Ultrastructural study of human glomerular capillary loops with IgA nephropathy using quick-freezing and deep-etching method

Immunoglobulin A (IgA) nephropathy shows great variability regarding the histological features of the lesions of human renal glomeruli. In the present study, the quick-freezing and deep-etching (QF-DE) method was used to analyze the glomerular ultrastructure of biopsied kidney tissues from children with IgA nephropathy. Biopsied renal tissues were routinely prepared for light microscopy, immunofluorescence microscopy, conventional electron microscopy, and replica electron microscopy. The three-dimensional ultrastructure of glomeruli of the kidney was clearly observed by using the QF-DE method. Three layers of glomerular basement membranes, i.e., middle, inner and outer layers, were clearly detected in the replica electron micrographs. The middle layer was 343.0+/-24.2 nm (n=20) in width and formed polygonal meshwork structures. We also observed slit diaphragms, electron-dense mesangial deposits, and increased amounts of mesangial matrix and foot process effacement. Many delicate filaments were found to be distributed from the apical to the bottom portions between neighboring foot processes. The ultrastructural difference between the replica electron micrographs and conventional electron micrographs was found to be especially marked in the appearance of foot processes and connecting filaments between the neighboring foot processes. The examination of extracellular matrix changes, as revealed at high resolution by the QF-DE method, gave us some morphofunctional information relevant to the mechanism of proteinuria with IgA nephropathy.     

The Determination of Axis Lengths of Dunaliella from the Image Analysis of TEM Sections through Randomly Orientated Cells

We present a method which improves the determination of geometric parameters of unicellular organisms with prolate ellipsoid geometry, such as Dunaliella. Approximately determined axis lenghts were obtained from transmission electron micrographs of ultrathin sections of microalgae. The density functions generated by these ‘axis lengths’ have poles which can be used to determine the short half axis length and ratio of axis lengths of the ellipsoid in order to determine the true cell volume.     

THE USE OF SHADOW-CASTING TECHNIQUE FOR MEASUREMENT OF THE WIDTH OF ELONGATED PARTICLES

A method is described for the estimation of the true width of fibrillar or rod-like structures from electron micrographs of metal-shadowed preparations. The method is based on variations in the image width as a function of the angle (β) between the long axis of the fibril and the direction of the shadow in the plane of the preparation. The image width when β = 0° practically represents the real width of the elongated particle but is often indistinguishable from the background. The fibril image width is conveniently measured at β values between 15° and 90°. The true width is obtained by plotting the image width versus sin β and extrapolating to β = 0°. Latex spheres are sprayed with the fibrils or rods to indicate the direction of shadow. Tobacco mosaic virus (TMV) was used as a model structure because of its known constant diameter of 150 A (5). The width (in the case of TMV equal to the diameter) found by the present method was 150 A ± 8 A.     

An electron microscope study of kidney basement membrane changes in the mouse by lipoteichoic acid from Streptococcus pyogenes

Mice injected repeatedly, intraperitoneally or intravenously, for approximately 1 month with a total of 1.04 mg lipoteichoic acid from a nephritogenic strain of Streptococcus pyogenes lost weight. Analysis by electron microscopy revealed that they also exhibited extensive kidney changes in basement membrane morphology which resembled, in part, those observed in human poststreptococcal glomerulonephritis. For example, the glomerular basement membrane became electron dense and exhibited at least a twofold increase in width sporadically within the same preparation after exposure to lipoteichoic acid. Also, whereas appreciable loss or reduction in epithelial foot processes as a result of fusion was clearly evident, epithelial slits and slit membranes or diaphragms between normal foot processes were not selectively affected. In addition, another mostly thickened, highly coiled or serpentinelike basement membrane with amorphous nodules appeared in these preparations. This type membrane was not observed surrounding the capillary lumina and was the most pronounced abnormality apparent in almost all preparations from mice exposed to lipoteichoic acid. Likewise, the proximal tubular basement membrane became variable in width and increased in electron density in mice given lipoteichoic acid as compared with controls. In addition, this membrane was often punctuated with various morphological protrusions originating from only its thickened areas and which extended away from, and not into, the capillary space. This change was only associated with the basement membrane of the proximal tubular capillaries. All membrane changes persisted but gradually subsided, with normal kidney membrane morphology reappearing on the 4th day following the last injection of lipoteichoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oleosomes (Spherosomes) from Daucus carota suspension culture cells

