Recovery of sliding ability in arm-depleted flagellar axonemes after recombination with extracted dynein I

1981 ◽  
Vol 48 (1) ◽  
pp. 223-239
Author(s):  
Y. Yano ◽  
T. Miki-Noumura
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Extraction Process ◽  
Sliding Velocity ◽  
Basal Segment ◽  
Edta Solution ◽  
Close Relationship

We compared sliding velocity between outer doublet tubules in demembranated axonemes of sea-urchin (Pseudocentrotus depressus) sperm flagella with that of arm-depleted axonemes recombined with extracted dynein I. The outer arm-depleted axonemes after extraction with 0.5 M NaCl had a velocity of 6.9 +/− I.0 micrometer/s, while the intact axonemes had a velocity of 14.3 +/− I.5 micrometer/s in the presence of I mM ATP and 2 microgram/ml trypsin at 25 degrees C. The sliding velocity was closely related to the number of remaining outer arms following the NaCl-extraction process. When the outer arm-depleted axonemes were recombined with dynein I, the sliding velocity increased to 11.3 +/− 1.3 micrometer/s. Electron microscopy confirmed the recovery of 94% of outer arms in the axonemes. After extraction with Tris-EDTA solution for 10 min, the axonemes lost their sliding ability completely, even in the presence of ATP and trypsin. Such axonemes lacked most of both inner and outer arms, although sometimes the basal segment of the arms appeared to remain. When the exogenous dynein I fraction extracted from other axonemes was added, the axonemes could extrude tubules, and both types of arms reappeared clearly and distinctly in the axonemes. The recombined axonemes with one-fold stoichiometric excess of dynein I recovered 58% of the total number of arms and had a velocity of 7.4 +/− 1.6 micrometer/s. Those with 2-fold stoichiometric excess had a velocity of 11.0 +/− 1.5 micrometer/s, up to 82% of the arms in these axonemes being restored. These results indicated that the exogenous dynein I fraction derived from the outer arms restored sliding ability to arm-depleted axonemes, recombining with th outer doublet tubules as inner and outer arms, and that the sliding velocity had a close relationship to the total number of arms in the axonemes, irrespective of their being inner or outer arms.

Sliding velocity between outer doublet microtubules of sea-urchin sperm axonemes

1980 ◽  
Vol 44 (1) ◽  
pp. 169-186
Author(s):  
Y. Yano ◽  
T. Miki-Noumura
Keyword(s):  
Polyethylene Glycol ◽  
Atpase Activity ◽  
Sea Urchin ◽  
High Efficiency ◽  
Dark Field ◽  
Video Tape ◽  
Sliding Velocity ◽  
Atp Concentration ◽  
Close Relationship ◽  
Hemicentrotus Pulcherrimus

Using a dark-field microscope equipped with a high-efficiency TV camera including a video tape-recorder, we recorded the sliding movement between outer doublet microtubules of the demembranated axonemes of sea-urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella by adding ATP and trypsin at 25 degrees C. The time and length of the sliding doublet microtubules from axonemes were measured directly from the image on the picture monitor from the video tape. The sliding velocity was almost constant in the range from 0 to 2% polyethylene glycol concentration in the reactivation medium and decreased a little at more than 2%. We prepared various lengths of axoneme fragments by homogenizing whole axonemes and found that the shorter fragments showed similar sliding velocity to that of longer ones at less than 200 microM ATP, but slightly decreased speed at more than 500 microM. ATP. The sliding movement sometimes stopped and the percentage of sliding axonemes was lower below 2 micrograms/ml trypsin. Above 3 micrograms/ml, the process appeared to be more like disintegration than sliding movement, which may be due to excess digestion by trypsin. Sliding speed was therefore measured in a reactivation medium containing 2% polyethylene glycol with the addition of ATP and 2 micrograms/ml trypsin. The velocity increased in proportion to the increase in ATP concentration. Vmax was approximately 14 micrograms/s at 2 mM ATP. In order to compare the Km for the sliding velocity with that of the ATPase activity of the axonemes, we measured ATPase activity of axonemes prepared and assayed under conditions in which sliding movement in the axonemes could be induced. Neither the curve of ATPase activity nor the curve of sliding velocity plotted against ATP concentration obeyed Michaelis-Menten kinetics. The close relationship between ATPase activity and sliding velocity suggested that ‘sliding-movement-coupled ATPase activity’ may well be reflected in the axoneme ATPase reported here.

