Characterization of Myelin Sheath F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATP Synthase and its Regulation by IF<Subscript>1</Subscript>

F_oF₁-ATP synthase is the nanomotor responsible for most of ATP synthesis in the cell. In physiological conditions, it carries out ATP synthesis thanks to a proton gradient generated by the respiratory chain in the inner mitochondrial membrane. We previously reported that isolated myelin vesicles (IMV) contain functional F_oF₁-ATP synthase and respiratory chain complexes and are able to conduct an aerobic metabolism, to support the axonal energy demand. In this study, by biochemical assay, Western Blot (WB) analysis and immunofluorescence microscopy, we characterized the IMV F_oF₁-ATP synthase. ATP synthase activity decreased in the presence of the specific inhibitors (olygomicin, DCCD, FCCP, valynomicin/nigericin) and respiratory chain inhibitors (antimycin A, KCN), suggesting a coupling of oxygen consumption and ATP synthesis. ATPase activity was inhibited in low pH conditions. WB and microscopy analyses of both IMV and optic nerves showed that the Inhibitor of F₁ (IF₁), a small protein that binds the F₁ moiety in low pH when of oxygen supply is impaired, is expressed in myelin sheath. Data are discussed in terms of the role of IF₁ in the prevention of the reversal of ATP synthase in myelin sheath during central nervous system ischemic events. Overall, data are consistent with an energetic role of myelin sheath, and may shed light on the relationship among demyelination and axonal degeneration.     

Optical characterization of orientation of collagen

The optical properties of reconstituted collagen were characterized in the frame-work of a two-phase model. The total birefringence is assumed to be the sum of form birefringence Δ_F, of amorphous birefringence Δ_a, and of crystalline birefringence Δ_c. The contribution of Δ_F has been determined as a function of strain. The intrinsic values of Δ_c and Δ_a were calculated to be 0.24 ± 0.06 and 0.055 ± 0.018, respectively. In the undeformed state, the observed birefringence is almost entirely due to the distortion of the electric field of the light at the phase boundary. Whereas there is a slight increase of form birefringence with increasing strain, the total birefringence shows much larger increments. The latter is attributable to orientation of the rodlike superstructures of the tropocollagen molecules.     

Supramolecular organization of the yeast F1Fo-ATP synthase

Background information. The yeast mitochondrial F₁F_o‐ATP synthase is a large complex of 600 kDa that uses the proton electrochemical gradient generated by the respiratory chain to catalyse ATP synthesis from ADP and P_i. For a large range of organisms, it has been shown that mitochondrial ATP synthase adopts oligomeric structures. Moreover, several studies have suggested that a link exists between ATP synthase and mitochondrial morphology. Results and discussion. In order to understand the link between ATP synthase oligomerization and mitochondrial morphology, more information is needed on the supramolecular organization of this enzyme within the inner mitochondrial membrane. We have conducted an electron microscopy study on wild‐type yeast mitochondria at different levels of organization from spheroplast to isolated ATP synthase complex. Using electron tomography, freeze‐fracture, negative staining and image processing, we show that cristae form a network of lamellae, on which ATP synthase dimers assemble in linear and regular arrays of oligomers. Conclusions. Our results shed new light on the supramolecular organization of the F₁F_o‐ATP synthase and its potential role in mitochondrial morphology.     

Tricarboxylic acid cycle-sustained oxidative phosphorylation in isolated myelin vesicles

The Central Nervous System (CNS) function was shown to be fueled exclusively by oxidative phosphorylation (OXPHOS). This is in line with the sensitivity of brain to hypoxia, but less with the scarcity of the mitochondria in CNS. Consistently with the ectopic expression of F_oF₁-ATP synthase and the electron transfer chain in myelin, we have reported data demonstrating that isolated myelin vesicles (IMV) conduct OXPHOS. It may suggest that myelin sheath could be a site for the whole aerobic degradation of glucose.     

