The role of the pro-apoptotic protein Siva in the pathogenesis of Familial Mediterranean fever: A structural and functional analysis

Familial Mediterranean fever (FMF) is an autosomal, recessive disease, attributed to mutations in MEFV gene encoding pyrin, which is characterized by recurrent, acute and self-limiting attacks of fever as well as an increased neutrophil and monocyte apoptosis. Most disease-associated mutations in MEFV gene reside on the C-terminal PRYSPRY (B30.2) domain of pyrin, an area found to interact with the pro-apoptotic protein Siva. Because apoptotic events may be contributing to endogenous inflammation we hypothesized that mutations in pyrin may affect Siva-mediated apoptosis. The confirmation of this hypothesis would be of a great biological significance since it would be demonstrated a connection between apoptosis and inflammation. We used homology modeling to construct a 3-D model of Siva protein and the constructed model of Siva defined structural elements with potential of binding other proteins to induce apoptosis. Given that Siva protein binds pyrin as shown by transfection and immunoprecipitation experiments, apoptosis was assessed by FACS and Western blotting. No differences in rates of apoptosis in myeloid cells (THP-1) upon transfection with either wt pyrin or mutant forms of pyrin were found. Patients with FMF did not display any mutations in the Siva-1 (full length) gene. Siva-1 was not linked to pyrin in the major predicted FMF gene network constructed using a literature-curated gene signature for FMF. These results suggest that Siva-mediated unprovoked apoptosis is not likely to be involved in the pathogenesis of FMF.     

The Risk of Familial Mediterranean Fever in MEFV Heterozygotes: A Statistical Approach

Background

Familial Mediterranean fever (FMF) is an autosomal recessive autoinflammatory disorder due to MEFV mutations and one of the most frequent Mediterranean genetic diseases. The observation of many heterozygous patients in whom a second mutated allele was excluded led to the proposal that heterozygosity could be causal. However, heterozygosity might be coincidental in many patients due to the very high rate of mutations in Mediterranean populations.

Objective

To better delineate the pathogenicity of heterozygosity in order to improve genetic counselling and disease management.

Methods

Complementary statistical approaches were used: estimation of FMF prevalence at population levels, genotype comparison in siblings from 63 familial forms, and genotype study in 557 patients from four Mediterranean populations.

Results

At the population level, we did not observe any contribution of heterozygosity to disease prevalence. In affected siblings of patients carrying two MEFV mutations, 92% carry two mutated alleles, whereas 4% are heterozygous with typical FMF diagnosis. We demonstrated statistically that patients are more likely to be heterozygous than healthy individuals, as shown by the higher ratio heterozygous carriers/non carriers in patients (p<10⁻⁷–p<0.003). The risk for heterozygotes to develop FMF was estimated between 2.1×10⁻³ and 5.8×10⁻³ and the relative risk, as compared to non carriers, between 6.3 and 8.1.

Conclusions

This is the first statistical demonstration that heterozygosity is not responsible for classical Mendelian FMF per se, but constitutes a susceptibility factor for clinically-similar multifactorial forms of the disease. We also provide a first estimate of the risk for heterozygotes to develop FMF.     

A combined scanning and transmission electron microscopic study and electron probe microanalysis of human pineal acervuli

Summary Untreated, decalcified and trypsinized acervuli from human pineal bodies were studied with the scanning and transmission electron microscope as well as by electron probe microanalysis. The mulberry-like acervuli are composed of a various number of spherical lobes (135–800 m) between which clustered groups of globuli (4–14 urn in diameter) are observed. The acervular lobes are very probably formed by an aggregation of these globuli. Small round particles 125–500 Å in diameter are observed on the surface of the pineal concretions. These are not influenced by either decalcification or trypsin treatment. The acervular mineral corresponds morphologically to hydroxyapatite. The electron probe microanalysis reveals the existence of calcium and phosphorus as main components of the acervuli. Small quantities of magnesium and strontium were also detected.Dedicated to Professor Berta Scharrer on the occasion of her 70th birthdayWith the technical assistance of Mr. P.A. MilliquetThe author wishes to thank Mr. Bauer and Mr. Fryder (Nestec SA, La Tour de Peilz) for the use of the Cambridge Stereoscan electron microscope and Dr. T. Jalanti (C.M.E., Lausanne) for his help with the use of the X-ray microanalyser     

