Possible role of calmodulin in the secretion of Entamoeba histolytica electron-dense granules containing collagenase

Entamoeba histolytica cells secrete electron-dense granules (EDGs) that have collagenase activity. To study the possible involvement of calmodulin (CaM) on EDG secretion, the effect of several CaM antagonists (TFP, R24571, W-7, W-5, dibucaine and DL-propranolol) was tested on this cellular function. Except for W-5 and dibucaine, the rest of these compounds inhibited EDG secretion. Transmission electron microscopy of collagen-activated trophozoites showed numerous EDGs located in or near the surface membrane. In contrast, trophozoites incubated with TFP showed no EDGs. Protein kinase C inhibitors (H-7, ML-9) had no effect on EDG secretion, suggesting that CaM antagonists acted by selectively inhibiting CaM. These results suggest that a CaM-dependent process is involved in EDG secretion.     

Cytoarchitectural features of Ucides cordatus (Crustacea Decapoda) hepatopancreas: structure and elemental composition of electron-dense granules

Hepatopancreal tissue of the crab Ucides cordatus was investigated by light and electron microscopy. The observed epithelial cells were: E-cells (embryonic), located in the distal portion of the hepatopancreal tubules, R-cells (resorptive) F-cells (fibrillar) and B-cells (blister or secretory), found in its intermediate and proximal regions. Two types of electron-dense granules (EDGs) were found frequently in the cells of the proximal portion of the hepatopancreal tubule. Both types of EDGs presented alternating concentric electron-dense and electron-lucent layers. In order to better characterize these granules, energy dispersive X-ray analysis (EDXA) and glucose-6-phosphatase (G6Pase) cytochemistry were performed. One type of spherical granule was seen inside vacuoles surrounded by an association of myelin-like membranes as well as some small membrane-bound vesicles. This type of granule neither presented detectable Ca and P on EDXA spectra nor G6Pase cytochemical reaction products. The second type of granule had O, P and Ca characteristic peaks. G6Pase cytochemical products were observed inside these structures and showed that this mineralized type was surrounded by endoplasmic reticulum membranes. This result suggests that in U. cordatus the endoplasmic reticulum is associated with the genesis of mineralized EDGs. While amorphous mineral granules may be associated with a storage of Ca and P for the new carapace synthesis, EDGs covered by the non-mineralized spherical multi-layered membranes may be associated with late endosomes. No specific secretory pathway however was determined for the EDGs at the epithelial proximal portion.     

Ca2+-binding sites and Ca2+-ATPase activity in barley root tip cells

Summary Different cytochemical methods were employed to demonstrate the existence of Ca²⁺-binding sites (Ca²⁺-bs) at the membranes of barley root tip cells, involving addition of CaCl₂ (10 mM or 1 mM) to all aqueous solutions used for tissue processing for electron microscopy, treatment of ultrathin sections by Ca-chelating agents, enzymic digestion of ultrathin sections and modification of Wachstein-Meisel procedure for localization of Ca²⁺-dependent ATPase activity. Addition of 10 mM CaCl₂ to the fixatives and rinsing solutions causes electron-dense globules (EDG) to be formed in a variety of cells, those in cortical cells being associated mainly with the plasma membranes, in root cap cells with the plasmalemma as well as with majority of intracellular membranes. The obligatory presence of EDG at the membranes of Golgi vesicles and secretory vesicles approaching plasmalemma was revealed in the secreting root cap cells. Besides, electron opaque connecting material was found between the plasmalemma and adjacent secretory vesicle membranes. In true meristematic cells Ca-supplemented solutions induce formation of EDG localized at the ER membranes, and nuclear and plastid envelopes. In root cells of seeds germinated in the presence of 1 mM CaCl₂ electron opaque deposits were found only in local areas of plasmalemma collars around plasmodesmata neck regions, contacting the terminals of subsurface ER channels. In control speciemens (germination, fixation and washing without added CaCl₂) EDG were absent in cortical and ground meristem cells, but present in root cap cells, although their number and average size were greatly reduced.Treatment of thin sections by 10mM EGTA or EDTA led to complete removing of EDGs, electron-transparent holes replacing them. Digestion by a variety of proteolytic enzymes and by phospholipase A induced partial destruction of EDG matrices, confirming the presence of protein as well as of phospholipid membrane components. Visualization of electron-dense granular product of cytochemical Ca-ATPase reaction at the same membrane areas where EDG were located suggests that one of the Ca-binding proteins in EDG may represent Ca-ATPase.It is proposed that EDG at plant cell membranes have a certain resemblance to the Ca²⁺-bs revealed by the same method on plasma membrane of a variety of animal cells. The data obtained are discussed regarding possible regulatory roles of calcium ions in plant cells, especially in exocytotic secretion.     