A model for the creep behaviour of tendon

A theoretical model is proposed to explain the viscoelastic behaviour of tendon. The model is based on the hypothesis that the mechanism of tendon deformation is one of shear of the mucopolysaccharide gel between the collagen ribbons in the ‘toe’ region¹ of the stress strain curve followed by fibrillar extension in the ‘linear’ region^2.3. Conventional linear viscoelastic parameters of the constituents of the tendon were used to describe the behaviour of the composite. Certain structural constants of the tendon and an appropriate form for the retardation spectrum of the composite also appear in the model. It was found that the following parameters play an important part in the viscoelastic deformation process; the mean value of the crimp angle of the strained fibres, 0_O; the broadness of the distribution of crimp angles $\text{α}$; the magnitude of the compliance difference for the gel, ΔJ_G and for the fibres, ΔD_F. Excellent agreement between experiment and theory has been observed for a variety of experimental circumstances. Values of 0_O, $\text{α}$ ΔJ_G and ΔD_F were determined for a number of specimens by fitting the model equations to the experimental data. The theory illustrates the expected influence on the viscoelastic properties of tendon which would result from changes in these parameters, which may arise from disease or ageing, for instance. The model also provides a challenge for future experimental work in that an independent determination of the parameters, 0_O, $\text{α}$ ΔJ_G and ΔD_F would confirm or refute the quantitative predictions of the theory presented here.     

Electrical activity and structural correlates of giant nerve fibers in Kuruma shrimp (Penaeus japonicus)

Morphology and recordings of electrical activity of Kuruma shrimp (Penaeus japonicus) giant medullated nerve fibers were carried out. A pair of giant fibers with external diameter of about 120 μ and 10 μ in myelin thickness were found in the ventral nerve cord. The diameter of the axon is about 10 μ. Thus there is a wide gap between the axon and the external myelin sheath. Each axon is doubly coated directly by Schwann cells and indirectly by the myelin sheath layer which is produced by those Schwann cells. Impulse conduction velocities of these giant fibers showed a range between 90–210 m/sec at about 22°C. Large action potentials (up to 113 mV, rise time of 0.16–0.3 msec, maximum rate of rise of 650–1250 V/sec, half decay time of 0.2–0.3 msec, maximum rate of fall of 250–450 V/sec and total duration of less than 1.5 msec) could be obtained by inserting microelectrodes or by longitudinal insertion of 25 μ diameter capillary electrodes into the gap but no DC-potential difference was observed across the myelin sheath. Transmyelin electrical parameters were very favorable for fast impulse conduction: myelin resistance of 3 × 10⁴ Ω cm²; time constant of 0.38 msec; myelin capacitance of 1.35 × 10^?8 F/cm²; gap fluid resistivity of 23 Ω cm. The existence of nodes of Ranvier could not be demonstrated morphologically, but electrophysiological evidence suggests that a type of saltatory conduction occurs in these giant fibers.     

The notochordal sheath in amphioxus--an ultrastructural and histochemical study

The notochord and notochordal sheath of 10 adult amphioxus were investigated ultrastructurally and histochemically. The notochord in amphioxus consists of parallel notochordal cells (plates) and each plate consists of parallel thicker and thinner fibrils and numerous profiles of smooth endoplasmic reticulum situated just beneath the cell membrane. Histochemical staining shows that the notochordal plates resemble neither the connective tissue notochordal sheath nor the typical muscular structure myotomes. The notochordal sheath has a complex three-layered organization with the outer, middle and inner layer The outer and middle layer are composed of collagen fibers of different thickness and course, that correspond to collagen type I and collagen type III in vertebrates, respectively, and the inner layer is amorphous, resembles basal lamina, and is closely attached to the notochord by hemidesmosome junctions. These results confirm the presence of collagen fibers and absence of elastic fibers in amphioxus.     