Isolated oleosomes from Daucus carota L. cells are lipid droplets consisting mainly of triacylglycerols (>97%) and very little protein (1–2%). The boundary between the lipid phase and the cytosol, which is visible on electron micrographs, is not built up by a true phospholipid-containing unit or half unit membrane. Enzymatic activities of lipid metabolism were not found to be associated with oleosomes with the exception of very low (contaminating) acyl-CoA:1,2-diacylglycerol acyltransferase (EC 2.3.1.20) and relatively high acyl-CoA hydrolase (EC 3.1.2.2) activities. The triacylglycerols exhibited a half life time of about 70 h, which is below the generation time of the cells (80–90 h). The fatty acid pattern of triacylglycerols was very similar to that of polar cellular membrane lipids.     

Toughness and Collagen Content of Abalone Muscles

Toughness and collagen content were measured for various muscle parts of the Japanese abalone, kuro-awabi (Haliotis discus), in relation to muscle structures. The dorsal surface of the foot was toughest, followed by the hard and soft part of the foot, then the upper and middle part of the adductor muscle, irrespective of being reared or wild specimens. When compared with other abalone species, kuro-awabi showed the highest toughness for all the muscle parts, followed by madaka (H. sieboldii) and megai-awabi (H. gigas), while ezo-awabi (H. discus hannai) was softest. Collagen content was parallel with muscle toughness: the higher the collagen content, the tougher the muscle. Light micrographs of kuro-awabi showed that foot and the dorsal surface of foot were dominated by connective tissues, while adductor muscle was mainly composed of myofibrils. Transmission electron micrographs demonstrated that myofibrils in the foot were surrounded by thick layers of collagen fibrils of about 1 μm, confirming light microscopic observations.     

On the precision and accuracy achieved by Escherichia coli cells at fission about their middle

Length and width of each of the prospective siblings of constricted Escherichia coli cells from different strains and culture conditions were measured from electron micrographs. The data were statistically analyzed to investigate how equally the length and volume of one cell was divided into two. The analysis showed that, for all cultures, bipartition is unbiased or very nearly so, i.e. sibling cells were on the average equally long and large. The precision of bipartition attained by the cells was usually high; it was related to the average cell shape (length/width): slender E. coli cells divided into two less precisely than squat cells. Absolute size, growth rate and strain specificity affected the precision of bipartition only indirectly, i.e. in as much as they influenced cell shape.     

The effects of orchidectomy and replacement therapy on the ultrastructure and gonadotropin-releasing hormone content of the median eminence of the rat

The effects of 2 weeks of orchidectomy and replacement therapy with testosterone upon the content and distribution of gonadotropin-releasing hormone (GnRH) in the median eminence were determined by means of radioimmunoassay and electron microscopy. Photographic montages were prepared from electron micrographs of the lateral median eminence at the point of deepest invagination of the tuberoinfundibular sulcus. Morphometric analysis of photographs of tissues immunohistochemically stained for GnRH was performed to determine changes in the volume density of GnRH-containing axon profiles following the experimental treatments. A decrease in GnRH content after orchidectomy was observed both by morphometric analysis of axon volume density and radioimmunoassay of total GnRH content. Testosterone treatment of orchidectomized animals prevented the postorchidectomy loss of GnRH. Morphometric analysis of conventional electron micrographs revealed an increase in the number of axons containing no dense-core vesicles following orchidectomy, but no decrease in volume density of the neuropil. The results indicate that the change in volume density of immunostained axons was related to the loss of immunostainable dense-core vesicles and not to a change in the size or number of axons. The area corresponding to the location of the highest concentration of GnRH-containing axons was observed to be largely avascular and separated from the vessels of the tuberoinfundibular sulcus by a "border zone" composed of glial foot processes. The unique morphology of the GnRH area has suggested the name "compact zone" to distinguish it from the palisade zone with which it is continuous medially. GnRH axons in this region are probably part of a tract extending farther caudally rather than a terminal field.     