Freeze-Etch Crystal-Like Structures in Membranous Glomerulosclerosis

1969 ◽  
Vol 27 ◽  
pp. 394-395
Author(s):  
Burton B. Silver
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Sodium Hypochlorite ◽  
Kidney Tissue ◽  
Extraction Process ◽  
Donor Kidney ◽  
Unknown Substance ◽  
Etched Surface ◽  
Standard Fixation ◽  
Diseased Kidney

Tissue from a non-functional kidney affected with chronic membranous glomerulosclerosis was removed at time of trnasplantation. Recipient kidney tissue and donor kidney tissue were simultaneously fixed for electron microscopy. Primary fixation was in phosphate buffered gluteraldehyde followed by infiltration in 20 and then 40% glycerol. The tissues were frozen in liquid Freon and finally in liquid nitrogen. Fracturing and replication of the etched surface was carried out in a Denton freeze-etch device. The etched surface was coated with platinum followed by carbon. These replicas were cleaned in a 50% solution of sodium hypochlorite and mounted on 400 mesh copper grids. They were examined in an Siemens Elmiskop IA. The pictures suggested that the diseased kidney had heavy deposits of an unknown substance which might account for its inoperative state at the time of surgery. Such deposits were not as apparent in light microscopy or in the standard fixation methods used for EM. This might have been due to some extraction process which removed such granular material in the dehydration steps.

scholarly journals Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy.

1975 ◽  
Vol 66 (1) ◽  
pp. 198-200 ◽  
Author(s):  
D Mazia ◽  
G Schatten ◽  
W Sale
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Mammalian Cells ◽  
Experimental Treatment ◽  
The Body ◽  
Transmission Electron ◽  
Coated Plate ◽  
Living Material ◽  
Coated Surfaces ◽  
Triton X

Cells of many kinds adhere firmly to glass or plastic surfaces which have been pretreated with polylysine. The attachment takes place as soon as the cells make contact with the surfaces, and the flattening of the cells against the surfaces is quite rapid. Cells which do not normally adhere to solid surfaces, such as sea urchin eggs, attach as well as cells which normally do so, such as amebas or mammalian cells in culture. The adhesion is interpreted simply as the interaction between the polyanionic cell surfaces and the polycationic layer of adsorbed polylysine. The attachment of cells to the polylysine-treated surfaces can be exploited for a variety of experimental manipulations. In the preparation of samples for scanning or transmission electron microscopy, the living material may first be attached to a polylysine-coated plate or grid, subjected to some experimental treatment (fertilization of an egg, for example), then transferred rapidly to fixative and further passed through processing for observation; each step involves only the transfer of the plate or grid from one container to the next. The cells are not detached. The adhesion of the cell may be so firm that the body of the cell may be sheared away, leaving attached a patch of cell surface, face up, for observation of its inner aspect. For example, one may observe secretory vesicles on the inner face of the surface (3) or may study the association of filaments with the inner surface (Fig. 1). Subcellular structures may attach to the polylysine-coated surfaces. So far, we have found this to be the case for nuclei isolated from sea urchin embryos and for the microtubules of flagella, which are well displayed after the membrane has been disrupted by Triton X-100 (Fig. 2).

Cytasters induced within unfertilized sea-urchin eggs

1983 ◽  
Vol 61 (1) ◽  
pp. 175-189
Author(s):  
R. Kuriyama ◽  
G.G. Borisy
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Sea Water ◽  
Light And Electron Microscopy ◽  
Sea Urchin Eggs ◽  
Microtubule Organizing Centres ◽  
Treatment Step

Conditions that induce the formation of asters in unfertilized sea-urchin eggs have been investigated. Monasters were formed by treatment of eggs with acidic or basic sea-water, or procaine- or thymol-containing sea-water. A second treatment step, incubation with D2O-containing, ethanol-containing or hypertonic sea-water induced multiple cytasters. The number and size of cytasters varied according to the concentration of agents and duration of the first and second treatments, and also upon the species of eggs and the season in which the eggs were obtained. Generally, a longer second treatment or a higher concentration of the second medium resulted in a higher number of cytasters per egg. Asters were isolated and then examined by light and electron microscopy. Isolated monasters apparently lacked centrioles, whereas cytasters obtained from eggs undergoing the two-step treatment contained one or more centrioles. Up to eight centrioles were seen in a single aster; the centrioles appeared to have been produced during the second incubation. Centrospheres prepared from isolated asters retained the capacity to nucleate the formation of microtubules in vitro as assayed by light and electron microscopy. Many microtubules radiated from the centre of isolated asters, whether they contained centrioles or not. This observation is consistent with many other reports that microtubule-organizing centres need not contain centrioles.

