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We performed a stream fertilisation experiment in an oligotrophic, forested, undisturbed stream by increasing nitrogen and phosphorus concentrations. The effect of nutrient loading on microfauna was studied over 6 months. A before after control impact (BACI) design was used to determine the outcome of the addition by comparing three upstream non-enriched reaches with an enriched one downstream. Ciliates such as Hymenostomata ( Frontonia, Lembadion, Paramecium, Cyclidium) and Pleurostomatida ( Litonotus) showed a positive response to fertilisation. No significant changes were detected for flagellates and testate amoebae. Mean density values observed (0.2–3×103 ind cm−2) were compared with those found in literature. [ABSTRACT FROM AUTHOR]

To investigate the relationship between the Japanese Paramecium bursaria host and its symbiont, we studied the effect of a host cell-free extract on carbon fixation and photosynthate release of the symbiont. The host extract enhanced symbiotic algal carbon fixation about 3-fold at an increased concentration; however, release of photosynthate hardly changed. Since the enhancing effect was not affected by elimination of carbon dioxide from the host extract, the existence of a host factor that stimulates algal carbon fixation was made clear. The host factor is a heat-stable, low molecular weight substance. In relation to the pH dependence, the extract improved carbon fixation at acidic and neutral pH and showed almost no effect at pH 9.0. Therefore, the stimulation of carbon fixation by the host factor is unlikely to be caused by intracellular pH change. The extract also improved carbon fixation of several Chlorella species, symbiotic and free-living, and apparently exhibited no species specificity. Therefore, the host seems to regulate the photosynthesis of the symbiont via a specific compound. [ABSTRACT FROM AUTHOR]

A particle inflow gun (PIG) was constructed and tested for its utility to transform Paramecium using tungsten or gold as the DNA carrier particle. In the first set of experiments we transformed Paramecium with a plasmid containing the neomycin-resistance gene, obtaining a transformation efficiency of 0.31±0.14% (mean±SD) for tungsten particles and 1.30±0.29% for gold particles. Plasmid DNA precipitated upon tungsten was shown to be stable for transformation purposes for up to 1 h prior to use and had no detectable effects on transformation efficiency. In addition, we demonstrated that at high frequency (71±20%) a Paramecium mutant strain could be phenotypically rescued by co-transformation with a second plasmid containing the selectable neomycin-resistance gene. The PIG coupled with tungsten particles as the carrier offers a low-cost alternative for biolistic transformation of Paramecium. [ABSTRACT FROM AUTHOR]

High-speed imaging was used to record the prey-tracking behavior of larval zebrafish as they fed upon paramecium. Prey tracking is comprised of a variable set of discrete locomotor movements that together align the larva with the paramecium and bring it into close proximity, usually within one body length. These tracking behaviors are followed by a brief capture swim bout that was previously described [Borla et al., 2002]. Tracking movements were classified as either swimming or turning bouts. The swimming bouts were similar to a previously characterized larval slow swim [Budick and O’Malley, 2000], but the turning movements consisted of unique J-shaped bends which appear to minimize forward hydrodynamic disturbance when approaching the paramecium. Such J-turn tracking bouts consisted of multiple unilateral contractions to one side of the body. J-turns slowly and moderately alter the orientation of the larva – this is in contrast to previously described escape and routine turns. Tracking behaviors appear to be entirely visually guided. Infra-red (IR) imaging of locomotor behaviors in a dark environment revealed a complete absence of tracking behaviors, even though the normal repertoire of other locomotive behaviors was recorded. Concomitantly, such larvae were greatly impaired in consuming paramecia. The tracking behavior is of interest because it indicates the presence of sophisticated locomotor control circuitry in this relatively simple model organism. Such locomotor strategies may be conserved and elaborated upon by other larval and adult fishes. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We propose a visual control system for motile cells. Our goal Is to control microorganisms as microscale smart robots for various applications. As a first step, we have developed a visual feedback control system for Paramecium caudatum cells. In order to ensure both detailed measurements and a large working space, "lock-on" tracking of a free-swimming cell with a high frame rate is essential. In our system, high-speed (1-kHz frame rate) tracking hardware and software are used for the continuous observation of moving cells with high magnification. Cells swim in a chamber, and their positions and other properties are measured and computed in real time. The chamber position is visually controlled automatically to track a specific cell. The cell motion is controlled electrically by utilizing the galvanotaxis (Intrinsic reaction to electrical stimulus) of microorganisms. Experimental results for open-loop control (periodic zigzag motion) and closed-loop control (trapping within a small region that is 1 mm wide) demonstrate the possibility of using microorganisms as micromachines. [ABSTRACT FROM AUTHOR]

Reports that abrupt increase in ultraviolet sensitivity at late log-phase of growth in Paramecium tetraurelia. Clonal life cycle of Paramecium; Developmental process of differentiation and aging that advances as the cell cycle rotates.