Comparative study of the corneal epithelium in some reptiles inhabiting different environments

El‐Bakry, A.M. 2011. Comparative study of the corneal epithelium in some reptiles inhabiting different environments. —Acta Zoologica (Stockholm) 92 : 54–61. The vertebrate cornea functions in either aquatic or aerial environments and in some cases in both. In terrestrial and aerial vertebrates, the cornea contributes most of the refractive powers of the eye because of the large variation in refractive index between the air and the cornea. The present study aimed to examine and compare the main features of the corneal epithelial surface of three reptilian species related to three different families (Caretta caretta, Varanus griseus and Mabuya quinquetaeniata) and inhabiting different environment, by light, scanning (SEM) and transmission electron microscopy. The mean epithelial cell densities of the species of the study were 8.670 ± 3.134, 5.945 ± 2.144 and 2.124 ± 713 respectively. The corneal epithelium of the three species observed by SEM showed a similarity to one another indicating that the apical cell surfaces possess regular polygonal cells with varieties of microprocesses. These microprocesses were represented by microplicae, numerous microvilli and some long microridges in C. caretta, microplicae and minute microholes in V. griseus and microplicae intermingled with short microvilli in M. quinquetaeniata. According to the densities of these microprocesses, three polymorphic cell types (light, medium and dark) appeared in C. caretta, light and medium cell types were observed in V. griseus and medium and dark cell types were noticed in M. quinquetaeniata. Different types of tight adhesions were observed by transmission electron microscopy between the cell borders of the epithelial cells which differ according to environment where the species occupy. In conclusion, variation in the structure of the corneal epithelial cells appears to be related to the living environment, such as aerial, terrestrial and aquatic ones, which is occupied by every species.     

Ultrastructural study of the epidermis in two free-living Platyhelminthes, Mesostoma viaregginum and M. productum (Rhabdocoela, Typhloplanida)

Abstract. The epidermis of the free-living typhloplanids Mesostoma viaregginum and M. productum (Mesostominae) is described. In both species, the epidermis has polarized cells with nuclei located at the basal part of the cell, whereas mitochondria are in the apical one. The epidermis is entirely covered by microvilli and locomotory cilia anchored in the cytoplasm by vertical and horizontal rootlets. Rootlets exhibit distinct length and periodic structure in the two species. Furthermore, in each species vertical and horizontal rootlets possess different periodic structure. The pattern of termination of microtubules in epidermal cilia is described for the first time in the Typhloplanida; central microtubules shift along one axonemal side, doublets 1 and 6–9 lose their microtubule B, and gradually peripheral doublets become singlets. Finally, an electron-dense material caps the tip of the cilia. This pattern of termination closely resembles that of Temnocephalida, Kalytorhynchia, and Dalyelliida examined so far, but differences exist.     

Frequency of MEFV gene mutations in Hatay province,Mediterranean region of Turkey and report of a novel missense mutation (I247V)

In the present study, 1000 patients with clinical suspicion of FMF were retrospectively reviewed to determine the spectrum of MEFV gene mutations by using DNA sequence analysis between September, 2008 and April, 2012. Sixteen different mutations and 55 different genotypes were detected in 618 of 1000 patients. Among 16 different mutations, R202Q (21.35%) was the most frequently observed mutation; followed by E148Q (8.85%), M694V (7.95%), M680I (2.40%), V726A (1.85%), M694I (0.95%), A744S (0.80%), R761H (0.55%), P283L (0.35%), K695R (0.20%), E230K (0.15%), L110P (0.10%), I247V (0.05%), G196W (0.05%) and G304R (0.05%). In the present study, a novel missense mutation (I247V) and a silent variant (G150G) were identified in the MEFV gene. On the other hand, P238L, G632A and G304R mutations are the first cases reported from Turkey. Our results indicated that MEFV mutations are highly heterogeneous in our study population as in other regions of Turkey and mutation screening techniques such as PCR-RFLP, amplification refractory mutation system or reverse hybridization do not adequately detect uncommon or novel mutations. Therefore, it was proven that sequence analysis of the MEFV gene could be useful for detection of rare or unknown mutations.     