ELECTRON MICROSCOPE AND LOW-ANGLE X-RAY DIFFRACTION STUDIES OF THE NERVE MYELIN SHEATH

1. A close correlation has been obtained between high resolution electron microscopy and low-angle x-ray diffraction studies of the myelin sheath of frog and rat peripheral and central nerves. Extensive studies were performed by application of both techniques to the same specimens, prepared for examination by OsO₄ or KMnO₄ fixation, and embedding either in methacrylate or in gelatin employing a new procedure. Controlled physical and chemical modifications of the myelin sheath prior to fixation were also investigated. 2. A correspondence was established between the layer spacings observed in electron micrographs and the fundamental radial repeating unit indicated by the low-angle x-ray diffraction patterns. The variations in relative intensities of the low-angle x-ray reflections could be related to the radial density distributions seen in the electron micrographs. 3. An analysis of the preparation procedures revealed that OsO₄ fixation introduces a greater shrinkage of the layer spacings and more pronounced changes in the density distribution within the layers than KMnO₄ fixation. The effects of methacrylate and gelatin embedding are described, and their relative merits considered in relation to the preservation of myelin structure by OsO₄ fixation. 4. The experimental modifications introduced by freezing and thawing of fresh whole nerve are described, particularly the enhancement of the intermediate lines and the dissociation of the layer components in the myelin sheath. A characteristic collapsing of the radial period of the sheath is observed after subjecting fresh nerve trunks to prolonged and intense ultracentrifugation. 5. Controlled extraction of fresh nerve with acetone at 0°C., which preferentially removes cholesterol, produces characteristic, differentiated modifications of the myelin sheath structure. Electron microscopy reveals several types of modifications within a single preparation, including both expanded and collapsed layer systems, and internal rearrangements of the layer components. Alcohol extraction leads to a more extensive structural breakdown, but in certain areas collapsed layer systems can still be observed. The components of the lipide extracts could be identified by means of x-ray diffraction. These modifications emphasize the importance of cholesterol in the myelin structure, and disclose a resistance of the dense osmiophilic lines to lipide solvents. 6. The significance of these structures is discussed in relation to present concepts of the molecular organization of myelin. The available evidence is consistent with the suggestion that the primary site of osmium deposition is at the lipoprotein interfaces and that the light bands probably represent regions occupied by lipide chains. The electron microscope and x-ray diffraction data also indicate the possibility of a regular organization within the plane of the layers, probably involving units of 60 to 80 A. The myelin sheath is regarded as a favourable cell membrane model for detailed analysis by combined application of x-ray diffraction and electron microscopy.     

The role of hydrophobic bonding in collagen fibril formation: a quantitative model

A model has been developed for approximating the free energy of collagen fibril formation (ΔF_f) and the equilibrium solubility of collagen under physiological conditions. The model utilizes an expression of Flory for rodlike polymers, with the modification that the “pure” anisotropic phase is defined as a collagen fibril containing about 0.3 g water/g collagen. The model also assumes that χ₁, the polymer–solvent interaction term, is entirely due to hydrophobic effects. χ₁ is estimated from hydrophobic bond energies of amino acid side chains, using the results of Némethy and Scheraga. The temperature dependence of χ₁ is utilized to calculate equilibrium solubilities and ΔF_f as a function of temperature.     

Evaluation of myelin sheath and collagen reorganization pattern in a model of peripheral nerve regeneration using an integrated histochemical approach