Describes the isolation and characterization of two additional Paramecium mutants that fail to excise an internal eliminated sequences (IES) due to a single base change. Differentiation of a new somatic macronuclear genome in ciliated protozoa; Analysis of many IES; Functional importance of nucleotides within the consensus sequence in Paramecium.

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APlasmodium falciparumcDNA expression clone,λPf9, had been identified earlier as a protective epitope, using anti-λPf9 antibodies and combinatorial phagotopes. A segment of the Pf9 gene showed homology withParameciumimmobilization surface antigens such as 51B, 51A and 156G. A synthetic Pf9-peptide was designed from this region, and specific antibodies were raised. Each of these anti-Pf9 antibodies and combinatorial reagents, as well as anti-Paramecium51B antibodies, recognized the Pf9-peptide on ELISA, and the same protein band in parasite immunoblots. TheP. falciparumprotein was released from the merozoite membrane fraction on treatment with PI-PLC, indicating the presence of a GPI anchor. Anti-Pf9-peptide antibodies specifically inhibited the growth ofP. falciparumin culture. Immunofluorescence assays showed the reactivity of anti-Pf9-peptide sera withP. falciparummerozoites and gametocytes, as well as on the surface ofParamecium tetraurelia. The Pf9-peptide was able to induce proliferation of splenic lymphocytes obtained from mice infected with the rodent malarial parasitesPlasmodium bergheiandPlasmodium yoelii.These results point towardsPlasmodiumPf9 as a conserved novel protective protein, sharing an epitope withParameciumsurface antigens. [ABSTRACT FROM AUTHOR]

This work deals with a comparative analysis of Stachybotrys chartarum strains isolated from various artificial cellulose-containing materials and natural substrates in geographically distant regions of Russia. The analysis included determination of the spore size; the strain toxicity to Paramecium caudatum; the strain resistance to the fungicides Benomil, Olilen, and Tilt; and the PCR study of the genome structure with the aid of a primer that was complementary to the core sequence of the SINE retrotransposon. It was found that some of the strains that were isolated from different areas and from different substrates differ in their toxicity, fungicide resistance, and genome structure. PCR analysis showed the absence of any correlation between the genome structure, the strain properties, the geographic area, and the substrates from which the strains were isolated. The pheno- and genotypic diversity of the strains and their different vegetative compatibility suggest the existence of an intraspecies diversity of the S. chartarum strains that were isolated in different geographic areas. The absence of any correlation between the pheno- and genotypic properties of the strains and the substrates from which they were isolated implies that the colonization of artificial substrates by S. chartarum occurred occasionally from natural habitats. The S. chartarum populations that live on artificial substrates are unlikely to have their own evolutionary history. [ABSTRACT FROM AUTHOR]

Assesses the potential role of G-proteins in chemokinenis, through a study conducted on a Paramecium tertaurelia that was pre-incubated with the G-protein modulator pertussis toxin. Results of whole cell behavioral and mortality assays; Effect of deltamethrin and modified guanosine nucleotide on calcium influx in ciliary vessels.

Summary In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl-BODIPY® FL C12 (BODIPY-CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red-shifted as the probe concentrates. In cells incubated with 25 µm BODIPY-CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10–15 min of continuous labelling, then it decreases until no food vacuoles are found in 30-min fed cells. BODIPY-CE accumulates in red-labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green-labelled vesicles at the apical pole. The inhibition of clathrin-mediated endocytosis does not interfere in P. primaurelia with BODIPY-CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol-binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH4 Cl reduces the lipid storage. Low temperature (4 °C) does not prevent the internalization of BODIPY-CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY-CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles,... [ABSTRACT FROM AUTHOR]

