Electron diffraction and high-resolution imaging on highly-crystalline β-chitin microfibril

The ultrastructure of β-chitin microfibrils from a centric diatom, Thalassiosira, and a tubeworm, Lamellibrachia, was studied using electron diffraction and high-resolution electron microscopy. Electron microdiffraction diagrams corresponding to each projection of the β-chitin crystals were obtained, and all the data support the structure model of anhydrous β-chitin crystals proposed by X-ray diffraction experiments. From high-resolution electron microscopy on ultrathin sections, the cross-sectional shapes of the microfibrils from Thalassiosira and Lamellibrachia were observed as a rectangular and parallelogram, respectively. The lattice fringes corresponding to the (0 1 0) plane of anhydrous β-chitin crystals were clearly observed in both cross-sections. Based on these observations, we have constructed a molecular packing model for β-chitin microfibrils.     

Synthesis of functionalized polyethylenimine-grafted mesoporous silica spheres and the effect of side arms on lipase immobilization and application

In the present study, silicate mesoporous materials (MCM-41), MCM-41-grafted polyethylenimine (MCM-41@PEI), and succinated PEI containing amine, amide, and acid groups were successfully synthesized and characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, Brunauer–Emmett–Teller analysis, and X-ray photoelectron spectroscopy. Thermomyces lanuginosa lipase (TLL) was then immobilized onto MCM-41 and polymer-grafted MCM-41 by physical adsorption. Besides, for enzyme immobilization via covalent bonding, glutaraldehyde (GLU), and hexamethylene diisocyanate (HMDI) were used as the bridges for binding the enzyme to supports. The best result was obtained with the immobilized lipase on MCM-41@PEI-GLU. In the study of the enzyme reusability, it was shown that about 83% of the initial activity could be retained after 12 cycles of uses. The immobilized lipase on the selected support was also applied for the synthesis of ethyl valerate. Following 24 h incubation in n-hexane and solvent free media, the esterification percentages were 79% and 67%, respectively.     

Characteristics of the Photosystem II reaction centre. II. Electron donors

The properties of Photosystem II electron donation were investigated by EPR spectrometry at cryogenic temperatures. Using preparations from mutants which lacked Photosystem I, the main electron donor through the Photosystem II reaction centre to the quinone-iron acceptor was shown to be the component termed Signal II. A radical of 10 G line width observed as an electron donor at cryogenic temperatures under some conditions probably arises through modification of the normal pathway of electron donation. High-potential cytochrome b-559 was not observed on the main pathway of electron donation. Two types of PS II centres with identical EPR components but different electron-transport kinetics were identified, together with anomalies between preparations in the amount of Signal II compared to the quinone-iron acceptor. Results of experiments using cells from mutants of Scenedesmus obliquus confirm the involvement of the Signal II component, manganese and high-potential cytochrome b-559 in the physiological process leading to oxygen evolution.     

Comparison of acellular and cellular bioactivity of poly 3-hydroxybutyrate/hydroxyapatite nanocomposite and poly 3-hydroxybutyrate scaffolds

Nanocomposites have recently been identified as a useful scaffolding material in tissue engineering applications. Poly (3-hydroxybutyrate)/hydroxyapatite nanoparticles (P3HB)/(nHA) porous scaffolds were successfully fabricated through a solvent casting and particulate leaching technique. P3HB/nHA and P3HB scaffolds were prepared by the same technique for comparison. The structure of the nanocomposite and P3HB scaffolds was observed by SEM. The Energy Disperssive X-ray Analysis (EDXA, map of Ca) results indicated that HA nanoparticles were homogeneously dispersed in the P3HB matrix. X-ray diffraction (XRD) analysis showed that P3HB and HA coexist in the nanocomposite. Transmission electron microscopy (TEM) images also showed that the particle size of HA was 30 ～ 40 nm. The porosity of the scaffolds was 84%, and macropores and micropores coexisted and interconnected throughout the scaffolds. Acellular bioactivity experiments showed that more HA crystals formed on the surface of the nanocomposite scaffold than on the P3HB scaffold after 4 weeks immersion in Simulated Body Fluid (SBF). Cell culture experiments demonstrated that the P3HB/nHA nanocomposite scaffold had a better tendency of proliferation and Alkaline Phosphatase (ALP) activity to MG 63 cells than the pure P3HB scaffold. It was found that nHA addition can improve acellular and cellular bioactivity of the P3HB scaffold.     

