments, microtubules, etc.) of the length of several hundreds angstroms and more, in which many-soliton coherent states can exist. Indeed, the correlated coherent model is shown to give better fit of the experimental data for this type of algae in a wide range of intensities of the stimulating light, as compared with the model of noncorrelated solitons. The nonlinearity of the dependence of delayed luminescence intensity on the level of excitation increases with the increase of correlation between solitons.

Analogous features of delayed luminescence from Acetabularia acetabulum and some solid state systems.

This paper compares some features of the delayed luminescence in the seconds range emitted from an unicellular alga Acetabularia acetabulum and from some solid state systems. Results lead us to think that such delayed luminescence is connected to the dimension of the ordered structures present in both biological and solid state systems. In particular, for the biological system examined, these structures are thought to be those which contribute to the dynamic formation of the cytoskeleton.

Changes in membrane potential and delayed luminescence of Acetabularia acetabulum.

This paper examines the effect of changes in membrane potential on the critical parameters of delayed luminescence of Acetabularia acetabulum. We show that these parameters are altered by changes in membrane potential in ways that may reflect concomitant changes in energy storage and energy coupling.

The roles of light and the nucleus in the regulation of reproductive onset in Acetabularia acetabulum.

At reproductive onset the marine green alga Acetabularia acetabulum (L.) P.C. Silva redirects growth from vertical elongation of the axis of the plant to lateral expansion of a disk-shaped reproductive structure, the "cap." We used amputation to synchronize cap initiation and to facilitate investigation of the light requirements during amputation-induced cap initiation. Following amputation of a nascent cap, most plants initiate one whorl of vegetative hairs and then a cap. Both hair and cap initiation required photosynthesis, as indicated by studies with 3-(3',4'-dichlorophenyl)-1, 1-dimethylurea, but did not require the nucleus. Amputation-induced hair initiation occurred in red light, but 10 min of blue light given in a background of red light significantly increased hair initiation, supporting previous studies that hair initiation is a blue-light-triggered photomorphogenic event. Amputation-induced cap initiation also occurred in red light, but daily 10-min flashes of blue light given in a background of red light did not significantly enhance cap initiation. We also examined the light requirements of intact plants at each phase of development. In the absence of blue light, juveniles and adults with /=13.7 +/- 4.3 whorls of hairs initiated caps in the absence of blue light, suggesting that there is a point in late adult development beyond which cap initiation does not require blue light. Several plausible interpretations of the role of light and the nucleus in the regulation of reproductive onset are discussed to try to reconcile these data with those in the literature.

The estimation of rapid rate constants from current-amplitude frequency distributions of single-channel recordings.

A method is described for estimating rapid rate constants from the distributions of current amplitude observed in single-channel electrical recordings. It has the advantages over previous, similar approaches that it can accommodate both multistate kinetic models and adjustable filtering of the data using an 8-pole Bessel filter. The method is conceptually straightforward: the observed distributions of current amplitude are compared with theoretical distributions derived by combining several simplifying assumptions about the underlying stochastic process with a model of the filter and electrical noise. Parameters are estimated by approximate maximum likelihood. The method was used successfully to estimate rate constants for both a simple two-state kinetic model (the transitions between open and closed states during the rapid gating of an outward-rectifying K(+)-selective channel in the plasma membrane of Acetabularia) and a complex multistate kinetic model (the blockade of the maxi cation channel in the plasma membrane of rye roots by verapamil). For the two-state model, parameters were estimated well, provided that they were not too fast or too slow in relation to the sampling rate. In the three-state model the precision of estimates depended in a complex way on the values of all rate parameters in the model.

Protein phosphatase 2A, a potential regulator of actin dynamics and actin-based organelle motility in the green alga Acetabularia.