Microstructure of matrix and mineral components of eggshells from White Leghorn chickens (Gallus gallus)

The avian eggshell is a composite structure of organic matrix and mineral (calcium carbonate) that is rapidly and sequentially fabricated in the oviduct in <24 hr. The eggshell is an excellent vehicle for the study of biomineralization processes and the role of the organic matrix in the mineral-matrix composite. The organic matrix components of eggshells from White Leghorn chickens (Gallus gallus) were examined by transmission electron microscopy (TEM) and optical microscopy. The mineral phase was analyzed by TEM, scanning electron microscopy (SEM), X-ray compositional microanalysis, and electron diffraction. Ultrastructural examination of the matrices within the calcified eggshell reveals a complex architecture that differs within each of the major zones of the eggshell: the shell membranes, the mammillary zone, the palisade region, and the cuticle. The mammillary layer consists of the calcium reserve assembly (CRA) and crown region, each with a unique substructure. TEM images show that the matrix of the CRA consists of a dense, flocculent material partially embedded within the outer shell membrane (a mostly noncalcified region of the shell). The mantle of the collagen fibers of the shell membranes is rich in polyanions (cuprolinic blue-positive), as is the CRA matrix. The CRA is capped by a centrally located calcium reserve body sac (CRB sac) that contains numerous 300–400 nm, electron-dense, spherical vesicles. Directly above the CRB sac is a zone of matrix consisting of stacks of interconnected vesicles (similar in morphology to CRA vesicles) that are interspersed with a granular material. The palisade region, the largest of the mineralized zones, contains hollow vesicles ∼450 nm (s.d. = 75 nm) in diameter, with a crescent-shaped, electron-dense fringe. An interconnecting matrix material is also found between the vesicles in the palisades region. The cuticle is composed of two layers, a mineralized inner layer and an outer layer consisting of only organic matrix. The bulk of the mineral within the eggshell is calcite, with small amounts of needlelike hydroxyapatite in the inner cuticle and occasionally, vaterite micro crystals found at the base of the palisade (cone) region. The well-crystallized calcite crystals within the palisade are columnar, typically ∼20 μm wide by 100–200 μm long; aside from numerous entrapped vesicles and occasional dislocations, they are relatively defect-free. The bulk of the matrix found in the palisade and crown regions are thought to be residual components of the rapid mineralization process. The unique matrix structure within the CRB corresponds to the region of preferentially solubilized calcite used by the developing embryo and the hydroxyapatite found in the inner cuticle may play a role in the cessation of mineral growth. © 1996 Wiley-Liss, Inc.     