Membrane potential responses of Paramecium caudatum to an application of K+-rich solution were examined to understand the mechanisms underlying K+-induced backward swimming. A wild-type cell impaled by a microelectrode produced action potentials followed by a sustained depolarization in response to an application of a K+-rich test solution. After termination of the application, a prolongation of the depolarization (depolarizing after-potential) took place. Behavioral mutants incapable of exhibiting K+-induced backward swimming did not show depolarizing afterpotentials. Upon short application of K+-rich solution, the timing and duration of the ciliary reversal of the wild-type cell coincided well with the K+-induced depolarization. The duration of the depolarizing afterpotential decreased as the duration of the application increased. The depolarizing afterpotential recovered slowly after it had been suppressed by a preceding application of the K+-rich solution. By injection of an outward current into the wild-type cell, the action potentials were evoked normally during the period when the K+-induced depolarizing afterpotential was suppressed. We concluded that the prolongation of the depolarizing membrane potential response following the application of the K+-rich solution represents the Ca2+ conductance responsible for the K+-induced backward swimming in P. caudatum and that the characteristics of the K+-induced Ca2+ conductance are distinct from those of the Ca2+ conductance responsible for the action potentials. [ABSTRACT FROM AUTHOR]

A non-discharge mutant of Paramecium tetraurelia (nd12-35 degrees C, lacking exocytotic response upon stimulation with the nonpermeable polycationic secretagogue aminoethyldextran, AED), in the pawnA genetic context (d4-500r, lacking ciliary voltage-dependent Ca2+ influx), was shown to lack (45)Ca2+ entry from outside upon AED stimulation. In contrast, cells grown at 25 degrees C behave like the wildtype. To check the functional properties in more detail, fluorochrome-loaded 35 degrees C cells were stimulated, not only with AED (EC(100) = 10(-6) M in wildtype cells), but also with 4-chloro-meta-cresol, (4CmC, 0.5 mM), a permeable activator of ryanodine receptor-type Ca2+ release channels, usually at extracellular [Ca2+] of 50 microM, and eventually with a Ca2+ chelator added. We confirm that pwA-nd12(35 degrees C) cells lack any Ca2+ influx and any exocytosis of trichocysts in response to any stimulus. As we determined by x-ray microanalysis, total calcium content in alveolar sacs (subplasmalemmal stores) known to be mobilized upon exocytosis stimulation in wild-type cells, contain about the same total calcium in 35 degrees C as in 25 degrees C cells, and Ca2+ mobilization from alveoli by AED or 4CmC is also nearly the same. Due to the absence of any AED-induced Ca2+ influx in 35 degrees C cells and normal Ca2+ release from stores found by x-ray microanalysis one can exclude a "CICR"-type mechanism (Ca2+-induced Ca2+ release) and imply that normally a store-operated Ca2+ ("SOC") influx would occur (as in 25 degrees C cells). Furthermore, 35 degrees C cells display a significantly lower basal intracellular [Ca2+], so that any increase upon stimulation may be less expressed or even remain undetected. Under these conditions, any mobilization of Ca2+ from stores cannot compensate for the lack of Ca2+ influx, particularly since normally both components have to cooperate to achieve full exocytotic response. Also striking is our finding that 35 degrees C cells are unable to perform membrane fusion, as analyzed with the Ca2+ ionophore, A23187. These findings were corroborated by cryofixation and freeze-fracture analysis of trichocyst docking sites after AED or 4CmC stimulation, which also revealed no membrane fusion. In sum, in nd12 cells increased culture temperature entails multiple defects, notably insensitivity to any Ca2+ signal, which, moreover, cannot develop properly due to a lower basal [Ca2+] level and the lack of Ca2+ influx, despite normal store activation. [ABSTRACT FROM AUTHOR]

Background: A breakthrough in the understanding of centriole assembly was provided by the characterization of the UNI3 gene in Chlamydomonas. Deletion of this gene, found to encode a novel member of the tubulin superfamily, delta-tubulin, results in the loss of the C-tubule, in the nine microtubule triplets which are the hallmark of centrioles and basal bodies. Delta-tubulin homologs have been identified in the genomes of mammals and protozoa, but their phylogenetic relationships are unclear and their function is not yet known. Results: Using the method of gene-specific silencing, we have inactivated the Paramecium deltatubulin gene, which was recently identified. This inactivation leads to loss of the C-tubule in all basal bodies, without any effect on ciliogenesis. This deficiency does not directly affect basal body duplication, but perturbs the cortical cytoskeleton, progressively leading to mislocalization and loss of basal bodies and to altered cell size and shape. Furthermore, additional loss of B- and even Atubules at one or more triplet sites are observed: around these incomplete cylinders, the remaining doublets are nevertheless positioned according to the native ninefold symmetry. Conclusions: The fact that in two distinct phyla, delta-tubulin plays a similar role provides a new basis for interpreting phylogenetic relationships among delta-tubulins. The role of delta-tubulin in C-tubule assembly reveals that tubulins contribute subtle specificities at microtubule nucleation sites. Our observations also demonstrate the existence of a prepattern for the ninefold symmetry of the organelle which is maintained even if less than 9 triplets develop. [ABSTRACT FROM AUTHOR]