A combined morphometric analysis of foot form and its association with sex,stature, and body mass

Objectives: Morphometric analysis of footprints is a classic means for orthopedic diagnosis. In forensics and physical anthropology, it is commonly used for the estimation of stature and body mass. We studied individual variation and sexual dimorphism of foot dimensions and footprint shape by a combination of classic foot measurements and geometric morphometric methods. Methods: Left and right feet of 134 healthy adult males and females were scanned twice with a 3D optical laser scanner, and stature as well as body mass were recorded. Foot length and width were measured on the 3D scans. The 2D footprints were extracted as the plantar‐most 2 mm of the 3D scans and measured with 85 landmarks and semilandmarks. Results: Both foot size and footprint shape are sexually dimorphic and relate to stature and body mass. While dimorphism in foot length largely results from dimorphism in stature, dimorphism in footprint shape partly owes to the dimorphism in BMI. Stature could be estimated well based on foot length (R² = 0.76), whereas body mass was more closely related to foot width (R² = 0.62). Sex could be estimated correctly for 95% of the individuals based on a combination of foot width and length. Discussion: Geometric morphometrics proved to be an effective tool for the detailed analysis of footprint shape. However, for the estimation of stature, body mass, and sex, shape variables did not considerably improve estimates based on foot length and width. Am J Phys Anthropol 157:582–591, 2015. © 2015 Wiley Periodicals, Inc.     

The reconstruction of structure from electron micrographs of randomly oriented particles

A new method for enhancing and reconstructing the three dimensional structure of randomly oriented particles from their electron micrographs is developed. The method requires as an input many pictures of randomly oriented identical particles. The analysis is based on the calculation and accumulation of the spatial correlation of the densities on the electron micrographs, from which the spherical harmonic coefficients of the structure can be found. The process of enhancement of the spatial correlation and the averaging out of background noise enables reconstructions by use of pictures with low signal-to-noise ratio. The theory is presented and implemented in a computer program package. Simulated electron micrographs of ellipses, rods and a model of hexameric glutamate dehydrogenase are analyzed to demonstrate reconstructions using the computer programs.     

Inhibition of Plastocyanin to P700+ Electron Transfer in Chlamydomonas reinhardtii by Hyperosmotic Stress

Oxygen electrode and fluorescence studies demonstrate that linear electron transport in the freshwater alga Chlamydomonas reinhardtii can be completely abolished by abrupt hyperosmotic shock. We show that the most likely primary site of inhibition of electron transfer by hyperosmotic shock is a blockage of electron transfer between plastocyanin (PC) or cytochrome c(6) and P(700). The effects on this reaction were reversible upon dilution of the osmolytes and the stability of plastocyanin or photosystem (PS) I was unaffected. Electron micrographs of osmotically shocked cells showed a significant decrease in the thylakoid lumen volume. Comparison of estimated lumenal width with the x-ray structures of plastocyanin and PS I suggest that lumenal space contracts during HOS so as to hinder the movement of docking to PS I of plastocyanin or cytochrome c(6).     

Scanning electron microscopical observations on the differentiating mesonephros of the chick embryo

Chick embryos were staged according to the method of Hamburger and Hamilton [1951] and fixed. Cross sections through the cephalic fourth of the mesonephric ridges were examined by scanning electron microscopy. The steps in glomerular differentiation could be observed with ease. The first foot processes to appear in podocytes arose directly from the basal surface of the cell body. In a second step, lateral branches appeared and gave off secondary or even tertiary branches that interdigitated with those from neighbouring podocytes, following a pattern that was very similar to the one previously described by other authors in metanephric nephrons. Endothelial pores appeared in the glomerular capillaries at very early stages of the glomerular differentiation. The differentiation of the epithelium of proximal tubules was characterized by the growth of apical microvilli and of finger-like evaginations from the lateral membranes. At stages 20 and 21, the most differentiated glomeruli had only basal foot processes; only after stage 25 did the first generation nephrons reach full maturity. Because during this period the mesonephros is known to produce urine, our results indicate that nephrons start to function before they have completed their differentiation.     

Contraction due to bimetallic, short-lived super-coiling in the helical, double stranded filaments of striated muscle.