Acid polysaccharides in the skeletal matrix and calicoblastic epithelium of the stony coral Mycetophyllia reesi

Like many corals the skeletal organic matrix and associated epithelium of Mycetophyllia reesi is physico-chemically unstable to preparative procedures for electron microscopy. Ethanol cryofracture of mineralized and demineralized material is accompanied by delamination of tissue and skeleton. Filamentous algae occur in the interface and account for some but not all of the separation artifact. Transmission microscopy accompanied by decalcification requires embedment in glycerol jelly to preserve the skeletal organic matrix. Even then, the matrix is not fixed and is not retained within the gel using standard double fixation with or without tannic acid as an additive. Ruthenium red, in combination with osmium, prevents the matrix from physical disruption, although positional artifacts relative to the calicoblastic epithelium are still evident. Inclusion of other glycan precipitating agents in the fixative sequence (Alcian blue, iron diamine or the detergent cetylpyridinium chloride) are more useful in preserving an acid polysaccharide-rich, fibrillar, extracellular matrix after demineralization. This material is not observed in SEM preparations. The calicoblast cells appear to be the source of this extracellular material that also appears to contribute to the composition of the mineralizing matrix. Moreover, a hyaluronan-like substance appears to play a significant role in matrix structure as suggested by its degradation by hyaluronidase.     

Proteomic analysis from the mineralized radular teeth of the giant Pacific chiton, Cryptochiton stelleri (Mollusca)

The biomineralized radular teeth of chitons are known to consist of iron-based magnetic crystals, associated with the maximum hardness and stiffness of any biomineral. Based on our transmission electron microscopy analysis of partially mineralized teeth, we suggest that the organic matrix within the teeth controls the iron oxide nucleation. Thus, we used Nano-LC-MS to perform a proteomic analysis of the organic matrix in radular teeth of the chiton Cryptochiton stelleri in order to identify the proteins involved in the biomineralization process. Since the genome sequence of C. stelleri is not available, cross-species similarity searching and de novo peptide sequencing were used to screen the proteins. Our results indicate that several proteins were dominant in the mineralized part of the radular teeth, amongst which, myoglobin and a highly acidic peptide were identified as possibly involved in the biomineralization process.     

Shell composition of Terreneuvian tubular fossils from north‐east Sichuan,China

Identification of the primary constituents of small shelly fossil (SSF) shells is important for explaining the evolution of SSF faunas. The characteristics and constituents of Terreneuvian tubular SSFs found in north‐east Sichuan, China, are revealed using light microscopy, scanning electron microscopy and energy dispersive X‐ray spectroscopy. Petrographic thin sections indicate that the chemical composition of the shells is mainly calcium carbonate with smaller amounts of phosphorus, silica and pyrite. Most of the tubular shells composed of calcium carbonate have a distinct layered structure. Evidence of replacement of the original shell by phosphatization, pyritization and silicification, and recrystallization of calcium carbonate have been found, all of which destroyed the shell's original layered structure. Most fossils treated with acetic acid are phosphatic casts or steinkerns, with some preserving organic textures of the shell as phosphatic casts. We conclude that the Terreneuvian tubular SSFs from north‐east Sichuan were originally composed mainly of calcium carbonate; indeed, most Terreneuvian small skeletal fossils appear to have had an originally calcareous composition. The fossil casts or internal core fossils are composed of phosphate, which is related to local taphonomic processes.     

Light‐harvesting superstructures of green plant chloroplasts lacking photosystems

The light‐harvesting antenna of higher plant photosystem II (LHCII) is the major photosynthetic membrane component encoded by an entire family of homologous nuclear genes. On the contrary, the great majority of proteins of photosystems and electron transport components are encoded by the chloroplast genome. In this work, we succeeded in gradually inhibiting the expression of the chloroplast genes that led to the disappearance of the photosystem complexes, mimicking almost total photoinhibition. The treated plants, despite displaying only some early signs of senescence, sustained their metabolism and growth for several weeks. The only major remaining membrane component was LHCII antenna that formed superstructures – stacks of dozens of thylakoids or supergrana. Freeze‐fracture electron microscopy revealed specific organization, directly displaying frequently bifurcated membranes with reduced or totally absent photosystem II (PSII) reaction centre complexes. Our findings show that it is possible to accumulate large amounts of light‐harvesting membranes, organized into three‐dimensional structures, in the absence of reaction centre complexes. This points to the reciprocal role of LHCII and PSII in self‐assembly of the three‐dimensional matrix of the photosynthetic membrane, dictating its size and flexible adaptation to the light environment.     

