Search PubMed⌕ Search

Biomedical subjects

P Satir

Publications and source records attributed to P Satir.

At least 55 records · Page 3Linked to original sources
PubMed1988

Ultrastructure and motion analysis of permeabilized Paramecium capable of motility and regulation of motility.

Structural and behavioral features of intact and permeabilized Paramecium tetraurelia have been defined as a basis for study of Ca2+ control of ciliary reversal. Motion analysis of living paramecia shows that all the cells in a population swim forward with gently curving spirals at speeds averaging 369 +/- 19 microns/second. Ciliary reversal occurs in 10% of the cell population per second. Living paramecia, quick-fixed for scanning electron microscopy (SEM), show metachronal waves and an effective stroke obliquely toward the posterior end of the cell. Upon treatment with Triton X-100, swimming ceases and both scanning and transmission electron microscopy reveal cilia that uniformly project perpendicularly from the cell surface. Thin sections of these cells indicate that the ciliary, cell, and outer alveolar membranes are greatly disrupted or entirely missing and that the cytoplasm is also disrupted. These permeabilized paramecia can be reactivated and are capable of motility and regulation of motility. Motion analysis of cells reactivated with Mg2+ and ATP in low Ca2+ buffer (pCa greater than 7) shows that 71% swim forward in straight or curved paths at speeds averaging 221 +/- 20 microns/second. When these cells are quick-fixed for SEM the metachronal wave patterns of living, forward swimming cells reappear. Motion analysis of permeabilized cells reactivated in high Ca2+ buffers (pCa 5.5) shows that 94% swim backward in tight spirals at a velocity averaging 156 +/- 7 microns/second. SEM reveals a metachronal wave pattern with an effective stroke toward the anterior region. Although the permeabilized cells do not reverse spontaneously, the pCa response is preserved and the Ca2+ switch remains intact. The ciliary axonemes are largely exposed to the external environment. Therefore, the behavioral responses of these permeabilized cells depend on interaction of Ca2+ with molecules that remain bound to the axonemes throughout the extraction and reactivation procedures.

Animals↗
PubMed1988

Dynein as a microtubule translocator in ciliary motility: current studies of arm structure and activity pattern.

The dynein arms of ciliary doublet microtubules cause adjacent axonemal doublets to slide apart with fixed polarity. This suggests that there is a unique mechanochemistry to the dynein arm with unidirectional force generation in all active arms and also that not all arms are active at once during a ciliary beat. Negative stain and thin-section images of arms in axonemes treated with beta, gamma methylene adenosine triphosphate (AMP-PCP) show a consistent subunit construction where the globular head of the arm interacts with subfiber B of doublet N+1. This interpretation differs from that provided by freeze etch and STEM interpretations of in situ arm construction and has implications for the mechanochemical cycle of the arm. A computer model of the arms in relation to other axonemal structures has been constructed to test these interpretations. Attachment of the head of the arm subfiber B is directly demonstrable in splayed axonemes in AMP-PCP. About half of the doublets in an axoneme show such attachments, while half do not. This might imply that about half the doublets in an axoneme are active at any given instant and can be identified as such. This information may be useful in probing questions of how active arms differ biochemically from inactive arms and of how microtubule translocators in general become active.

Adenosine Triphosphatases↗
PubMed1988

Immunogold localization of the regulatory subunit of a type II cAMP-dependent protein kinase tightly associated with mammalian sperm flagella.

We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.

Adenosine Triphosphatases↗
PubMed1987

Dynein arm attachment probed with a non-hydrolyzable ATP analog. Structural evidence for patterns of activity.

The dynein arms that power ciliary motility are normally permanently attached by one end exclusively to subfiber A of each axonemal doublet (N) while the other (head) end transiently attaches to the subfiber B of the adjacent doublet (N + 1) to produce sliding of the doublets. In Tetrahymena axonemes, sliding of contiguous groups of doublets is induced by ATP suggesting that, in the absence of exogenous protease, there may be sets of potentially active and potentially inactive or refractory arms in a single axoneme. In the presence of a non-hydrolyzable analog of ATP, beta,gamma-methylene adenosine 5'-triphosphate (AMP-PCP), about half the doublets in an axonemal preparation retain all arms bound to subfiber A, but half the doublets show long regions where some arms are pulled away from subfiber A of doublet N and attached to subfiber B of doublet N + 1 by their head ends. In AMP-PCP-induced splaying, positional information regarding arm state is retained. Analysis reveals that throughout regions where B subfiber attachment is found, small groups of about four subfiber B attached arms alternate with groups of about four arms that remain attached to subfiber A. This unique pattern of attachment suggests that arms function co-operatively in groups of four. Further, the repetition of the pattern is reminiscent of metachronal activity seen at higher levels of biological organization. This suggests that in these regions we have instantaneously preserved groups of arms capable of attaching to and detaching from doublet N + 1 in rapid succession. This appearance could be used to delineate the potentially active sets of arm, primed for mechanochemical activity, within an axoneme.