Oxidation of Manganese and Formation of Mn3O4 (Hausmannite) by Spore Coats of a Marine Bacillus sp

Isolated spore coats of a marine Bacillus species were incubated in 25 mM MnCl₂ at pH 7.5. Manganese precipitates, formed on the coat surfaces, were analyzed by transmission electron microscopy, electron diffraction, and energy-dispersive X-ray spectroscopy. Initially, an amorphous manganese oxide was observed on the coats which recrystallized to hausmannite after prolonged incubation in the MnCl₂ solution. The spore coats catalyze the oxidation of Mn(II) and have no structural influence on the final mineral phase precipitated.     

Macromolecular crowding and its influence on possible reaction mechanisms in photosynthetic electron flow

The diffusion of plastoquinol and its binding to the Qo site of the cyt bf complex in the course of photosynthetic electron transport was studied by following the sigmoidal flash-induced re-reduction kinetics of P700 after previous oxidation of the intersystem electron carriers. The data resulting from these experiments were matched with a simulation of electron transport using Monte Carlo techniques. The simulation was able to account for the experimental observations. Two different extreme cases of reaction mechanism at the Qo site were compared: a diffusion limited collisional mechanism and a non-diffusion limited tight binding mechanism. Assuming a tight binding mechanism led to best matches due to the high protein density in thylakoids. The varied parameters resulted in values well within the range of published data. The results emphasise the importance of structural characteristics of thylakoids in models of electron transport.     

Electron-counting MicroED data with the K2 and K3 direct electron detectors

Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å resolution. Even though a beam stop was not used with the K3 studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.     

Antioxidant Capacity of Ugni molinae Fruit Extract on Human Erythrocytes: An In Vitro Study

Ugni molinae is an important source of molecules with strong antioxidant activity widely used as a medicinal plant in Southern Chile–Argentina. Total phenol concentration from its fruit extract was 10.64 ± 0.04 mM gallic acid equivalents. Analysis by means of HPLC/MS indicated the presence of the anthocyanins cyanidin and peonidin, and the flavonol quercitin, all in glycosylated forms. Its antioxidant properties were assessed in human erythrocytes in vitro exposed to HClO oxidative stress. Scanning electron microscopy showed that HClO induced an alteration in erythrocytes from a normal shape to echinocytes; however, this change was highly attenuated in samples containing U. molinae extracts. It also had a tendency in order to reduce the hemolytic effect of HClO. In addition, X-ray diffraction experiments were performed in dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine bilayers, classes of lipids preferentially located in the outer and inner monolayers, respectively, of the human erythrocyte membrane. It was observed that U. molinae only interacted with DMPC. Results by fluorescence spectroscopy on DMPC large unilamellar vesicles and isolated unsealed human erythrocyte membranes also showed that it interacted with the erythrocyte membrane and DMPC. It is possible that the location of U. molinae components into the membrane outer monolayer might hinder the diffusion of HClO and of free radicals into cell membranes and the consequent decrease of the kinetics of free radical reactions.     

Dehydration of nigeran crystals: crystal structure and morphological aspects.

The crystal structure of anhydrous nigeran, poly[(1 → 3)-α-d-maltose], obtained from Penicillium crustosum has been determined by a combined electron diffraction. X-ray diffraction and packing analysis. The electron diffraction pattern from solution-grown single crystals shows some (h k I) reflections in the baseplane pattern. The polymer crystallizes with two chains passing through the cell of dimensions:$\text{a = 17.76, b = 6.0}\text{and}\text{c = 14.62}\text{A}\text{?}$ (fiber repeat). The space group is P2₁2₁2₁. The glycosidic torsion angles of this poly(disaccharide) were determined using an iteration process. The results are analyzed on the basis of the effect of side-group orientations on backbone conformation, and are compared with those obtained from a survey of relevant linear and cyclic oligosaccharides.The crystallographic reliability index was R_e = 0.25 and R_x = 0.30 when the model was tested against electron and X-ray diffraction data, respectively. The poly(disaccharide) chain is a 2-fold helix stabilized by an intrachain hydrogen bond between contiguous α-(1 → 4) residues. The hydroxymethyl side-chains are in the gauche-gauche form. Two such chains pack with anti-parallel polarity and the 2-fold screw axis coincides with the macromolecular axis. A network of hydrogen bonds holds the chains together in the crystal.In the condensed state, the nigeran chains are more or less extended depending on the state of hydration. The two extremes correspond to the “dry” and “wet” crystal forms. The transition between the two conformations takes place reversibly in the crystalline state. This is observed on solution-grown crystals, as well as in vivo where nigeran is found to occur in crystalline domains. Such a transition is analyzed in terms of the conformational flexibility of the backbone and in terms of the affine deformation concept. Morphological and textural transformations occurring as a result of drying are suggested to have important consequences on the subsequent microbiology such as enzymic digestion.     