In vitro differentiation and mineralization of cartilaginous nodules from enzymatically released rat nasal cartilage cells

Nasal cartilage cells from 21-day-old rat fetuses were cultured at high density in the presence of ascorbic acid and β-glycerophosphate over a 12-day period. Immediately after plating, the cells exhibited a fibroblastic morphology, lost their chondrocyte phenotype and expressed type I collagen. On day 3, clusters of enlarged polygonal cells were found. These cell clusters synthetised type II collagen and formed an alcian-blue-positive matrix. The following days, a progressive increase in the number of cells positive for type 11 collagen was noted and, on day 8, typical cartilaginous nodules were formed. These nodules increased in size and number, spreading outward, laying down a dense matrix which mineralized. Light and electron microscopy observations of cross-sections of nodules confirmed the cartilaginous nature of this tissue formed in vitro with typical chondrocytes embedded in a hyaline matrix. Furthermore, at the electron microscopic level, matrix vesicles were seen in extracellular matrix associated with the initiation of mineralization. Typical rod-like crystals were present in the intercellular spaces along the collagen fibers. These results indicated that in a specific environment, dedifferentiated chondrocytes were able to redifferentiate and to form nodular structures with morphological ultrastructure of calcified cartilage observed in vivo.     

Poly(vinyl amine)-silica composite nanoparticles: models of the silicic acid cytoplasmic pool and as a silica precursor for composite materials formation

The role of polymer (poly(vinylamine)) size (238-11000 units) on silicic acid condensation to yield soluble nanoparticles or composite precipitates has been explored by a combination of light scattering (static and dynamic), laser ablation combined with aerosol spectrometry, IR spectroscopy, and electron microscopy. Soluble nanoparticles or composite precipitates are formed according to the degree of polymerization of the organic polymer and pH. Nanoparticles prepared in the presence of the highest molecular weight polymers have core-shell like structures with dense silica cores. Composite particles formed in the presence of polymers with extent of polymerization below 1000 consist of associates of several polymer-silica nanoparticles. The mechanism of stabilization of the "soluble" silica particles in the tens of nanometer size range involves cooperative interactions with the polymer chains which varies according to chain length and pH. An example of the use of such polymer-poly(silicic acid) nanoparticles in the generation of composite polymeric materials is presented. The results obtained have relevance to the biomimetic design of new composite materials based on silica and polymers and to increasing our understanding of how silica may be manipulated (stored) in the biological environment prior to the formation of stable mineralized structures. We suspect that a similar method of storing silicic acid in an active state is used in silicifying organisms, at least in diatom algae.     

Mineralized soft-tissue structure and chemistry in a mummified hadrosaur from the Hell Creek Formation,North Dakota (USA)

An extremely well-preserved dinosaur (Cf. Edmontosaurus sp.) found in the Hell Creek Formation (Upper Cretaceous, North Dakota) retains soft-tissue replacement structures and associated organic compounds. Mineral cements precipitated in the skin apparently follow original cell boundaries, partially preserving epidermis microstructure. Infrared and electron microprobe images of ossified tendon clearly show preserved mineral zonation, with silica and trapped carbon dioxide forming thin linings on Haversian canals within apatite. Furthermore, Fourier transform infrared spectroscopy (FTIR) of materials recovered from the skin and terminal ungual phalanx suggests the presence of compounds containing amide groups. Amino acid composition analyses of the mineralized skin envelope clearly differ from the surrounding matrix; however, intact proteins could not be obtained using protein mass spectrometry. The presence of endogenously derived organics from the skin was further demonstrated by pyrolysis gas chromatography mass spectrometry (Py-GCMS), indicating survival and presence of macromolecules that were in part aliphatic (see the electronic supplementary material).     