Adenosine Triphosphatases↗
PubMed1986

Spreading ciliary arrest in a mussel gill epithelium: characterization by quick fixation.

Spreading ciliary arrest, induced by local laser microinjury, in freshwater mussel (e.g., Elliptio) gill lateral (L) cell cilia, has been characterized by quick fixation with osmium tetroxide, which permits the correlation of known features of the response with structural features of the gill epithelium. Quick fixation reliably preserves the state of the epithelium including the activity state of the L cilia at the moment of fixation. From a disrupted region, the stimulus that triggers arrest spreads outward along an undamaged filament preferentially from L cell to L cell for more than 300 microns to either side of the lesion. In physiological salt solutions transverse spread across the filament via heterologous cells is insufficient to elicit L ciliary arrest on the opposite side of the filament. The spread of arrest is dependent upon the structural integrity of the L epithelium, normally terminates at a boundary between adjacent L cells, and does not spread past a focal break. Arrest occurs asynchronously because cilia in different stroke positions respond to the stimulus with different time courses. The cilia stop in a uniform "hands up" position, i.e., pointing frontally. The arrest response is inhibited by reducing the concentration of extracellular Ca2+ (less than 10(-7) M) or by adding extracellular La3+ (1 mM) or K+ (15 mM). Recovery begins at the margin of a segment of arrested L cilia and spreads back toward the lesion at a constant initial velocity of ca. 60 microns/sec. About 300 microns from the lesion the recovery velocity rapidly falls to ca. 5 microns/sec. Recovery of ciliary beat precedes the recovery of metachronal coordination. Neither spread of the stimulus nor recovery require ciliary beat. The data support the hypothesis that the microinjury-induced arrest is initiated by an injury potential that triggers a graded regenerative depolarization that is propagated electrotonically along the epithelium from L cell to L cell, triggering Ca2+ influx into the axoneme and consequent Ca2+-induced L ciliary arrest as it spreads. A temporary non-linear gradient of intracellular Ca2+ concentration is established along the injured L epithelial tract. As individual cells recover, they lower their intracellular Ca2+ concentration from pCa 5 to pCa 7 in about 10 seconds.

Animals↗
PubMed1986

Structures attached to doublet microtubules of cilia: computer modeling of thin-section and negative-stain stereo images.

With a single set of positional coordinates for longitudinal and transverse attachment of the inner and outer rows of dynein arms with respect to the doublet microtubules of Tetrahymena ciliary axonemes, a computer model has been constructed at 4-nm resolution that reconciles negative-stain en face stereo images of arm and spoke positions to traditional images of tannic acid/glutaraldehyde-fixed sections. In this model, inner and outer arms correspond in substructure; both repeat with a 24-nm periodicity without stagger between rows, and a pair of arms is in exact alignment with the first spoke (S1) in each doublet spoke group. The model and the supporting micrographs suggest that each arm cycles in three dimensions and that, during cycling, the inner and outer arms move in opposite directions with respect to the center of subfiber A of the doublet (N). Attachment is off-center with respect to subfiber B of the adjacent doublet (N + 1), causing the sliding doublets to skew with respect to one another.

Adenosine Triphosphatases↗
PubMed1985

Alloaffinity filtration: a general approach to the purification of dynein and dynein-like molecules.