不同温度下黄曲霉菌菌丝超微形态的比较研究

为探究黄曲霉菌的毒素合成是否影响菌丝超微形态,本研究结合扫描和透射电镜技术比较观察产毒(28℃和30℃)和不产毒(37℃和40℃)温度下培养的不同发育阶段的黄曲霉菌菌丝形态和超微结构.扫描电镜结果显示:28℃下,在24h和44h菌丝体表面有丝状粘性分泌物附着,48-72h之间菌丝体逐渐出现皱缩、塌陷和扭曲现象,而37℃...     

Renal microvasculature in the adult pipid frog,Xenopus laevis: A scanning electron microscope study of vascular corrosion casts

We studied the opisthonephric (mesonephric) kidneys of adult male and female Xenopus laevis using scanning electron microscopy (SEM) of vascular corrosion casts and light microscopy of paraplast embedded tissue sections. Both techniques displayed glomeruli from ventral to mid-dorsal regions of the kidneys with single glomeruli located dorsally close beneath the renal capsule. Glomeruli in general were fed by a single afferent arteriole and drained via a single thinner efferent arteriole into peritubular vessels. Light microscopy and SEM of vascular corrosion casts revealed sphincters at the origins of afferent arterioles, which arose closely, spaced from their parent renal arteries. The second source of renal blood supply via renal portal veins varied interindividually in branching patterns with vessels showing up to five branching orders before they became peritubular vessels. Main trunks and their first- and second-order branches revealed clear longish endothelial cell nuclei imprint patterns oriented parallel to the vessels longitudinal axis, a pattern characteristic for arteries. Peritubular vessels had irregular contours and were never seen as clear cylindrical structures. They ran rather parallel, anastomosed with neighbors and changed into renal venules and veins, which finally emptied into the ventrally located posterior caval vein. A third source of blood supply of the peritubular vessels by straight terminal portions of renal arteries (vasa recta) was not found.     

Electron microscopic study of Burkholderia cepacia biofilms

Using the methods of transmission electron microscopy, the structure of the biofilms formed by the bacterium Burkholderia cepacia (clinical isolate and mutants with an increased and decreased ability to produce biofilm) were investigated. The biofilms were obtained on a liquid nutrient medium or on an abiotic surface (polystyrene). It has been demonstrated that the cultures of the studied strains differ in some morphological and functional characteristics. In biofilms, changes in the size and submicroscopic organization of all the components of bacterial cells occur. Staining biofilms with ruthenium red revealed the presence of exopolysaccharides in the intercellular space. The differences in the ultrastructure of bacterial films formed on nutrient medium and abiotic surfaces were demonstrated.     

Culture of cells in beem capsules: A new technique for electron microscopic study of monolayer cultures

Summary A method is described for the monolayer cultivation of primary cell suspensions and established cell lines directly in carbon-coated BEEM capsules. BEEM capsules are routinely employed by electron microscopists in tissue embedding procedures; growing monolayer cultures directly on the lids of inverted BEEM capsules presents the obvious advantage of maintaining cell to cell to substratum contacts with a minimum of stress and damage in the preparative steps for electron microscopy. This work was supported by grant AM 17631 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, grant CA 11339 from the National Cancer Institute. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Structure and composition of influenza virus. A small-angle neutron scattering study