Peripheral nerves are complex histological structures that can be affected by a variety of conditions with different degree of axonal degeneration and demyelination. For the study of peripheral nerve regeneration in pathology and tissue engineering, it is necessary to evaluate the regeneration, remyelination and extracellular matrix reorganization of the neural tissue. Currently, different histochemical techniques must be used in parallel, and a correlation among their findings should be further performed. In this work, we describe a new histochemical method for myelin and collagen fibers based on luxol fast blue and picrosirius methods, for the evaluation of the morphology, the myelin sheath and the collagen fiber reorganization using a model of peripheral nerve regeneration. Whole brain, normal sciatic nerve and regenerating peripheral nerve samples were fixed in 10% neutral buffered formalin and paraffin-embedded, for the performance of the hematoxylin-eosin stain, the Luxol fast blue method and the new histochemical method for myelin and collagen. The results of this technique revealed that this new histochemical method allowed us to properly evaluate histological patterns, and simultaneously observe the histochemical reaction for myelin sheath and collagen fibers in normal tissue, and during the regeneration process. In conclusion, this new method combines morphological and histochemical properties that allowed us to determine with high accuracy the degree of remyelination and collagen fibers reorganization. For all these reasons, we hypothesize that this new histochemical method could be useful in pathology and tissue engineering.     

Effect of hydration upon the thermal stability of tropocollagen and its dependence on the presence of neutral salts

The endotherm enthalpy changes ΔH_D and temperatures T_D of thermal denaturation of tropocollagen fibers were measured by DSC calorimetry as functions of water content. The denaturation temperatures decrease with increasing water content. The enthalpy change values increase sharply in the range 0–28% of water content, where a maximum of 14.3 cal g^?1 is reached. The effect of water uptake on the enthalpy term is explained by water bridge formation within the collagen triple helix. Evidence is given for the existence of approximately three intercatenary water bridges per triplet at the enthalpy maximum, their H-bond energy amounting to approximately 4000 kcal/mol of protein. In the 30–60% range of water content, ΔH_D decreases by 2 cal^?1 probably due to interactions between secondary water structures and the stabilizing intrahelical water bonds. The influence of two neutral potassium salts, with a structure-stabilizing and a structure-breaking anion (F^? and I^?), on the hydration dependence of ΔH_D and T_D was also studied. It was shown that the primary hydration is not influenced by these ions, but that T_D and ΔH_D are altered in an ion specific way in the presence of interface and bulk water. Hydrophobic interactions do not explain the experimental results. A reaction mechanism of the effects of ions upon the structural stability of collagen is proposed and discussed in terms of interactions of the medium water molecules with the intrahelical water bonds, and in terms of proton-donor/proton-acceptor equilibria between peptide groups, hydrated ions, and intrahelical water molecules.     

Structural Origins of Chiral Second-Order Optical Nonlinearity in Collagen: Amide I Band

The molecular basis of nonlinear optical (NLO) chiral effects in the amide I region of type I collagen was investigated using sum-frequency generation vibrational spectroscopy; chiral and achiral tensor elements were separated using different input/output beam polarization conditions. Spectra were obtained from native rat tail tendon (RTT) collagen and from cholesteric liquid crystal-like (LC) type I collagen films. Although RTT and LC collagen both possess long-range order, LC collagen lacks the complex hierarchical organization of RTT collagen. Their spectra were compared to assess the role of such organization in NLO chirality. No significant differences were observed between RTT and LC with respect to chiral or achiral spectra. These findings suggest that amide I NLO chiral effects in type I collagen assemblies arise predominantly from the chiral organization of amide chromophores within individual collagen molecules, rather than from supramolecular structures. The study suggests that sum-frequency generation vibrational spectroscopy may be uniquely valuable in exploring fundamental aspects of chiral nonlinearity in complex macromolecular structures.     

The cell organization underlying structural colour is involved in Flavobacterium IR1 predation

Flavobacterium IR1 is a gliding bacterium with a high degree of colonial organization as a 2D photonic crystal, resulting in vivid structural coloration when illuminated. Enterobacter cloacae B12, an unrelated bacterium, was isolated from the brown macroalga Fucus vesiculosus from the same location as IR1. IR1 was found to be a predator of B12. A process of surrounding, infiltration, undercutting and killing of B12 supported improved growth of IR1. A combination of motility and capillarity facilitated the engulfment of B12 colonies by IR1. Predation was independent of illumination. Mutants of IR1 that formed photonic crystals less effectively than the wild type were reduced in predation. Conversely, formation of a photonic crystal was not advantageous in resisting predation by Rhodococcus spp. PIR4. These observations suggest that the organization required to create structural colour has a biological function (facilitating predation) but one that is not directly related to the photonic properties of the colony. This work is the first experimental evidence supporting a role for this widespread type of cell organization in the Flavobacteriia.Subject terms: Water microbiology, Microbial ecology     