Electron microscopy study of the enzymic hydrolysis of Valonia cellulose

The enzymic degradation of cellulose from Valonia macrophysa was observed by electron microscopy and evaluated by electron diffraction. Two types of Valonia samples were subjected to digestion; firstly, intact fragments cut from cell wall material and, secondly, cellulose microcrystals resulting from the acid hydrolysis of entire vesicles. These two substrates, when subjected to the action of crude cellulase complexes either from Trichoderma reesei or Schizophyllum commune were readily degraded. During the degradation, each microfibril or microcrystal became fibrillated into longitudinal crystalline sub-elements having widths ranging from below 2 nm to the full size of the initial Valonia microfibrillar width. These observations are evaluated in term of current theories concerning the topological action of cellulases.     

Structural studies on triacetates of mannan and glucomannan

Mannan triacetates prepared from material extracted from ivory nut and Tubera salep were studied by means of electron and X-ray diffraction. The former is uniquely constituted of acetylated d-mannopyranosyl units linked by a (1 → 4)-β-linkage whereas the latter contains acetylated (1 → 4)-β-d-glucopyranosyl randomly distributed in the backbone with a ratio of mannose to glucose of about 3:1. However, there seems to be no effect on crystallisation due to the presence of the glucosidic units on the conformation of the chain.Single crystals of ivory nut triacetate were prepared by slowly cooling a dilute solution of nitromethane and butanol. The crystals were long narrow laths which provide electron diffraction data after annealing at 190°C in a vacuum.Two different unit cells were derived from the acetylated Tubera salep X-ray data. A first unit cell with a = 1·18 nm, b = 1·54 nm and c = 1·60 nm contains eight sugar units, whereas the second unit cell with a = 0.369 nm, b = 0·96 nm and c = 1·58 nm would accommodate 16 residues. The latter agrees best with the base-plane parameters derived from electron diffraction of single crystals.The X-ray fibre diagram was interpreted in terms of a two-fold helix and an asymmetric unit composed of two triacetyl mannopyranosyl units. This means that two chemically identical mannose units would not be conformationally equivalent along the backbone.The presence of glucose units in the backbone does not seem to perturb the crystalline conformation. The ‘isomorphous replacement’ hypothesis was invoked to explain this observation. The helical parameters derived herein for Tubera salep mannan triacetate are different from those reported earlier for the same acetylated glucomannan but crystallised using a different technique. This is attributed to the occurrence of polymorphism in this material.     

Possible mechanisms underlying copepod grazing responses to levels of toxicity in red tide dinoflagellates

A previous study indicated that rates of ingestion exhibited by adult female copepods of Acartia hudsonica (Pinhey) and Pseudocalanus spp. were lowered by increasing levels of toxicity in clonal cultures of the bloom-causing dinoflagellate Protogonyaulax tamarensis (Taylor). In the present study, three types of laboratory grazing experiments were performed to determine which of two contending hypotheses —behavioural rejection or physiological incapacitation - could explain the observed relationship best. The experimental results consistently supported the postulated mechanism of physiological incapacitation, and not the mechanism of behavioural rejection, as the reason for the lowered rates of ingestion on the more toxic dinoflagellate clones.     

Three-dimensional electron diffraction of plant light-harvesting complex

Electron diffraction patterns of two-dimensional crystals of light-harvesting chlorophyll a/b-protein complex (LHC-II) from photosynthetic membranes of pea chloroplasts, tilted at different angles up to 60°, were collected to 3.2 Å resolution at -125°C. The reflection intensities were merged into a three-dimensional data set. The Friedel R-factor and the merging R-factor were 21.8 and 27.6%, respectively. Specimen flatness and crystal size were critical for recording electron diffraction patterns from crystals at high tilts. The principal sources of experimental error were attributed to limitations of the number of unit cells contributing to an electron diffraction pattern, and to the critical electron dose. The distribution of strong diffraction spots indicated that the three-dimensional structure of LHC-II is less regular than that of other known membrane proteins and is not dominated by a particular feature of secondary structure.     