Presents a study on the contractile vacoule complex (CVC) of Paramecium. Information on the two CVCs of paramecium; Dynamics of the smooth membranes of the CVC; Electrophysiology of the CVC.

Studies the time-dependent transfection of Paramecium cells with a vector containing the gene of green fluorescent protein (GFP). Retention of the number of sarcoendoplasmic reticulum calcium (SERCA) molecules in the endoplasmic reticulum (ER); Insertion of GFP in the major cytoplasmic loop; Delivery of SERCA molecules from ER to the alveolar sacs.

The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca2+] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca2+ signals also occur when AED is applied in presence of the fast Ca2+ chelator, BAPTA. (ii) Extracellular La3+ application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca2+]i transient (while injected La3+ causes a sustained fluorescence signal). (iii) Simply increasing [Ca2+]o causes a similar rapid, short-lived [Ca2+]i transient. All these phenomena, (i–iii), are compatible with activation of an extracellular ``Ca2+/(polyvalent cation)-sensing receptor'' known from some higher eukaryotic systems, where this sensor (responding to Ca2+, La3+ and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores (``alveolar sacs'') are physically linked to the cell membrane and they can also be activated by the Ca2+ releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca2+ signal also in absence of Ca2+o we largely exclude a ``Ca2+-induced Ca2+ release'' (CICR) mechanism. Our finding of increased cortical Ca2+ signals after store depletion and re-addition of extracellular Ca2+ can be explained by a ``store-operated Ca2+ influx'' (SOC), i.e., a Ca2+ influx superimposing store activation. AED stimulation in presence of Mn2+o causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOC-type Ca2+ influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca2+/polyamine-sensing receptor, (ii) release of Ca2+ from subplasmalemmal stores, (iii) and Ca2+ influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca2+] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca2+] microdomains (≤0.5 μm, ≤33 msec) upon stimulation. [ABSTRACT FROM AUTHOR]

Chloroviruses are large viruses that replicate in chlorella-like green algae and normally exist as mutualistic endosymbionts (referred to as zoochlorellae) in protists such as Paramecium bursaria. Chlorovirus populations rise and fall in indigenous waters through time; however, the factors involved in these virus fluctuations are still under investigation. Chloroviruses attach to the surface of P. bursaria but cannot infect their zoochlorellae hosts because the viruses cannot reach the zoochlorellae as long as they are in the symbiotic phase. Predators of P. bursaria, such as copepods and didinia, can bring chloroviruses into contact with zoochlorellae by disrupting the paramecia, which results in an increase in virus titers in microcosm experiments. Here, we report that another predator of P. bursaria, Bursaria truncatella, can also increase chlorovirus titers. After two days of foraging on P. bursaria, B. truncatella increased infectious chlorovirus abundance about 20 times above the controls. Shorter term foraging (3 h) resulted in a small increase of chlorovirus titers over the controls and more foraging generated more chloroviruses. Considering that B. truncatella does not release viable zoochlorellae either during foraging or through fecal pellets, where zoochlorellae could be infected by chlorovirus, we suggest a third pathway of predator virus catalysis. By engulfing the entire protist and digesting it slowly, virus replication can occur within the predator and some of the virus is passed out through a waste vacuole. These results provide additional support for the hypothesis that predators of P. bursaria are important drivers of chlorovirus population sizes and dynamics. [ABSTRACT FROM AUTHOR]

Interviews anesthesiologist Stuart Hameroff concerning microtubules, anesthetics and quantum consciousness. Overview of pan-protopsychism; Difference between a fundamentalist and an emergentist; Means that microtubules control the paramecium sensory input and motor output.