A detailed analysis is presented of the small-angle neutron scattering curves of homogeneous solutions of influenza B virus, both intact and after treatment with bromelain, which removes the external glycoprotein spikes. The two sets of data are consistent with the following low-resolution structure: the virus particles are spherical, about 1200 A in diameter and of Mr about 180 X 10(6). The lipid bilayer is centred at a radius of 425 A, is 40 A to 50 A thick and constitutes 25% to 28% of the virus mass. The surface glycoproteins, predominantly haemagglutinin, contribute 40% to 46% of the total mass. Surprisingly little protein is found in the interior of the virus. It is suggested that the reason for this is that many particles do not contain the full complement of ribonucleoprotein complexes. These results are in good agreement with recent scanning transmission electron microscopic measurements of molecular mass and cryo-electron microscopic observations of the same preparations. Appendix 1 describes a new method of deriving spherical shell models from contrast variation neutron scattering data on viruses, in which scattering curves from all measured contrasts are used simultaneously. There is also a discussion of the assumptions and limitations implicit in the structural interpretation of such models, with emphasis on viruses containing lipid bilayers. Appendix 2 examines the effect on the scattering curves of various arrangements of the surface glycoproteins.     

Scanning electron microscopic study of spermatozoa from gossypol-treated rats

Summary Spermatozoa from the cauda epididymidis of gossypol-treated rats exhibit distinctive departures from the morphology of spermatozoa from control rats: wrinkled and disorganized cell membrane in the head and tail regions, cell membrane missing from segments of the tail midpiece and principal piece regions, malformed heads, decapitate spermatozoa, retention of a cytoplasmic droplet at variable loci along tail midpieces, and looped tails. The observations suggest that gossypol exerts its contraceptive effect during spermatocytogenesis and spermiogenesis, including the posttesticular development and maturation of spermatozoa in the epididymis.     

Electron microscopic study of mouse embryonic stem cell-derived cardiomyocytes

Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.     

Lymphocytocidal lymphocyte trapping by human lymph node cells: A tissue culture-ultrastructural study

Summary An ultrastructural study was carried out on 25 lymphocyte-trapping cells selected from tissue cultures of human axillary lymph nodes. The trapping cells contained several hundred intravacuolar lymphocytes, most of which showed degenerative changes. The principal findings are: (a) a broad spectrum of lymphocyte degeneration; (b) a consistent pattern of lymphocyte degeneration beginning with perinuclear vacuoles and ending with breakdown of the nuclear envelope; (c) the viable lymphocytes tended to be located in a juxtanuclear region; (d) a lysosomal relationship was suggested for lymphocyte degeneration but not for lymphocyte trapping; and (e) degeneration of the trapping cell, or lymphocytes associated with other cells, was not observed. The sequence of degenerative changes differs from those reported for several classes of lymphocytocidal agents. There were no morphologic properties of the trapping cell which served to identify it more specifically. The findings, together with previous time-lapse film observations, warrant further investigation of the hypothesis that lymphocytocidal lymphocyte trapping may be involved in the control of lymphocyte populations. Supported by contract G-72-3860 from the National Cancer Institute and grant HL-17412-01 of the National Heart and Lung Institute. An erratum to this article is available at .     

Electron microscopic visualization of the filament binding mode of actin-binding proteins

A large number of actin-binding proteins (ABPs) regulate various kinds of cellular events in which the superstructure of the actin cytoskeleton is dynamically changed. Thus, to understand the actin dynamics in the cell, the mechanisms of actin regulation by ABPs must be elucidated. Moreover, it is particularly important to identify the side, barbed-end or pointed-end ABP binding sites on the actin filament. However, a simple, reliable method to determine the ABP binding sites on the actin filament is missing. Here, a novel electron microscopic method for determining the ABP binding sites is presented. This approach uses a gold nanoparticle that recognizes a histidine tag on an ABP and an image analysis procedure that can determine the polarity of the actin filament. This method will facilitate future study of ABPs.     