X-ray diffraction analysis of the 3D organization of collagen fibrils in the wall of the dogfish egg case

The wall of the egg case of the dogfish,Scyliorhinus canicula, contains a network-forming collagen assembled into a regular three-dimensional (3D) structure. It accomplishes supportive, protective and filtering functions for the embryo contained within it. The collagen molecules in the egg case are organized into a body-centred unit cell of dimensions (mean ± s.d.) (11.6 ± 1.0) nm X (11.6 ± 1.0) nm X (81.6 ± 3.2) nm, which belongs to the I422 space group. At a higher hierarchical level, the collagen molecules assemble into parallel arrays of fibrils, ca. 100 nm in diameter, which aggregate to form laminae ca. 0.5 μm thick. These laminae are organized into a plywood-like structure and account for 98% of the thickness of the wall of the egg case. X-ray diffraction patterns of the wall of the egg case were taken along mutually perpendicular directions, one being perpendicular to the surface of the egg case. Three different kinds of diffraction pattern were observed. One of the patterns was characteristic of an X-ray direction perpendicular to the laminae in the egg case (along the x-direction). The two other patterns were obtained with the X-rays directed parallel to the plane of the laminae, either along the capsule long axis (z) or perpendicular to this (y). These two patterns were observed interchangeably in either of the x- or y-directions depending on the specimen. The diffraction patterns were analysed and interpreted taking into consideration the 3D electron microscope data of the egg case. The results confirm and extend previous findings from transmission electron microscopy and low-angle X-ray diffraction and they suggest that there is only one major type of ordered collagen arrangement in the wall of the egg case.     

Evidence for a mixed lattice in microtubules reassembled in vitro

An extensive structural analysis of microtubules assembled in vitro has been carried out using electron microscopy in conjunction with computer analysis based on Fourier transforms and helical diffraction theory. Microtubules assembled in vitro displayed a range of numbers of protofilaments from 12 to 16, with 14 the most abundant (84% in one large sampling). In almost all structures observed protofilaments are staggered to form a characteristic 3-start shallow helix. The presence of the 3-start helix was confirmed by fiber tilting experiments to correct the effects of microtubule flattening. Since α and β tubulin subunits alternate along the protofilaments, continuous helical lattices can be constructed with interactions between adjacent protofilaments involving unlike subunits (type A lattice) or like subunits (type B lattice). However, the 14-protofilament, 3-start microtubules are incompatible with either the A or B-type continuous helical lattice. Evidence is presented which indicates that lattice discontinuities are present which generate features of both the A and B-types, with the latter predominating.     

In Situ D-periodic Molecular Structure of Type II Collagen

Collagens are essential components of extracellular matrices in multicellular animals. Fibrillar type II collagen is the most prominent component of articular cartilage and other cartilage-like tissues such as notochord. Its in situ macromolecular and packing structures have not been fully characterized, but an understanding of these attributes may help reveal mechanisms of tissue assembly and degradation (as in osteo- and rheumatoid arthritis). In some tissues such as lamprey notochord, the collagen fibrillar organization is naturally crystalline and may be studied by x-ray diffraction. We used diffraction data from native and derivative notochord tissue samples to solve the axial, D-periodic structure of type II collagen via multiple isomorphous replacement. The electron density maps and heavy atom data revealed the conformation of the nonhelical telopeptides and the overall D-periodic structure of collagen type II in native tissues, data that were further supported by structure prediction and transmission electron microscopy. These results help to explain the observed differences in collagen type I and type II fibrillar architecture and indicate the collagen type II cross-link organization, which is crucial for fibrillogenesis. Transmission electron microscopy data show the close relationship between lamprey and mammalian collagen fibrils, even though the respective larger scale tissue architecture differs.     

Physico-chemical and biological properties of a nano-hydroxyapatite powder synthesized at room temperature

ObjectiveThe aim of this work was to synthesize and to characterize chemically and biologically (in vitro and in vivo) a nano-sized hydroxyapatite (nHA).Materials and methodsWet chemical precipitation at room temperature was performed, then chemical structure was explored using transmission electron microscopy, X-ray diffraction and Fourier transformed infrared spectroscopy. In vitro biological characterization was done using MG63 osteoblastic cell line cultured onto the material, and characterization was done for morphology (scanning electron microscopy), viability (live/dead assay) and proliferation (MTT test). Finally, nHA powder was tested in vivo in a study involving C57 Black mice for bone repair in a calvarial bone critical sized defect.ResultsMorphological, physico-chemical and cristallographic analyses revealed specific features of hydroxyapatite. Biological in vitro experiments revealed high affinity and proliferative ability of MG63 cells cultured onto the material. In vivo study displayed that in this model, the material allowed to repair bone continuity after 1 month healing.Discussion and conclusionsThe different ways of nHA synthesis are discussed regarding the potential application of the material. The obtained material should find applications in bone tissue engineering experiments.     