Induction of non-bilayer lipid structures by functional signal peptides.

Using 31P NMR and freeze-fracture electron microscopy we investigated the effect of several synthetic signal peptides on lipid structure in model membranes mimicking the lipid composition of the Escherichia coli inner membrane. It is demonstrated that the signal peptide of the E. coli outer membrane protein PhoE, as well as that of the M13 phage coat protein, strongly promote the formation of non-bilayer lipid structures. This effect appears to be correlated to in vivo translocation efficiency, since a less functional analogue of the PhoE signal peptide was found to be less active in destabilizing the bilayer. It is proposed that signal sequences can induce local changes in lipid structure that are involved in protein translocation across the membrane.     

Molecular indicators of microbial diversity in oolitic sands of Highborne Cay,Bahamas

Microbialites (stromatolites and thrombolites) are mineralized mat structures formed via the complex interactions of diverse microbial‐mat communities. At Highborne Cay, in the Bahamas, the carbonate component of these features is mostly comprised of ooids. These are small, spherical to ellipsoidal grains characterized by concentric layers of calcium carbonate and organic matter and these sand‐sized particles are incorporated with the aid of extra‐cellular polymeric substances (EPS), into the matrix of laminated stromatolites and clotted thrombolite mats. Here, we present a comparison of the bacterial diversity within oolitic sand samples and bacterial diversity previously reported in thrombolitic and stromatolitic mats of Highborne Cay based on analysis of clone libraries of small subunit ribosomal RNA gene fragments and lipid biomarkers. The 16S‐rRNA data indicate that the overall bacterial diversity within ooids is comparable to that found within thrombolites and stromatolites of Highborne Cay, and this significant overlap in taxonomic groups suggests that ooid sands may be a source for much of the bacterial diversity found in the local microbialites. Cyanobacteria were the most diverse taxonomic group detected, followed by Alphaproteobacteria, Gammaproteobacteria, Planctomyces, Deltaproteobacteria, and several other groups also found in mat structures. The distributions of intact polar lipids, the fatty acids derived from them, and bacteriohopanepolyols provide broad general support for the bacterial diversity identified through analysis of nucleic acid clone libraries.     

Ultrastructural study of encystation and excystation in Acanthamoeba castellanii

Encystation and excystation of Acanthamoeba castellanii were studied by transmission electron microscopy. The differentiation process was induced in asynchronous cultures grown axenically. Cytoplasmic vesicles containing a dense fibrous material very similar in appearance to the cyst wall were observed in trophozoites induced to encyst. When these trophozoites were incubated with calcofluor white m2r, fluorescence was observed in cytoplasmic vesicles, suggesting that the material contained in these vesicles corresponded to cyst wall precursors. Semithin cryosections of mature cysts with the same treatment showed fluorescence in the ectocyst and a less intense fluorescence in the endocyst, suggesting the presence of cellulose in both structures of the cyst wall. In mature cysts induced to excystation, small structures very similar to electron-dense granules (EDG) previously described in other amoebae were frequently observed. The EDGs were either sparsely distributed in the cytoplasm or associated with the cytoplasmic face of the plasma membrane. Many of them were located near the ostiole. In advanced phases of excystation, endocytic activity was suggested by the formation of endocytic structures and the presence of vacuoles with fibrous content similar to that of the cyst wall. Electron-dense granules in the process of dissolution were also observed in these vacuoles. Furthermore, the formation of a pseudopod suggests a displacement of the amoeba toward the ostiole.     