Initiation of replication of the linear mitochondrial DNA from Paramecium occurs at a unique cross-linked terminus of the monomer molecule. Dimer length molecules, containing head-to-head monomers, are replicative intermediates. Previous studies have been with cloned dimer initiation region fragments but here we have isolated and sequenced isomeric forms of restriction fragments located at the initiation end of the monomer. The sequence isomers are inverted complements of each other in the region identified as the single-stranded DNA terminal loop. The unusual electrophoretic behaviour of these terminal restriction fragments supports the sequence data result that the loop is single stranded. These structural features are discussed in regard to mechanisms for the processing of dimer to monomer molecules. [ABSTRACT FROM AUTHOR]

Discusses deoxyribonucleic acid (DNA) rearrangements and mating-type determination in Paramecium tetraurelia. Paramecium tetraurelia genetics; Sexual processes in Paramecium; Determination of mating type; Effects of macronuclear development.

The cooling of deciliated Paramecium cells induced a transient Ca current and its amplitude depended on the rate of the temperature drop. The amplitude of the Ca current was increased by the addition of Ca2+ to the bath solution in a concentration-dependent manner, whereas Ni2+, Co2+, Mn2+ and Mg2+ each reversibly inhibited the Ca current in a concentration-dependent manner with apparent dissociation constants of 0.52, 0.66, 0.67 and 2.17 mmol · l−1, respectively. The Ca current was also inhibited reversibly by amiloride, with a dissociation constant of 0.32 mmol · l−1. The Ca current was desensitized by repetitive cooling. The amplitude of the Ca current at the second cooling was smaller than that at the first cooling when the interval was short, but recovered as the interval increased. Replacing extracellular Ca2+ with equimolar Sr2+ or Ba2+ did not significantly affect the amplitude of the current response to cooling, but it accelerated the rate of recovery from desensitization and slowed the decay of the current response. These results suggest that the desensitization and the inactivation of the Ca current may involve a Ca2+-dependent pathway. [ABSTRACT FROM AUTHOR]

The ionic mechanisms of the depolarizing and the hyperpolarizing quinine receptor potentials in the ciliate Paramecium caudatum were examined by using a behavioral mutant strain. The depolarizing receptor potential was induced by stimulating the anterior end of the specimen, and the hyperpolarizing receptor potential by stimulating the posterior end. The amplitude of both the depolarizing and the hyperpolarizing receptor potentials increased linearly with logarithmic increase in quinine concentration applied. Threshold concentration for inducing the depolarizing receptor potential was lower than that for the hyperpolarizing one. The peak level of the depolarizing receptor potential shifted towards the depolarizing direction with increasing external Ca2+ concentration while that of the hyperpolarizing receptor potential shifted in the depolarizing direction with increasing external K+ concentration. Under voltage-clamp conditions, the specimen produced an inward current in response to anterior stimulation, and an outward current in response to posterior stimulation. Both the peak inward and the peak outward currents showed a linear relationship with membrane potential. Current-voltage relationships of the receptor currents indicated conductance increase during the application of quinine. The depolarizing quinine receptor potential appears to be produced by an activation of Ca2+ channels, and the hyperpolarizing quinine receptor potential by an activation of K+ channels. [ABSTRACT FROM AUTHOR]

The antimalarial drugs, quinacrine, chloroquine, quinine, primaquine, and mefloquine, share structural similarities with W-7, a compound that inhibits calcium-dependent backward swimming and calcium currents in Paramecium. Therefore, we tested whether antimalarial drugs also inhibit backward swimming and calcium currents in P. calkinsi. When the Paramecium is depolarized in high potassium medium, voltage-dependent calcium channels in the ciliary membrane open causing the cell to swim backward for 30 to 70 s. Application of calcium channel inhibitors, such as W-7, reduce the duration of backward swimming. In 0.05 mM calcium, quinacrine, mefloquine, quinine, chloroquine, primaquine and W-7 all reduced the duration of backward swimming. These effects were seen in sodium-containing and sodium-free high potassium solutions as well as sodium-free depolarizing solutions containing potassium channel blockers. In these low calcium solutions, backward swimming was inhibited by 50% at concentrations ranging from 100 n M to 30 μ M. At higher calcium concentrations (1 m M or 15 m M), the effects of the antimalarials and W-7 were reduced. The effects of quinacrine and W-7 were tested directly on calcium currents using the two microelectrode voltage clamp technique. In 15 mM calcium, 100 μ M quinacrine and 100 μM W-7 reduced the peak calcium current by 51% and 42%, respectively. Thus, antimalarial drugs reduce calcium currents in Paramecium calkinsi. [ABSTRACT FROM AUTHOR]