The psbO mutant of Chlamydomonas reinhardtii is capable of assembling stable, photochemically active reaction center of photosystem II

The functional status of photosystem II (PSII) complex in the dark-grown PsbO-deficient mutant of green alga Chlamydomonas reinhardtii was studied. It was found that ΔpsbO mutant cells of C. reinhardtii grown under heterotrophic conditions (dark + acetate) were capable of assembling stable, photochemically-competent reaction centers of PSII (as confirmed by immunological analysis of D1 protein level, pigments content and photoinduced changes of PSII chlorophyll fluorescence yield), while O₂-evolution activity was not revealed. The ratio F _v/F _m for the dark-grown ΔpsbO mutant C. reinhardtii was 0.37 and that for the dark-grown wild type cells was 0.56. Analysis of chlorophyll fluorescence induction curve indicated that the absence of oxygen-evolving activity could be due to some defects in the organization of the PSII catalytic manganese cluster. Decrease of the rate of the electron donation from water-oxidizing complex to the PSII reaction center as well as the appearance of an additional transient fluorescence peak during the dark relaxation of F _v testify to the damages to the PSII donor side. The data obtained suggest that the dark-grown PsbO-deficient cells of C. reinhardtii are able to form stable, photochemically active PSII reaction center, unable to oxidize water due to probable defects in the assembly of the manganese cluster.     

A hill-type muscle model expansion accounting for effects of varying transverse muscle load

Recent studies demonstrated that uniaxial transverse loading (F_G) of a rat gastrocnemius medialis muscle resulted in a considerable reduction of maximum isometric muscle force (ΔF_im). A hill-type muscle model assuming an identical gearing G between both ΔF_im and F_G as well as lifting height of the load (Δh) and longitudinal muscle shortening (Δl_CC) reproduced experimental data for a single load.Here we tested if this model is able to reproduce experimental changes in ΔF_im and Δh for increasing transverse loads (0.64 N, 1.13 N, 1.62 N, 2.11 N, 2.60 N). Three different gearing ratios were tested: (I) constant G_c representing the idea of a muscle specific gearing parameter (e.g. predefined by the muscle geometry), (II) G_exp determined in experiments with varying transverse load, and (III) G_f that reproduced experimental ΔF_im for each transverse load.Simulations using G_c overestimated ΔF_im (up to 59%) and Δh (up to 136%) for increasing load. Although the model assumption (equal G for forces and length changes) held for the three lower loads using G_exp and G_f, simulations resulted in underestimation of ΔF_im by 38% and overestimation of Δh by 58% for the largest load, respectively. To simultaneously reproduce experimental ΔF_im and Δh for the two larger loads, it was necessary to reduce F_im by 1.9% and 4.6%, respectively. The model seems applicable to account for effects of muscle deformation within a range of transverse loading when using a linear load-dependent function for G.     

Oxydative phosphorylation in sciatic nerve myelin and its impairment in a model of dysmyelinating peripheral neuropathy

