Estimation of flattening coefficient for absorption and circular dichroism using simulation

The absorbance and circular dichroism (CD) of suspensions is lower than if the same amount of chromophore were uniformly distributed throughout the medium. Several mathematical treatments of this absorption flattening phenomenon have been presented using various assumptions and approximations. This article demonstrates an alternative simulation approach that allows relaxation of assumptions. On current desktop computers, the algorithm runs quickly with enough particles and light paths considered to get answers that are usually accurate to better than 3%. Results from the simulation agree with the most popular analytical model for 0.01 volume fraction of particles, showing that the extent of flattening depends mainly on the absorbance through a particle diameter. Unlike previous models, the simulation can show that flattening is significantly lower when volume fraction increases to 0.1 but is higher when the particles have a size distribution. The simulation can predict the slope of the nearly linear relationship between flattening of CD and the absorbance of the suspension. This provides a method to correct experimental CD data where volume fraction and particle size are known.     

Spreading-independent growth of normal fibroblasts in three-dimensional cultures

Changes of cell shape resulting from cellular flattening on culture substratum have previously been demonstrated to correlate with mitotic activity of normal animal cells in monolayer cultures. Here, we compared the shapes and proliferation of chick embryo fibroblasts cultured either in multicellular, multilayered sheets extended between glass fibres, or in standard monolayers. Fibroblasts in sheets retained the mitotic activity characteristic of that observed in sparse monolayer cultures, i.e. considerably higher that in confluent monolayers. Morphometric analyses revealed, however, that the cells in sheets were considerably less flattened than in monolayer cultures. These observations indicate that the modulation of culture conditions resulting in multidirectional cell stretching leads to the dissociation of flattening and mitotic activity of normal animal cells, so long as an intracellular stress field, generated by contractile cytoskeleton and stabilised by intercellular contacts, is maintained.     

Differential absorption flattening optical effects are significant in the circular dichroism spectra of large membrane fragments

The circular dichroism spectra of membrane particles are distorted by effects of differential absorption flattening, which are a consequence of the nonrandom distribution of chromophores in these samples. We have shown that this phenomenon is not significant in small unilamellar vesicles with high lipid to protein ratios [Mao, D., & Wallace, B. A. (1984) Biochemistry 23, 2667-2673]. It has recently been claimed [Glaeser, R. M., & Jap, B. K. (1985) Biochemistry 24, 6398-6401] that absorption flattening effects are also inconsequential in large membrane fragments with high protein concentrations, such as purple membrane sheets. This paper will demonstrate that absorption flattening is significant in these samples and that it causes substantial distortion of the calculated protein secondary structures derived from the uncorrected circular dichroism data.     

Photon dose at the maze entrance door: The comparison of flattening filter and flattening filter free working modes

In recent years, field flattening free accelerators have been introduced in therapy practice. One of the objective of these measurements was to establish if the maze door, designed for accelerators operating with flattening filter can provide adequate shielding in field flattening free mode of operation. Linac installed in this standard one band maze vault is equipped to operate at 6 MV with field flattening filter and in field flattening free mode of operation. Series of measurements of the photon dose at the maze door (with different jaws openings and gantry positions) were performed in both operation modes with and without water canister to mimic standard therapy conditions. In this paper results of photon dose measurements, performed at the maze door of the therapy linear accelerator vault are presented in order to compare photon dose in flattening filter and flattening filter free operation modes. It was obtained that in field flattening free mode of operation, the dose at the maze door is always lower than the dose measured in standard mode of operation with the field flattening filter. In the case when FFF therapy practice should start in some existing therapy vault, no additional shielding measures need to be added at the existing maze door.     

A modified adenosine polyphosphatase histochemical method to demonstrate Langerhans cells in fragile epidermis

Adenosine polyphosphatase enzymes provide useful markers for epidermal Langerhans cells. Established adenosine polyphosphatase histochemical methods were refined and applied to demonstrate Langerhans cells in thin sheets of murine dorsal epidermis. The skin was supported during staining by attaching the keratinized surface to polyallyl diglycol carbonate "plastic" slides with cyanoacrylate adhesive and flattening it with pressure from a glass slide on the dermal surface. Optimal specific staining of dendritic Langerhans cells occurred after fixation of ethylenediaminetetraacetic acid-separated epidermal sheets in cacodylate buffered formaldehyde for 20 min and incubation, in the presence of magnesium and lead ions, with 9.36 X 10(-4) M adenosine diphosphate (ADP) for 45 min. Better definition of the cells was obtained with ADP as a substrate than with any concentration of adenosine triphosphate.     

The cellular basis of wing size modification in Drosophila: The effects of the miniature gene,crowding, and temperature

To elucidate the mechanisms whereby genes and environment influence wing size, we investigated the effects of various rearing temperatures and larval crowding conditions on the wings of the mutant miniature and wild-type fruit flies. In adults we monitored wing size, cell number, wing thickness, cell density; in larval imaginal discs we looked for cell death. Cell density was inversely proportional to wing size. Of particular interest was the finding that smaller wings tend to be thicker. Electron microscope studies showed that the miniature wing layers are grossly abnormal. We hypothesize that these abnormalities are due to abnormal cell flattening of the wing epithelial cells, and we conclude that gene and environmental effects on cell flattening may be an important component in determining cell density and hence organ size.     