Effect of imposed head vibration on the stability and waveform of flagellar beating in sea urchin spermatozoa

1991 ◽  
Vol 156 (1) ◽  
pp. 63-80 ◽  
Author(s):  
C. Shingyoji ◽  
I. R. Gibbons ◽  
A. Murakami ◽  
K. Takahashi
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Distal Region ◽  
Proximal Region ◽  
Bend Angle ◽  
Vibration Frequencies ◽  
Sliding Velocity ◽  
Additional Energy ◽  
The Stability ◽  
Cyclic Changes

The heads of live spermatozoa of the sea urchin Hemicentrotus pulcherrimus were held by suction in the tip of a micropipette mounted on a piezoelectric device and vibrated either laterally or axially with respect to the head axis. Within certain ranges of frequency and amplitude, lateral vibration of the pipette brought about a stable rhythmic beating of the flagella in the plane of vibration, with the beat frequency synchronized to the frequency of vibration [Gibbons et al. (1987), Nature 325, 351–352]. The sperm flagella, with an average natural beat frequency of 48 Hz, showed stable beating synchronized to the pipette vibration over a range of 35–90 Hz when the amplitude of vibration was about 20 microns or greater. Vibration frequencies below this range caused instability of the beat plane, often associated with irregularities in beat frequency. Frequencies above about 90 Hz caused irregular asymmetrical flagellar beating with a marked decrease in amplitude of the propagated bends and a skewing of the flagellar axis towards one side; the flagella often stopped in a cane shape. In flagella that were beating stably under imposed vibration, the wavelength was reduced at higher frequencies and increased at lower frequencies. When the beat frequency was equal to or lower than the natural beat frequency, the apparent time-averaged sliding velocity of axonemal microtubules, obtained as twice the product of frequency and bend angle, decreased with beat frequency in both the proximal and distal regions of the flagella. However, at vibration frequencies above the natural beat frequency, the sliding velocity increased with frequency only in the proximal region of the flagellum and remained essentially unchanged in more distal regions. This apparent limit to the velocity of sliding in the distal region may represent an inherent limit in the intrinsic velocity of active sliding, while the faster sliding observed in the proximal region may be a result of passive sliding or elastic distortion of the microtubules induced by the additional energy supplied by the vibrating pipette. Axial vibration with frequencies either close to or twice the natural beat frequency induced cyclic changes in the waveform, compressing and expanding the bends in the proximal region, but did not affect bends in the distal region or alter the beat frequency.

Study on Nano- and Microcrystallites in Urines of Uric Acid Stone Patients

2013 ◽  
Vol 655-657 ◽  
pp. 1927-1930 ◽  
Author(s):  
Guang Na Zhang ◽  
Zhi Yue Xia ◽  
Jian Ming Ouyang ◽  
Li Kuan
Keyword(s):  
Electron Microscopy ◽  
Uric Acid ◽  
X Ray Diffraction ◽  
Uric Acid Stone ◽  
Urine Ph ◽  
Stone Formers ◽  
Transmission Electron ◽  
Close Relationship ◽  
Ft Ir ◽  
Ft Ir Spectroscopy

The presence of crystallites in urine is closely related to stones formation. In this article, the components, morphology of nano- and micro-crystallites in urines of 20 uric acid (UA) stone formers as well as their relationship with the formation of UAstones were comparatively studied using X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The main constituent of urinary crystallites was uric acid. Their particle size distribution was highly uneven, ranging from several nanometers to several tens of micrometers, and obvious aggregation was observed. These results showed that there was close relationship among stone components, urinary crystallites composition and urine pH.

scholarly journals Membranous Nephropathy Associated with Atheroembolism

2017 ◽  
Vol 10 (1) ◽  
pp. 29-33
Author(s):  
Kiyotaka Uchiyama ◽  
Tamiko Takemura ◽  
Yoshitaka Ishibashi
Keyword(s):  
Electron Microscopy ◽  
Renal Function ◽  
Catheter Ablation ◽  
Disease Activity ◽  
Membranous Nephropathy ◽  
Hypereosinophilic Syndrome ◽  
Cholesterol Crystal ◽  
Chronic Infections ◽  
Unusual Manifestation ◽  
Close Relationship