The possibility of a super-coiling of the thin filament is studied. The bimetallic super-coiling might contribute to the power-stroke. The calculation of the axial shortening of the proposed super-coiling leads to a very surprising geometric fitting: the maximal axial shortening of the super-coiling, without any loop, of the thin filament segment (38.5 nm) is equal to the axial repeat of cross-bridges (14 nm). According to the present model, the sarcomere shortening is caused by the axial shortening of the super-coiled actin filament. The top-view of the super-coiled segment shows a dual half-circle form. There are experimental evidences for such a type of formation in electron micrographs. According to this model, the widely accepted sterically hidrance model on association of actin and myosin can be neglected. One of the main roles of calcium is to make the thin filament segment flexible enough for the association.     

Structural organization of collagen in Metridium senile

The structure and distribution of collagen fibres in Metridium senile mesoglea has been investigated using high and small angle X-ray diffraction techniques on conventional and synchrotron sources. The mesoglea collagen axial spacing appears very close to that of rat tail tendon, which is at variance with the values previously obtained from electron microscopic observations. The different intensity distribution of the small angle X-ray diffraction maxima recorded for mesoglea and rat tail tendon indicates a different distribution of electron density inside the repeating period. Furthermore the absence of the first order, the weak second order and the strong third and sixth orders in the patterns of wet and dry mesogleal collagen could explain that only a periodicity of 20–22 nm corresponding to one-third of the true axial period observed in the electron micrographs. The analysis of the reflections at 0.29 and 1.1–1.4 nm characteristics of the collagen molecular structure have been used to determine the distribution and orientation of the collagen fibres in unstretched and stretched samples     

Observations on the solitary cilium of rabbit oviductal epithelium: its motility and ultrastructure

Solitary cilia have been observed on rabbit oviductal epithelial cells. In tissue cultures of fimbrial epithelium of 3- and 4-day-old animals observed by phase microscopy, most of these single cilia exhibited a vortical or funnel-type movement while others had the usual to-and-fro motility. Primary cilia are usually considered immotile. Transmission electron microscopy of specifically identified single cilia revealed differences between the ciliary shafts and basal bodies of the single cilia as compared to those of mature oviductal ciliated cells. The basal body of the solitary cilium often had at least two triangular, striated, basal foot processes, lacked electron-dense satellite material around its basal end, and occasionally had striated rootlets. In contrast, the cilia of mature ciliated cells had only one basal foot, exhibited much electron-dense satellite material, and lacked rootlets. Cross sections of the single cilia showed patterns of microtubules different from the usual 9 + 2 axonemal complexes of normal cilia and included 9 + 0, 10 + 2 singlets, 7 + 2 doublets, and 8 + 1 doublet and 2 singlets; one did have the usual 9 + 2 arrangement. We postulate that the presence of more than one basal foot process may be responsible for the vortical motility observed. The primary cilia are shorter than normal cilia; the longest one measured was 1.86 micron in length, 0.28 micron in width at its base, and 0.14 micron at its tip. Based on the light-microscopic, scanning-electron-microscopic and transmission-electron-microscopic observations, such solitary cilia were observed more frequently in the oviductal tissues of the 3- to 4-day postnatal rabbits grown in tissue culture and in ovariectomized and ovariectomized/progesterone-treated adult animals than in estrous, ovulatory, or ovariectomized/estradiol-treated rabbits.     

Role of the polarity protein Scribble for podocyte differentiation and maintenance

The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their complex foot process network. While deficiency of the polarity proteins Crumbs and aPKC result in impaired podocyte foot process architecture, the function of basolateral polarity proteins for podocyte differentiation and maintenance remained unclear. Here we report, that Scribble is expressed in developing podocytes, where it translocates from the lateral aspects of immature podocytes to the basal cell membrane and foot processes of mature podocytes. Immunogold electron microscopy reveals membrane associated localisation of Scribble predominantly at the basolateral site of foot processes. To further study the role of Scribble for podocyte differentiation Scribble(flox/flox) mice were generated by introducing loxP-sites into the Scribble introns 1 and 8 and these mice were crossed to NPHS2.Cre mice and Cre deleter mice. Podocyte-specific Scribble knockout mice develop normally and display no histological, ultrastructural or clinical abnormalities up to 12 months of age. In addition, no increased susceptibility to glomerular stress could be detected in these mice. In contrast, constitutive Scribble knockout animals die during embryonic development indicating the fundamental importance of Scribble for embryogenesis. Like in podocyte-specific Scribble knockout mice, the development of podocyte foot processes and the slit diaphragm was unaffected in kidney cultures from constitutive Scribble knockout animals. In summary these results indicate that basolateral polarity signaling via Scribble is dispensable for podocyte function, highlighting the unique feature of podocyte development with its significant apical membrane expansions being dominated by apical polarity complexes rather than by basolateral polarity signaling.     