The Crystal Structure of Human Pyrin B30.2 Domain: Implications for Mutations Associated with Familial Mediterranean Fever

The inherited autoinflammatory syndrome familial Mediterranean fever (FMF) is characterized by recurrent episodes of fever, which are independent of any bacterial or viral infections. This disease is associated with point mutations in the mefv gene product pyrin. Although the precise molecular functions of pyrin are unknown, it seems to be involved in the maturation and secretion of interleukin-1β. Approximately two thirds of all FMF-associated mutations cluster in the C-terminal B30.2 domain of pyrin. To investigate the molecular consequences of FMF-associated mutations, we determined the crystal structure of the pyrin B30.2 domain at 1.35-Å resolution. The comparison with other B30.2/ligand complex structures revealed a shallow cavity, which seems to be involved in binding the pyrin ligand. The bottom of this cavity is covered mainly with hydrophobic amino acids, suggesting that pyrin recognizes its ligand by hydrophobic contacts and surface complementarities. FMF-associated mutations cluster around two sites on the B30.2 surface. Approximately two thirds, including those mutations with the most severe disease outcomes, are observed in the vicinity of the predicted peptide binding site, suggesting that they will have a direct impact on ligand binding. A second mutational hot spot was observed on the opposite side of the B30.2 domain in the neighbourhood of its artificial N-terminus. Although most FMF-associated mutations are solvent exposed, several will modify the main-chain conformation of loops. The experimental crystal structure of the pyrin B30.2 domain serves as a basis for an accurate modelling of these mutations.     

The three-dimensional cytoarchitecture of the interstitial tissue in the rat kidney

Summary The cytoarchitecture of the interstitial tissue of the rat kidney was studied by combined scanning and transmission electron microscopy. The renal interstitium is composed of an elaborate network of stellate sustentacular cells. In the cortex, sustentacular cells radiate thin branching processes to form a fine reticulum, which supports intertubular spaces. In the medulla, these cells extend thick processes horizontally along the basal surfaces of the thin limbs or vasa recta, reinforcing their attenuate walls. The horizontal processes connect with each other at their terminals, compartmentalizing the interstitial space into thin layers. The medullary sustentacular cells contain abundant small lipid droplets. The network of sustentacular cells houses vasa recta, keeping them in parallel position to each other and to the tubules. The arterial vasa recta are accompanied by pericytes, which frequently contain lipid droplets larger in size than those in the sustentacular cells. Venous vasa recta extend numerous basal microvilli, which anchor the venous wall to adjacent tubules or vessels. Numerous free cells, round in shape, are found in the sustentacular cell network, especially in the cortex. They consist of macrophages and occasional lymphocytes. Some macrophages extend long pseudopodia, while others make intimate contact with lymphocytes, suggesting their high level of activity.     

A scanning electron microscopic study of germ cell maturation in the reproductive tract of the male soupfin shark (Galeorhinus galeus)

Germ cell maturation in the reproductive tract of the soupfin shark (Galeorhinus galeus) was studied using scanning electron microscopy (SEM). The SEM showed changes in Sertoli cytoplasm volume during spermatogenic development. In immature spermatocysts in the germinal zone, spermatogonia were embedded in Sertoli cytoplasm. In spermatogonial spermatocysts, Sertoli cells were adluminally located in the spermatocyst, with spermatogonia enveloped in the basal portions of the cytoplasm. During the round spermatid stage, Sertoli cytoplasm was very scanty. Spermatid elongation was accompanied by a progressive increase in the volume of Sertoli cytoplasm, notably around the spermatid heads. In the mature spermatocyst, bundles of spermatozoa are totally enveloped by Sertoli cytoplasm. Spermatozoa occurred randomly in the epididymis. However, in the ampulla ductus deferentis, spermatozoa reaggregated and were embedded in a mucoid substance to form highly ordered spherical bundles. In the sperm bundle, the spermatozoa heads were arranged such that the helical turns of adjacent spermatozoa were precisely aligned, and all the heads in the bundle formed a distinct apex. This study demonstrates the utility of exploring the relationship between germ cells and Sertoli cells in an evolutionarily ancient vertebrate, such as the shark.