Structural transitions and oligomerization along polyalanine fibril formation pathways from computer simulations

The results of a computer simulation study of the aggregation kinetics of a large system of model peptides with particular focus on the formation of intermediates are presented. Discontinuous molecular dynamic simulations were used in combination with our intermediate-resolution protein model, PRIME, to simulate the aggregation of a system of 192 polyalanine (KA(14) K) peptides at a concentration of 5 mM and a reduced temperature of T* = 0.13 starting from a random configuration and ending in the assembly of a fibrillar structure. The population of various structures, including free monomers, beta sheets, amorphous aggregates, hybrid aggregates, and fibrils, and the transitions between the structures were tracked over the course of 30 independent simulations and averaged together. The aggregation pathway for this system starts with the association of free monomers into small amorphous aggregates that then grow to moderate size by incorporating other free monomers or merging with other small amorphous aggregates. These then rearrange into either small beta sheets or hybrid aggregates formed by association between unstructured chains and beta sheets, both of which grow in size by adding free monomer chains or other small aggregates, one at a time. Fibrillar structures are formed initially either by the stacking of beta sheets, rearrangement of hybrid aggregates or association between beta sheets and hybrid aggregates. They grow by the addition of beta sheets, hybrid aggregates, and other small fibrillar structures. The rearrangement of amorphous aggregates into beta sheets is a critical and necessary step in the fibril formation pathway.     

Circular dichroism analyses of membrane proteins: an examination of differential light scattering and absorption flattening effects in large membrane vesicles and membrane sheets

The circular dichroism spectra of membrane suspensions are distorted by differential light scattering and absorption flattening effects, which arise as a consequence of the large size of the membrane particles relative to the wavelength of light and the high concentration of proteins in the membranes. In this paper, the consequences of these phenomena on the protein spectra of large membrane particles are discussed, and methods for eliminating them are examined. The distortions due to differential light scattering are relatively small in membrane systems, and can be compensated for by use of a large detector acceptance angle geometry. Several methods for correcting for differential flattening, which introduces a substantial distortion, have been evaluated, and a new method, the flattening quotient approach, which produces by far the best results, is described. Since the secondary structures calculated from circular dichroism spectra are highly dependent on accurate spectral shape and magnitude, this method for correcting the spectra may find general application in circular dichroism studies of membrane proteins.     

A Modified Adenosine Polyphosphatase Histochemical Method to Demonstrate Langerhans Cells in Fragile Epidermis

Adenosine polyphosphatase enzymes provide useful markers for epidermal Langerhans cells. Established adenosine polyphosphatase histochemical methods were refined and applied to demonstrate Langerhans cells in thin sheets of murine dorsal epidermis. The skin was supported during staining by attaching the keratinized surface to polyallyl diglycol carbonate “plastic” slides with cyanoacrylate adhesive and flattening it with pressure from a glass slide on the dermal surface. Optimal specific staining of dendritic Langerhans cells occurred after fixation of ethylenediaminetetraacetic acid-separated epidermal sheets in cacodylate buffered formaldehyde for 20 min and incubation, in the presence of magnesium and lead ions, with 9.36 × 10^-4 M adenosine diphosphate (ADP) for 45 min. Better definition of the cells was obtained with ADP as a substrate than with any concentration of adenosine triphosphate.     

The tail flattening reflex in Lumbricus: reconstitution after tail amputation and modifications in segmental nerve roots

On stimulation of the head, the caudal 20-30 segments of intact earthworms exhibit a marked dorso-ventral flattening reflex which is an element of the escape response. When the tail is removed this reflex is reconstituted gradually in segments anterior to the amputation level without regeneration of new tail segments. It takes longer to reestablish the flattening if larger parts of the body are cut off, but after 60 days p.o. all animals regained the full response independent of the number of segments amputated. Extirpation of three consecutive ganglia from the ventral nerve cord also suffices to develop flattening anterior to the operated segments, but the reaction is less pronounced and appears later than after tail amputation. Moreover, it vanishes at the same time the normal tail flattening is observed again, presumably after regeneration across the gap in the cord. In different regions of the body, area and perimeter distributions of transversely sectioned axons in the three pairs of segmental nerves (SN I-III) of the cord were measured. Characteristic changes along the length of the animal were found for SN I and SN III. In tail segments the axonal size distributions are more variable than "typical" segments. The flattening reflex is mediated by axons in SN III (Pallas and Drewes, 1981). In body segments having reconstituted behavioral characteristics of the lost tail segments, the area of the largest axons and the general size distribution in SN III approximate those found in normal tail segments.     