Eco-friendly synthesis of silver nanoparticles from the whole plant of Cleome viscosa and evaluation of their characterization,antibacterial, antioxidant and antidiabetic properties

The current research is to develop an easy and eco-friendly method for the synthesis of three different concentrations of silver nanoparticles (1mMCvAgNPs, 2mMCvAgNPs and 3mMCvAgNPs) using aqueous whole plant extract of Cleome viscosa and to evaluate their antibacterial, antioxidant and antidiabetic properties. CvAgNPs were characterized by Using UV–vis spectrophotometer, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM) and transmission electron microscope (TEM). The formation of CvAgNPs was confirmed by the observation of band between 250 nm to 600 nm UV–vis spectrum. The crystalline structure of CvAgNPs with a face-centered cubic (FCC) was confirmed by XRD. The responsible phytochemicals for the reduction and capping material of CvAgNPs were observed with FT-IR. The SEM analysis confirmed the size and shapes of CvAgNPs. The CvAgNPs have shown the rich content of total phenolic and total flavonoid components. The CvAgNPs have shown significant antibacterial activity on multi drug resistance Gram-negative and Gram-positive bacteria and also have shown significant strong antioxidant activities (DPPH, ABTS, H₂O₂ scavenging, Phosphomolybdenum assay and reducing power). The inhibitory action of CvAgNPs on α-glucosidase and α-amylase was stronger than the inhibitory action of acarbose. To best of our knowledge, this is the first attempt on the synthesis of AgNPs using C. viscosa whole plant aqueous extract. The synthesized CvAgNPs exhibited good antimicrobial, antioxidant and antidiabetic properties. Hence, to validate our results, the in vivo studies at the molecular level are needed to develop Cleome viscosa as an antibacterial, antioxidant and anti-diabetic agent.     

Electron transport and triplet formation in membranes of the photosynthetic bacterium Heliobacterium chlorum

The spectroscopic and thermodynamic properties of the electron-transport components of the photosynthetic bacterium Heliobacterium chlorum were studied by means of absorbance-difference spectroscopy. Upon flash illumination of membranes of H. chlorum photooxidation of the primary electron donor, P-798, was observed. In about 15% of the reaction centers P-798⁺ was reduced by cytochrome c-553, while in the remaining reaction centers P-798⁺ reduction occurred via a back reaction with a reduced electron acceptor. Titration experiments indicated a midpoint potential of −440 mV for the electron acceptor. At low redox potentials the formation of the triplet of P-798 was observed after a flash. The triplet was formed in about 30 ns by a back reaction with a reduced electron acceptor and decayed with a time constant of 35 μs. The yield of triplet formed in a flash was 30%. Upon continuous illumination at low redox potentials the accumulation in the reduced state of an electron acceptor was observed. The difference spectrum of this acceptor indicates that it is an iron-sulfur center. The yield of triplet formation was independent of the redox state of the iron-sulfur center, which indicates that the center is not located in the main electron-transport chain. A scheme with three acceptors in the main electron-transport chain is presented to accomodate our results and those of others.     

Crystalline sheets of tropomyosin

In the presence of spermine tropomyosin forms sheets having two-dimensional crystallinity and tactoids. The most common form of sheet has cmm symmetry with a = 80 nm and b = 5 nm. The structure of this sheet has been solved in projection to a nominal resolution of 1.5 nm by combining data from electron diffraction and electron microscopy. Analysis of this pattern and that of rarely observed sheets having p2 symmetry (a = 40 nm, b = 5 nm and γ = 80 °) indicated that the cmm structure was formed by superposition of two p2 sheets. The tropomyosin molecules in each p2 sheet were arranged in rows directed along the p2 (0, 1) lattice lines, with all the molecules in one row having the same polarity and lying antiparallel to the molecules in adjacent rows. These rows associated in pairs, possibly by the supercoiling of the molecules in one row about those in the neighbouring row.