The giant, unicellular alga Acetabularia is a well known experimental model for the study of actin-dependent intracellular organelle motility. In the cyst stage, however, which is equivalent to the gametophytic stage, organelles are immobile, even though an actin cytoskeleton is present. The reason for the lack of organelle motility at this stage has not been known. To test the hypothesis that organelle motility could be under the control of posttranslational modification by protein phosphorylation, we have treated cysts with submicromolar concentrations of okadaic acid or calyculin A, both potent inhibitors of serine/threonine protein phosphatases (ser/thr-PPases). The effects were dramatic: Instead of linear actin bundles typical for control cysts, circular arrays of actin bundles formed in the cortical cyst cytoplasm. Concomitant with the formation of these action rings, the cytoplasmic layers beneath the rings began to slowly rotate in a continuous and uniform counter-clockwise fashion. This effect suggests that protein phosphorylation acts on the actin cytoskeleton at two levels: (1) It changes the assembly properties of the actin filament system to the extent that novel cytoskeletal configurations are formed and (2) it raises the activity of putative motor proteins involved in the rotational movements to levels sufficiently high to support motility at a stage when organelle motility does not normally occur. Northern blot analysis of cyst stage-mRNA using probes specific to protein phosphatase type 1 (PP1) and type 2A (PP2A) reveals that PP2A is strongly expressed at this developmental stage whereas PP1 is not detectable, suggesting that PP2A is the likely target to the protein phosphatase inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

In defense of proper cosinor analysis.

Parallaxes can originate from different sources, from bias towards proving a favored hypothesis, but also from bias against existing evidence in support of an unwanted hypothesis. One approach to avoiding either bias is to shift emphasis from hypothesis testing to parameter estimation. Complementary statistical approaches applied to replicates of given experiments can then serve to further a given field of science by providing converging or diverging results depending on whether the original hypothesis was true or false.

Dynamics and pharmacological perturbations of the endoplasmic reticulum in the unicellular green alga Acetabularia.

The giant unicellular green alga Acetabularia was labeled with the lipophilic fluorochrome DiOC6 (3,3'-dihexyloxacarbocyanine) and examined by confocal laser scanning microscopy to study the distribution of the endoplasmic reticulum (ER) and its dynamic changes after the application of inhibitors. In control cells, a two-dimensional polygonal network of ER sheets and tubulus is suspended between parallel, longitudinally oriented bands. These bands coincide with the main physical tracks of organelle transport. All treatments that inhibited organelle motility caused a transformation of the polygonal network into confluent large patches of lamellar ER sheets. The shape of the lamellar sheets and residual activities of the ER were dependent on the inhibitors used. The largest ER lamellae were obtained after cytochalasin D (CD) treatment which effectively stopped cytoplasmic streaming. CD also caused the formation of a network of fine tubules overlapping with the lamellar sheets. Okadaic acid, a specific inhibitor of serine/threonine-protein phosphatases, also caused inhibition of organelle movement and enlargement of lamellar areas. Tension in the cytoplasm appeared to be reduced, as judged from the convexly curved lamellar rims and wavy connecting ER tubules. In contrast, N-ethylmaleimide, a sulfhydryl group blocking reagent, rapidly stopped streaming and halted all activities of the ER in a rigor-like state. These effects are interpreted in the context of actin-based motility phenomena prevalent in Acetabularia, and regulatory principles are discussed that might underlie ER dynamics.

High-resolution measurement of circadian periodicities in Acetabularia.

Well-expressed endogenous circadian rhythms in Acetabularia acetabulum were spectrally analyzed and recorded in time-period distributions. The stability of the circadian periods under constant conditions and their changes could be monitored continually in step sizes close to the circadian period length. The resolution of period estimates of the circadian component was increased by a factor of approximately 4-10 by adapting analyzed interval lengths to full period sizes of the corresponding main component. Methodological aspects of the applied algorithms are discussed by means of examples that measure the temperature dependency of the circadian period.

A re-examination of the role of the nucleus in generating the circadian rhythm in Acetabularia.

The role of the nucleus in the generation of the circadian rhythm in Acetabularia has been nuclear. Early experiments showed that the plant could exhibit a circadian rhythm in the absence of a nucleus. However, other experiments appeared to show that the nucleus could impart phase information to the rhythm, and so therefore must be a part of the system that generates the rhythm. We have conducted experiments similar to these--in particular, one in which the nuclear end of the plant was entrained on a light-dark cycle that was opposite that of the rest of the plant. The phase of the free-running rhythm of this type of plant is not consistent with the conclusion that the nucleus is part of the circadian oscillator. We have also tried entraining opposite ends of plants with no nuclei on opposite light-dark cycles. The ultimate phases of these plants appear to be nearly random. A possible interpretation of these experiments is discussed.