Biomineralization of endolithic microbes in rocks from the McMurdo Dry Valleys of Antarctica: implications for microbial fossil formation and their detection

In some zones of Antarctica's cold and dry desert, the extinction of cryptoendolithic microorganisms leaves behind inorganic traces of microbial life. In this paper, we examine the transition from live microorganisms, through their decay, to microbial fossils using in situ microscopy (transmission electron microscopy, scanning electron microscopy in back-scattered electron mode) and microanalytical (energy dispersive X-ray spectroscopy) techniques. Our results demonstrate that, after their death, endolithic microorganisms inhabiting Commonwealth Glacier sandstone from the Antarctica McMurdo Dry Valleys become mineralized. In some cases, epicellular deposition of minerals and/or simply filling up of empty moulds by minerals leads to the formation of cell-shaped structures that may be considered biomarkers. The continuous deposition of allochthonous clay minerals and sulfate-rich salts fills the sandstone pores. This process can give rise to microbial fossils with distinguishable cell wall structures. Often, fossilized cell interiors were of a different chemical composition to the mineralized cell walls. We propose that the microbial fossil formation observed was induced by mineral precipitation resulting from inorganic processes occurring after the death of cryptoendolithic microorganisms. Nevertheless, it must have been the organic template that provoked the diffusion of mineral elements and gave rise to their characteristic distribution pattern inside the fossilized cells.     

Immunological and biophysical alteration of hepatitis B virus antigens by sodium hypochlorite disinfection.

Sodium hypochlorite (NaOCl) was examined as an effective disinfectant in hepatitis laboratories. Concentrations of NaOCl containing 5,600 ppm (5,600 microgram/ml) of available chlorine were found to be effective in destroying the antigenicity of hepatitis B surface antigen (HBsAg) in virion-rich plasma after an exposure time of 1 min or more. In the treatment of protein-deficient solutions containing HBsAg, smaller concentrations of available chlorine (less than 500 pm) are equally effective. Neither 17-to 25-nm HBsAg particles nor 45-nm virion particles could be detected by electron microscopy after treatment. chemical interaction of protein and NaOCl was confirmed by isoelectrofocusing of 125I-labeled HBsAg. More than 90% of the labeled material was found at pH 3.0 or lower, indicating complete antigen oxidation. Labeled HBsAg was reduced in density from 1.21 g/cm3 in CsCl to approximately 1.07 g/cm3 after treatment with NaOCl. Both hepatitis B core antigen and deoxyribonucleic acid polymerase activity were significantly reduced after interaction with hypochlorite solutions. These results show that NaOCl destroys hepatitis B antigenicity and virus structures and therefore may be utilized as a disinfectant for the virus.     

Evidence of π‐stacking interactions in the self‐assembly of hIAPP22‐29

The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self‐assembly of human islet amyloid polypeptide (hIAPP)_22‐29, peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe‐23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe‐23 virtually abolish the ability of hIAPP_22‐29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in β‐sheet secondary structure. Transmission electron microscopy images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the ? C?C? ring mode (ca. 1600 cm^?1) upon aggregation that has previously been shown to be indicative of π‐stacking. While previous work has demonstrated that π‐stacking is not an absolute requirement for fibrillization, our findings indicate that Phe‐23 also contributes to fibril formation through π‐stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self‐assembly of hIAPP_22‐29. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Immunohistochemical localization of type IV collagen fibronectin and laminin in the juxtaglomerular apparatus of the rat kidney

The juxtaglomerular apparatus (JGA) is a complex structure containing several components: the vessels, the extraglomerular mesangium and the distal tubule. These structures include cellular elements and an extracellular matrix (ECM). Collagenous (type IV collagen) and noncollagenous components of the basement membranes were studied. The localization of type IV collagen and of two extracellular glycoproteins (laminin and fibronectin) was investigated using immunofluorescent and immunoperoxidase labelled antibodies. Type IV collagen and laminin have the same localization on the JGA basement membranes. On the other hand, fibronectin is limited to the entrance of the glomerular stalk. On electron microscopy, type IV collagen is found in the basement membrane while fibronectin is restricted to certain areas of the extracellular matrix. These findings confirm data concerning the distribution of these three components in basement membranes and allow a better understanding of the histoarchitecture of the juxtaglomerular apparatus.