A stopped-flow spectrophotometric technique was used to study the kinetics of Ca flux into ciliary membrane vesicles from Paramecium tetraurelia wild-type and several 'pawn' mutants with defective Ca conductances. 15 mm Arsenazo III was used as metallochromic indicator and as intravesicular Ca trap. The absolute amount of Ca-permeable vesicles was significantly reduced in preparations from the 'pawn' mutants compared to wild-type. However, influx kinetics were identical for vesicles from wild-type and 'pawn' mutant Paramecia when the fraction of Ca-permeable vesicles was taken into account. Ca influx was rapid with a time constant of about 1.5 sec and an initial saturation rate of arsenazo III of about 50%/single vesicle ×sec. Ca influx rates were half-maximal at approximately 20 μ m Ca. Comparisons of Ba toxicity tested with a behavioral assay, Ca inward conductances under voltage-clamp conditions and Ca influx kinetics between wild-type and the 'pawn' mutants pwA (d4-94), leaky pwB (d4-96) and the double mutant pwA/pwB indicated that Ca transport in all types of ciliary membrane vesicles occurred through similar Ca gates. [ABSTRACT FROM AUTHOR]

A model is presented for one type of membrane tubulation. In this model large transmembrane protein complexes link together to form helical bands. To form the helical bands the individual complexes within the band would need to form a slight bend and twist with adjacent complexes as they link together. Such linkages would lead to the band and lipid bilayer extending out from the plane of the membrane to form a tube of a diameter equal to or smaller than the radial diameter of the helix. This model is supported by two examples of membrane tubulation found in Paramecium, in which proton pumps are the transmembrane complexes that are associated together to form the helically coiled bands. These include the helically linked FF complexes of the mitochondrial inner membrane and the VV complexes of the decorated tubules of the contractile vacuole complex. Such tubules result in very high surface-to-volume ratios and may be important in efficient ATP production or in forming proton gradients for transporting ions across membranes. [ABSTRACT FROM AUTHOR]

Review of the last decade has generally confirmed the presence of nine major kinetid patterns of ciliates. These patterns form the basis of several recent classification schemes that are not able, however, to recognize several variant patterns: Phacodinium, Transitella, Plagiopyla, Lechriopyla, Mesodinium, and Schizocaryum are examples of taxa whose position remains unclear even with ultrastructural evidence. The development of cell structures has provided some striking insights into the phylogenetic relationship of some taxa, notably the stomatogenesis of Coleps and kinetid replication of Eudiplodinium. Systematic implications of these data are also discussed. [ABSTRACT FROM AUTHOR]

BSTRACT [ABSTRACT FROM AUTHOR]

The major components of the internal flagellar apparatus of Chilomonas paramecium Ehr. are two large microtubular roots and a striated root paralleled by three microtubules. The two microtubular roots follows a curved path in the anterior of the cell, and the other extends straight to the posterior passing through a groove in the nucleus. The striated root extends laterally from the basal bodies. Except that it is smaller, the posteriorly directed root bears a strong resemblance to the axostyle of oxymonads. The overall arrangement and structure of the flagellar roots is similar to the pelta, axostyle and costa of trichomonads and the pelta and axostyle of oxymonads, groups of mitochondrion-less, largely parasitic or symbiotic protozoans. An affinity between cryplomonads and oxymonads or trichomonads would have many phylogenetic implications, some of which are discussed. [ABSTRACT FROM AUTHOR]

We had previously shown that an influx of extracellular Ca2+ (Ca2+), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocytotic membrane fusion in Paramecium. We now analyze, by quenched-flow/freeze-fracture, to what extent Ca e ), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocytotic membrane fusion in Paramecium. We now analyze, by quenched-flow/freeze-fracture, to what extent Ca2+ e contributes to exocytotic and exocytosis-coupled endocytotic membrane fusion, as well as to detachment of ``ghosts'' — a process difficult to analyze by any other method or in any other system. Maximal exocytotic membrane fusion (analyzed within 80 msec) occurs readily in the presence of [Ca2+] e ≥ 5 × 10−6 m, while normally a [Ca2+] e = 0.5 m m is in the medium. A new finding is that exocytosis and endocytosis is significantly stimulated by increasing [Ca2+] e even beyond levels usually available to cells. Quenching of [Ca2+] e by EGTA application to levels of resting [Ca2+] i or slightly below does reduce (by ∼50%) but not block AED-triggered exocytosis (again tested with 80 msec AED application). This effect can be overridden either by increasing stimulation time or by readdition of an excess of Ca2+ e . Our data are compatible with the assumption that normally exocytotic membrane fusion will include a step of rapid Ca2+-mobilization from subplasmalemmal pools (``alveolar sacs'') and, as a superimposed step, a Ca2+-influx, since exocytotic membrane fusion can occur at [Ca2+] e even slightly below resting [Ca2+] i . The other important conclusion is that increasing [Ca2+] e facilitates exocytotic and endocytotic membrane fusion, i.e., membrane resealing. In addition, we show for the first time that increasing [Ca2+] e also drives detachment of ``ghosts'' — a novel aspect not analyzed so far in any other system. According to our pilot calculations, a flush of Ca2+, orders of magnitude larger than stationary values assumed to drive membrane dynamics, from internal and external sources, drives the different steps of the exo-endocytosis cycle. [ABSTRACT FROM AUTHOR]