Alloaffinity filtration simply and specifically separates certain axonemal dyneins and dynein arm components from crude mixtures on the basis of their ability to bind and decorate Tetrahymena axonemal microtubules on a filter in the absence of ATP and to detach and pass into the eluate when 0.5 mM ATP is added. The procedure, which may be performed repetitively, is successful in purifying a Tetrahymena dynein that has characteristics of 30 S dynein prepared by conventional methods, while other dyneins originally present in the mixture, e.g., 14 S Tetrahymena dynein, are not found in the ATP eluate. A relatively homogeneous population of dynein oligomers is obtained. Alloaffinity-purified 30 S Tetrahymena dynein consists of heavy-, intermediate-, and light-chain polypeptides that cosediment in a sucrose gradient in fixed molar ratios and that have structural features of in situ Tetrahymena arms. Dyneins from other species will bind to Tetrahymena microtubules and can be purified by this method. Alloaffinity-purified Chlamydomonas dynein is a set of polypeptides including the four heavy chains that characterize the outer arm.

Adenosine Triphosphatases↗
PubMed1985

The antagonistic effects of 5-hydroxytryptamine and methylxanthine on the gill cilia of Mytilus edulis.

The laterofrontal (LF) cirri on isolated gill filaments of Mytilus edulis, prepared in natural seawater, are active and initially beat with an average frequency of about 8 Hz (with a range of 6-14 Hz). However, the lateral (L) cilia on these filaments are arrested in a position at the end of their recovery stroke. Perfusion of the filament with artificial seawater (ASW), with or without 1% ethanol, has little or no biological effect on the activity of the LF cirri, although a transitory decrease in frequency often accompanies the perfusion process. The L cilia remain arrested during perfusion with ASW. The exposure of the gill to low levels of 5-hydroxytryptamine (5HT) (10(-8) less than 5HT less than 10(-7) M) has no effect on the activity of the LF cirri but stimulates the L cilia to beat. Exposure to higher concentrations of 5HT (greater than 10(-7) M) elevates the beat frequency of the L cilia and simultaneously inhibits the activity of the LF cirri, leading to their arrest in a position at the end of the effective stroke. This arrest of the LF cirri occurs as the L cilia attain a 5HT-induced beat frequency between 12 to 14 Hz. The influence of 5HT on the L cilia and the LF cirri can be reversibly mimicked or enhanced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). A concentration of 0.5 mM IBMX mimics low 5HT concentrations (about 10(-7) M) by stimulating the L cilia to beat without affecting the beat frequency of the LF cirri. A combination of 10(-7) M 5HT and 0.5 mM IBMX in ASW mimics high (greater than 10(-6) M) 5HT concentrations by arresting the LF cirri and increasing the beat frequency of the L cilia. Under these conditions, the threshold of the LF cirri arrest response is again found to occur as the L cilia attain a beat frequency of 12-14 Hz. These results suggest that the mechanisms of LF cirri arrest and L cilia activation are mediated by 5HT-induced changes in intracellular cyclic AMP levels.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Dynein arm substructure and the orientation of arm-microtubule attachments.

In the presence of AMP-PCP (beta, gamma-methyleneadenosine 5'-triphosphate), a non-hydrolyzable analog of ATP, negative stain images of increased morphological detail indicate that the dynein arm, attached to ciliary doublet microtubules, is composed of subunits including a cape, an elongated body and a head. The arrangement of these subunits makes it possible to distinguish A from B subfiber binding sites on a single arm and to demonstrate that the head of an extended arm on subfiber A of one ciliary doublet is capable of binding to subfiber B of an adjacent doublet in a specific orientation, which supports a key step in a current model of the mechanochemical cycle by which the arm produces microtubule sliding in the ciliary axoneme.

Adenosine Triphosphatases↗
PubMed1984

Trifluoperazine-induced changes in swimming behavior of paramecium: evidence for two sites of drug action.