Myelin sheath is the proteolipid membrane wrapping the axons of CNS and PNS. We have shown data suggesting that CNS myelin conducts oxidative phosphorylation (OXPHOS), challenging its role in limiting the axonal energy expenditure. Here, we focused on PNS myelin. Samples were: (i) isolated myelin vesicles (IMV) from sciatic nerves, (ii) mitochondria from primary Schwann cell cultures, and (iii) sciatic nerve sections, from wild type or Charcot‐Marie‐Tooth type 1A (CMT1A) rats. The latter used as a model of dys‐demyelination. O₂ consumption and activity of OXPHOS proteins from wild type (Wt) or CMT1A sciatic nerves showed some differences. In particular, O₂ consumption by IMV from Wt and CMT1A 1‐month‐old rats was comparable, while it was severely impaired in IMV from adult affected animals. Mitochondria extracted from CMT1A Schwann cell did not show any dysfunction. Transmission electron microscopy studies demonstrated an increased mitochondrial density in dys‐demyelinated axons, as to compensate for the loss of respiration by myelin. Confocal immunohistochemistry showed the expression of OXPHOS proteins in the myelin sheath, both in Wt and dys‐demyelinated nerves. These revealed an abnormal morphology. Taken together these results support the idea that also PNS myelin conducts OXPHOS to sustain axonal function.     

Transformation of amyloid-like fibers, formed from an elastin-based biopolymer, into a hydrogel: an X-ray photoelectron spectroscopy and atomic force microscopy study

Previous studies have revealed the propensity of elastin-based biopolymers to form amyloid-like fibers when dissolved in water. These are of interest when considered as "ancestral units" of elastin in which they represent the simplest sequences in the hydrophobic regions of the general type XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu). We normally refer to these biopolymers based on elastin or related to elastin units as "elastin-like polypeptides". The requirement of water for the formation of amyloids seems quite interesting and deserves investigation, the water representing the natural transport medium in human cells. As a matter of fact, the "natural" supramolecular organization of elastin is in the form of beaded-string-like filaments and not in the form of amyloids whose "in vivo" deposition is associated with some important human diseases. Our work is directed, therefore, to understanding the mechanism by which such hydrophobic sequences form amyloids and any conditions by which they might regress to a non-amyloid filament. The elastin-like sequence here under investigation is the ValGlyGlyValGly pentapeptide that has been previously analyzed both in its monomer and polymer form. In particular, we have focused our investigation on the apparent stability of amyloids formed from poly(ValGlyGlyValGly), and we have observed these fibers evolving to a hydrogel after prolonged aging in water. We will show how atomic force microscopy can be combined with X-ray photoelectron spectroscopy to gain an insight into the spontaneous organization of an elastin-like polypeptide driven by interfacial interactions. The results are discussed also in light of fractal-like assembly and their implications from a biomedical point of view.     

Conduction velocities and fiber diameters in a cutaneous nerve of the pigeon

Summary The characteristics of fibers of a cutaneous nerve supplying the wing skin of the pigeon have been investigated with electrophysiological and electron microscopic techniques.Recordings of the compound action potential showed four distinct peaks with conduction velocities of about 30 m/s, 12 m/s, 4 m/s and 0.5 m/s.From electron micrographs both fiber diameters and thickness of myelin sheath were assessed and used as criteria for segregating various fiber populations. Altogether four groups could be discerned: large thickly myelinated fibers, small thickly myelinated fibers, small thinly myelinated fibers, and unmyelinated or C-fibers. The subdivision of the thickly myelinated fibers into two populations is evidenced mainly by corresponding peaks in the compound action potential. The thinly myelinated fibers with a mean diameter of 2 m contributed about 90% of all myelinated fibers in this nerve.When comparing fiber dimensions and conduction velocities of this avian nerve with those of mammalian cutaneous nerves, the lower CV's of avian nerve fibers can be explained by smaller diameters and thinner myelin sheaths.The results of this investigation are a prerequisite for latency considerations in central somatosensory pathways in birds.Abbreviations CAP compound action potential - CV conduction velocity - D fiber diameter - d axon diameter - g ratio d/D - m thickness of myelin sheath     

High-performance Opening-up Dual-core Photonic Crystal Fiber Sensors Based on Surface Plasmon Resonance

Plasmonics - Nowadays, plasmonic sensors based on photonic crystal fiber (PCF) attracted a great deal of attention in the field of optical sensing. Opening-up dual-core photonic crystal fibers...