The concentration of intracellular calcium, [Ca2+]i, in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red. Cooling of a deciliated Paramecium caused a transient increase in [Ca2+]i at the anterior region of the cell. Increase in [Ca2+]i was not observed at any region in Ca(2+)-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K+ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing extracellular Ca2+. The cold-induced inward current was lost upon replacing extracellular Ca2+ with equimolar concentration of Co2+, Mg2+ or Mn2+, but it was not affected significantly by replacing with equimolar concentration of Ba2+ or Sr2+. These results indicate that Paramecium cells have Ca2+ channels that are permeable to Ca2+, Ba2+ and Sr2+ in the anterior soma membrane and the channels are opened by cooling. [ABSTRACT FROM AUTHOR]

Using combined intracellular recordings and behavioral bioassays, it was found that lysozyme has two different effects in Paramecium, depending upon the concentrations used. At low concentrations (0.5 n m to 1.0 μ m) it acts as an effective chemorepellent that causes reliable electrophysiological changes. Lysozyme-induced somatic depolarizations, isolated by blocking K channels with Cs-TEA, showed concentration dependencies that were well correlated with chemorepulsion. Ion dependency experiments showed that these were Ca based depolarizations. Addition of either Na or Mg improves chemorepulsion by providing additional depolarizations. Both the depolarizations and chemorepulsion were blocked by 10 μ m neomycin, suggesting that the depolarization is necessary for this chemosensory transduction event. At higher concentrations (100 μ m), lysozyme is a secretagogue. A transient inward current, recorded in Ca alone solutions with Cs-TEA present, was seen in response to high lysozyme concentrations. The amplitude of this inward current was well correlated with exocytosis. Addition of neomycin (1.0 m m) eliminated both the inward current and exocytosis, suggesting a causal relationship. Neither amiloride or W-7, compounds previously suggested to affect the electrophysiological responses to secretagogues, had any significant effects. The mucopolysaccharide hydrolysis activity of lysozyme was not required for any of these responses. We propose that Paramecium have a high affinity receptor on the body plasma membrane that responds to either lysozyme or a related compound to cause an increase in a novel body Ca conductance. This receptor-operated Ca conductance causes membrane depolarization and chemorepulsion at low concentrations and triggers a sufficient Ca influx at high concentrations to cause exocytosis. [ABSTRACT FROM AUTHOR]

Toward isolating channel proteins from Paramecium, we have explored the possibility of functionally reconstituting ion channels in an artificial system. Proteins from Paramecium cortex reconstituted with soybean azolectin retained several channels whose activities were readily registered under patch clamp. The most commonly encountered activities were three: (i) a 71-pS cation channel that opens at all voltages unless dior trivalent cations were added to close them, (ii) a 40 pS monovalent cation channel, and (iii) a large-conductance channel that prefers anions and exhibits many subconductance states. These channels survived mild detergent treatments without observable functional alterations. The possible origin of these channels from internal membranes, the possible role of 71-pS channel in internal Ca release, and the prospects of their purification are discussed. [ABSTRACT FROM AUTHOR]