Trifluoperazine (TFP), a drug that binds to Ca2+-calmodulin (CaM) complexes, altered swimming behavior not only in living paramecia, but also in reactivated, Triton-extracted "models" of the ciliate. By comparing the responses of living cells and models, we have ascertained that two sites of drug action exist in paramecium cilia. Swimming movements were recorded in darkfield stroboscopic flash photomicrographs; this permitted accurate quantitation of velocities and body-shape parameters. When living paramecia were incubated in a standard buffer containing 10 microM TFP, their speed of forward swimming fell over several minutes and their bodies shortened. Untreated paramecia backed up repeatedly and frequently upon transfer to a solution containing barium ions (the "barium dance"), but cells preincubated in TFP did not "dance." Instead they swam forward slowly for long periods of time without reversing and occasionally then exhibited abnormally prolonged reversals. W7 effects on swimming mimicked low doses of TFP, and the analog W5 did not visibly alter normal swimming patterns. These results suggest that TFP induces a decrease in the intracellular pCa of living paramecia, perhaps by reducing the efficiency of a calmodulin-activated calcium pump in the cell membrane. Paramecia extracted with Triton X-100 and reactivated to swim forward (7 greater than or equal to pCa greater than or equal to 6) were not affected by addition of up to 40 microM TFP to the reactivation medium. We conclude that the main drug effect in living cells is probably not at the axoneme. However, at low pCa, TFP directly affected the ciliary axoneme to shift its behavior to one characteristic of a higher pCa: TFP inhibited backward swimming in models reactivated at pCa less than 6; instead they swam forward or rocked in place. The mechanism of ciliary reversal in paramecium may therefore depend on an axonemal Ca2+-sensor, possibly bound CaM, which is affected by TFP only at low pCa, as has been postulated for other types of cilia.

Animals↗
PubMed1984

The cytoskeleton of the apical border of the lateral cells of freshwater mussel gill: structural integration of microtubule and actin filament-based organelles.

The coexistence of densely packed microtubule- and microfilament-based elements in the apex of ciliated epithelial cells, such as the lateral (L) cells of freshwater mussel gill, suggests that with this system it may be possible to define structural connections and interactions that permit integrated cytoskeletal responses to known physiological stimuli. In this study we examine the structure of the L cell apex in detail. The central elements of the elaborate cytoskeleton of the cortex are the basal bodies whose specialized accessory processes are points of integration and the focus of cortical microtubule and microfilament networks. Each basal body supports a cilium and interacts with adjacent basal bodies and with the cell periphery via a dual set of fibre-containing flat trabeculae, both of which are attached to a special organizing centre, the basal foot cap. The distal trabecula is composed of microtubules and the proximal of microfilaments. Connecting the trabeculae, at vertices in the filamentous grids, are core bundles of microfilaments from apical microvilli. In this way, a zig-zag pattern that characterizes microvillar organization at the cell surface is generated. At the cell periphery, the microfilaments from basal foot caps join a peripheral band of microfilaments that underlies the cell border and is associated with four special sites, one in each corner of the cell. Mussel gill epithelial cells contain a polypeptide that resembles actin in its mobility in sodium dodecyl sulphate/10% (w/v) polyacrylamide gels and its affinity for DNase I. Decoration with heavy meromyosin demonstrates that many microfilaments of the L cell apex contain actin, including the microvillar core and peripheral band microfilaments. Actin-associated proteins are also present in these epithelial cells. The actin filaments of the peripheral band are organized to support contraction of the cell border, which would also affect each element of the cortex. This structural complexity, combined with the limited number of modes of interaction between various elements, suggests that the L cell apical cytoskeleton endows the cell with significant positional and morphogenetic information that could be used to compute organellar and cytoskeletal lengths, spacing and changes upon stimulation.

Actins↗
PubMed1982

The ultrastructure of the larval malpighian tubules of a saline-water mosquito.

The larval Malpighian tubules of the saline-water mosquito Aedes taeniorhynchus were examined using light and electron microscopy. The tubules contain two cell types; primary cells and stellate cells. Primary cells are characterized by their size (70 microns x 70 microns x 10 microns) and an abundance of intracellular membrane-bound crystals. Two types of microvilli are found on the luminal surface of the primary cells: (1) small microvilli containing core microfilaments and extensions of endoplasmic reticulum, and (2) larger microvilli (approximately equal to 3 microns in length) which in addition to the above components contain a mitochondrion along their entire length. Both microvillar types have abundant knobs lining the cytoplasmic surface of the microvillar membrane. These knobs, which are often found in insect ion transporting tissues, have been termed 'portasomes' by Harvey (1980). The possible role of these structures in ion transport and mitochondrial positioning is discussed. The stellate cells are much smaller than the primary cells, and lack intracellular crystals. Their microvilli are smaller as well (approximately equal to 0 x 6 microns in length) and contain no endoplasmic reticulum, mitochondria or knobs. The cells types found in the saline-water mosquito larva, Aedes taeniorhynchus, are identical to those found in Aedes aegypti, indicating that the unique capacity of saline-water mosquito larvae to transport Mg2+ and SO42 is not associated with the presence of an additional cell type.

Aedes↗