Various morphological aspects of Escherichia coli lysis by RNA bacteriophage MS2 observed by transmission and scanning electron microscopes

Escherichia coli cell lysis profiles induced by the cloned lysis gene from RNA bacteriophages MS2 were presented. Transmission electron micrographs showed that ballooning structures appeared on the cell surfaces and the others were leaking materials through the cell wall. Harsh rupture of the host cell was also observed. Scanning electron micrographs revealed many extruded structures from the cells. For the scanning electron microscopy, plastic sheets coated with calf dermis collagen and SEMPORE filters were successfully used to collect the samples and for various chemical treatments. The lysing cells by bacteriophage MS2 lysis gene observed in transmission and scanning electron micrographs showed various morphological aspects of the intermediates in the lysing process.     

Idealization of pericellular fluid space geometry and dimension results in a profound underprediction of nano-microscale stresses imparted by fluid drag on osteocytes

To date, no published study has examined quantitatively the effect of geometric and dimensional idealization on prediction of the mechanical signals imparted by fluid drag to cell surfaces. We hypothesize that this idealization affects the magnitude and range of imparted forces predicted to occur at a subcellular level. Hence, we used computational fluid dynamics to predict magnitudes and spatial variation of fluid velocity and pressure, as well as shear stress, on the cell surface in two- and three-dimensional models of actual and idealized pericellular canalicular geometries. Furthermore, variation in actual pericellular space dimensions was analyzed statistically based on high-resolution transmitted electron micrographs (TEM). Accounting for the naturally occurring protrusions of the pericellular space delineating lamina limitans resulted in predictions of localized stress spikes on the cell surface, up to five times those predicted using idealized geometries. Predictions accounting for actual pericellular geometries approached those required to trigger cell activity in in vitro models. Furthermore, statistical analysis of TEM-based dimensions showed significant variation in the width of the canalicular space as well as the diameter of the cell process, both of which decrease with increasing distance from the cell body. For the first time to our knowledge, this study shows the influence of physiologic geometry per se on the nano-scale flow regimes in bone, and the profound influence of physiologic geometry on force magnitudes and variations imparted locally to cells through load-induced fluid flow.     

v-Bodies in mitotic chromatin

Electron micrographs of mitotic chromosomal fibers from Chinese hamster ovary cells resemble very closely published electron micrographs of chromatin from interphase nuclei. The mitotic fibers are composed of particles tandemly aligned along the fiber length, with short connecting strands between the particles. The average diameter of the particles or v-bodies is 89 Å.     

Semi-automated methods for simulation and measurement of amyloid fiber distributions obtained from transmission electron microscopy experiments

Transmission electron microscopy (TEM) is the standard procedure for qualitatively confirming the presence of amyloid fibers in a protein aggregation reaction product. However, extracting quantitative information about the amyloid size distribution from the electron micrographs is a nontrivial problem. Here we describe methods for (i) the simulation of pseudo-TEM images of amyloid fiber distributions having known characteristic properties and (ii) the semi-automated processing of experimental TEM images of amyloid fibers to produce two-dimensional histogram plots reflecting either the distribution of amyloid length and width or, alternatively, the distribution of width and fiber rigidity/persistence. The processing method is fully automatic when the density of fibers on the grid is sufficiently low (such that the adsorbed fibers do not touch) and is semi-automatic (requiring some user decision making) when the fibers are overlapping. Termed "ADM" (for Amyloid Distribution Measurement), the program suite is written in MATLAB code and is available on request from the author.