The effects of proteolysis on a hyperpolarization- and Ca-dependent K channel from the surface membrane of Paramecium tetraurelia were examined in the inside-out excised patch mode. Treatment with trypsin, pronase or thermolysin removed the Ca-dependence of the channel activation, yielding an increase in channel activity greater than 2.5-fold at all Ca concentrations between 10 and 10 m. Thermolysin addition-ally removed the voltage dependence of channel opening and gave the most activation among the three proteases tested. Proteolysis did not affect the single-channel conductance. In an analogy to the mechanism of activation of many Ca-dependent enzymes it is suggested that this Paramecium channel has a cytoplasmic inhibitory domain which can be removed by proteolysis, and that the physiological activation by Ca is due to a temporary removal of this inhibition. Moreover, these findings indicate structural differences between depolarization-, Ca-dependent K channels (BK channels) and the hyperpolarization-, Ca-dependent K channels in Paramecium. [ABSTRACT FROM AUTHOR]

The Ca-inward current of Paramecium is related to cGMP production by a Ca-dependent guanylate cyclase. Excitation with Ba increases cGMP levels about ninefold to 45 pmol/ mg within 15 sec. Inhibition of cGMP hydrolysis reveals a large rate of synthesis of up to 25 pmol cGMP/mg·sec, or about 1.2 ·10 molecules/cell·sec. Because no other factors than the Ca-inward current were found to affect cGMP formation in Paramecium, we used it as a quantitative measure of Ca channel activity. After a transient stimulation of cGMP formation by 1 mm Ba, an additional increase of Ba to 5 mm did not result in a renewed elevation of cGMP levels. The extent of desensitization towards a second stimulus was graded with the strength of the first stimulus. Termination of the first stimulus after various time intervals and restimulation after 3 min with 1 mm Ba revealed a time-dependent inactivation of the Ca channel, which could be fitted by a single exponential. The inactivated form of the channel was stable for a few minutes at room temperature. The partial desensitization of Paramecium reduced the maximal response, but did not shift the dose-response curve for Ba. Veratridine, which activates the Ca channel, was also used as a first stimulus. It effectively and transiently inactivated the channel resulting in a complete loss of both a behavioral response of Paramecium and cGMP elevation towards a second stimulus. The time course of reactivation of channel excitability was studied at different temperatures. Half times of recovery were 51 and 7.5 min at 12 and 25°C, respectively. Reactivation curves can be described by a single exponential, indicating a first order reaction. The activation energy was 100 kJ/mol. The extremely high rate of cGMP turnover in Paramecium is reminiscent of findings in visual cells. A model for regulation of the voltage-dependent Ca channel of Paramecium is proposed. [ABSTRACT FROM AUTHOR]

The membrane currents of wild type Paramecium tetraurelia and the behavioral mutant teaA were analyzed under voltage clamp. The teaA mutant was shown to have a greatly increased outward current which was blocked completely by the combined use of internally delivered Cs+ and external TEA+. This, along with previous work (Satow, Y., Kung, C., 1976, J. Exp. Biol. 65:51-63) identified this as a K+ current. It was further found to be a calcium-activated K+ current since this increased outward K+ current cannot be elicited when the internal calcium is buffered with injected EGTA. The mutation pwB, which blocks the inward calcium current, also blocks this increased outward K+ current in teaA. This shows that this mutant current is activated by calcium through the normal depolarization-sensitive calcium channel. While tail current decay kinetic analysis showed that the apparent inactivation rates for this calcium-dependent K+ current are the same for mutant and wild type, the teaA current activates extremely rapidly. It is fully activated within 2 msec. This early activation of such a large outward current causes a characteristic reduction in the amplitude of the action potential of the teaA mutant. The teaA mutation had no effect on any of the other electrophysiological parameters examined. The phenotype of the teaA mutant is therefore a general decrease in responsiveness to depolarizing stimuli because of a rapidly activating calcium-dependent K+ current which prematurely repolarizes the action potential. [ABSTRACT FROM AUTHOR]

The membrane of Paramecium generates a Ca-dependent Na current upon depolarization. There is, however, also a Na current upon hyperpolarization in this membrane. The second Na current was analyzed under voltage clamp and found to have properties identical to those of the first. Both currents could be carried by Na and Li ions and not by K, Cs or choline ion. They were eliminated by either EGTA injection into the cell or Ca removal from the bath. Both currents were eliminated by a single-gene mutation, fast-2, that had no effect on Ca currents. These findings strongly suggest that these two currents are through the same Ca-dependent Na conductance. A hyperpolarization-induced Ca current was also identified, which served to activate the second Na current. These observations support a model that the Paramecium membrane has two Ca channels with different voltage dependencies and only one Na channel, which is elicited by a rise of the itternal free Ca concentration. The function of the Ca-dependent Na conductance is discussed. [ABSTRACT FROM AUTHOR]