Membranous nephropathy (MN) is one of the most common biopsy diagnoses in adults, and it has been associated with chronic infections, autoimmune diseases, malignancies, and drugs. However, MN associated with cholesterol crystal emboli has never been reported. Here we present a patient with MN as an unusual manifestation of atheroembolism. A 75-year-old man with worsening renal function after catheter ablation developed moderate proteinuria and underwent a renal biopsy. Findings on light, immunofluorescence, and electron microscopy were all compatible with membranous nephropathy. Moreover, one occluded interlobular artery contained a pathognomonic, biconvex, needle-shaped cleft, which indicated a cholesterol crystal emboli. The degree of proteinuria was in parallel with the number of eosinophils, which indicated a close relationship between MN disease activity and renal atheroembolism. Hypereosinophilic syndrome secondary to atheroembolism may cause MN; thus, corticosteroid therapy was likely to be effective.

scholarly journals Effect of beat frequency on the velocity of microtubule sliding in reactivated sea urchin sperm flagella under imposed head vibration.

1995 ◽  
Vol 198 (3) ◽  
pp. 645-653 ◽  
Author(s):  
C Shingyoji ◽  
K Yoshimura ◽  
D Eshel ◽  
K Takahashi ◽  
I R Gibbons
Keyword(s):  
Sea Urchin ◽  
Vibration Frequency ◽  
Beat Frequency ◽  
Distal Region ◽  
Bend Angle ◽  
Vibration Frequencies ◽  
Sliding Velocity ◽  
Flagellar Beat ◽  
Tripneustes Gratilla ◽  
Sea Urchin Sperm

The heads of demembranated spermatozoa of the sea urchin Tripneustes gratilla, reactivated at different concentrations of ATP, were held by suction in the tip of a micropipette and vibrated laterally with respect to the head axis. This imposed vibration resulted in a stable rhythmic beating of the reactivated flagella that was synchronized to the frequency of the micropipette. The reactivated flagella, which in the absence of imposed vibration had an average beat frequency of 39 Hz at 2 mmol l-1 ATP, showed stable beating synchronized to the pipette vibration over a range of 20-70 Hz. Vibration frequencies above 70 Hz caused irregular, asymmetrical beating, while those below 20 Hz induced instability of the beat plane. At ATP concentrations of 10-100 mumol l-1, the range of vibration frequency capable of maintaining stable beating was diminished; an increase in ATP concentration above 2 mmol l-1 had no effect on the range of stable beating. In flagella reactivated at ATP concentrations above 100 mumol l-1, the apparent time-averaged sliding velocity of axonemal microtubules decreased when the imposed frequency was below the undriven flagellar beat frequency, but at higher imposed frequencies it remained constant, with the higher frequency being accompanied by a decrease in bend angle. This maximal sliding velocity at 2 mmol l-1 ATP was close to the sliding velocity in the distal region of live spermatozoa, possibly indicating that it represents an inherent limit in the velocity of active sliding.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs.

1979 ◽  
Vol 82 (1) ◽  
pp. 212-226 ◽  
Author(s):  
A Spudich ◽  
J A Spudich
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Actin Filaments ◽  
Cell Types ◽  
Optical Diffraction ◽  
Muscle Actin ◽  
Sea Urchin Eggs ◽  
Inner Surface ◽  
Low Ionic Strength ◽  
Helical Parameters

Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.

Optimization of dry sliding wear parameters of recursive friction stir processed aluminium 7075 alloy

2020 ◽  
pp. 135065012094161
Author(s):  
G Girish ◽  
V Anandakrishnan
Keyword(s):  
Electron Microscopy ◽  
Wear Rate ◽  
Sliding Wear ◽  
Wear Testing ◽  
Wear Behaviour ◽  
Dry Sliding Wear ◽  
Dry Sliding ◽  
Sliding Velocity ◽  
Friction Stir ◽  
Sliding Distance

In this work, the dry sliding wear behaviour of recursively friction stir processed AA7075 was investigated using a pin-on-disc wear testing apparatus. The microstructure of the processed specimen was probed using optical microscopy, transmission electron microscopy and atomic force microscopy. Experiments were conducted using Taguchi experimental design by varying three different parameters like load, sliding velocity and sliding distance, and the analysis of variance was performed to identify the influence of the parameters over the wear rate. From the main effect plot, the combination of 9.81 N of load, 2 m/s of sliding velocity and a sliding distance of 2000 m was identified as the optimum levels that minimize the wear rate. The regression model was developed to calculate the wear rate, and the validation test was performed with the optimum parameter combination and compared with the experimental results. Wear tracks were examined using field-emission scanning electron microscopy to identify the type of wear mechanism.

Sign in / Sign up

Export Citation Format

Share Document