Obstruction of Photoinduced Electron Transfer from Excited Porphyrin to Graphene Oxide: A Fluorescence Turn-On Sensing Platform for Iron (III) Ions

A comparative reaserch of the assembly of different porphyrin molecules on graphene oxide (GO) and reduced graphene oxide (RGO) was carried out, respectively. Despite the cationic porphyrin molecules can be assembled onto the surfaces of graphene sheets, including GO and RGO, to form complexes through electrostatic and π-π stacking interactions, the more obvious fluorescence quenching and the larger red-shift of the Soret band of porphyrin molecule in RGO-bound states were observed than those in GO-bound states, due to the differenc of molecular flattening in degree. Further, more interesting finding was that the complexes formed between cationic porphyrin and GO, rather than RGO sheets, can facilitate the incorporation of iron (III) ions into the porphyrin moieties, due to the presence of the oxygen-contained groups at the basal plane of GO sheets served as auxiliary coordination units, which can high-efficiently obstruct the electron transfer from excited porphyrin to GO sheets and result in the occurrence of fluorescence restoration. Thus, a fluorescence sensing platform has been developed for iron (III) ions detection in this contribution by using the porphyrin/GO nanohybrids as an optical probe, and our present one exhibited rapid and sensitive responses and high selectivity toward iron (III) ions.     

Cellular reactions of the polychaete annelid Nereis diversicolor against coelomic parasites

We studied the cellular reactions of the polychaete Nereis diversicolor against different natural coelomic parasites: a yeast, a coccidian, and a supposed orthonectid. The coelomocytes engaged in these reactions are type 1 and type 2 granulocytes. We observed recognition processes, accumulation and flattening of coelomocytes into concentric sheets around the parasites, and then formation of thick capsules which become brown bodies. The parasites are first paralyzed (in case of motile stages), then show degeneration symptoms of the cell organelles, and are finally killed and turn into necrosed nodules. Encysted stages of coccidians may remain alive in the capsules. Life of small coccidian sporozoites is possible inside the coelomocytes.     

Obelionic cranial deformation in the Puebloan Southwest

As a form of cranial deformation, obelionic flattening is rare. Originally named and described by Stewart (J Wash Acad Sci 29 ( 1939 ) 460–465), based on a small sample from Florida, it has been little noted since. Previously [Nelson and Madimenos, Paper presented at the Paleopathology Association annual meeting (2007)], we reported the discovery of two individuals from the Pueblo III Gallina site of Cañada Simon I who exhibit flattening of this type. Although technically undescribed in the Southwest before now, there are tantalizing clues in the literature that it occurred in low frequencies throughout the Ancestral Pueblo world. To determine whether the obelionic flattening found at Cañada Simon I was isolated or an indication of a more widespread phenomenon, we undertook a survey of crania from other Gallina sites, Chaco Canyon, and the literature (type of deformation can be determined on lateral photographs of crania properly positioned along the Frankfort Horizontal). We examined 146 crania (78 firsthand) of which seven exhibit obelionic flattening. Our results indicate that obelionic flattening should be added to the suite of cranial deformations that occur in the Southwest. Here, we propose parameters by which obelionic flattening can be described and differentiated from the more common lambdoidal and occipital forms and suggest that the three types of flattening form a continuum of cradleboard induced deformation, although the exact mechanism for obelionic flattening remains elusive. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Absorption flattening in the circular dichroism spectra of small membrane fragments

The inhomogeneous distribution of chromophore occurring in a particulate suspension can result in a reduction in the apparent molar ellipticity recorded in circular dichroism (CD) spectra. The possibility of such a systematic error has often been a matter of concern when CD spectra of cell membrane proteins are recorded. The recent publication of CD spectra for bacteriorhodopsin in native and sonicated membranes, in detergent-solubilized form, and reconstituted into small unilamellar vesicles [Mao, D., & Wallace, B. A. (1984) Biochemistry 23, 2667-2673] gives a unique opportunity to apply the theoretical analysis of Gordon and Holzwarth [Gordon, D. J., & Holzwarth, G. (1971) Arch. Biochem. Biophys. 142, 481-488] so as to provide a definitive answer to the question of whether absorption flattening is significant for membrane particles. We show here that the data of Mao and Wallace can be combined with the theoretical analysis of Gordon and Holzwarth to rule out significant absorption flattening effects over the range 200-240 nm for submicrometer-sized membranes. In addition, the results show that absorption flattening can be disregarded even at 190 nm for membranous material in the size range below 100 nm. The demonstration that there are no major flattening effects in the CD spectra of bacteriorhodopsin, particularly in the region of 200-240 nm, means that the experimental spectra are incompatible with the proposal that this transmembrane protein contains seven transmembrane helices.     

NUCLEAR CHANGES DURING SPERMIOGENESIS IN A PULMONATE SNAIL

Changes in both external form and internal arrangement of nuclear material have been investigated in the differentiation of the sperm of the pulmonate snail, Otala lactea. Sperm head differentiation begins with a flattening of the previously spherical nucleus and a thickening of the nuclear envelope covering the anterior and posterior surfaces of that nucleus. Tail filaments can be seen in the cytoplasm at this time. At a slightly later period the mitochondria begin to form the tail filament sheath and at this time the nucleus begins to elongate in a direction parallel to the future axis of the sperm. At the same time the nuclear material begins to orient itself at right angles to the nuclear surface which lines the pit occupied by the centriole. As nuclear elongation proceeds, this orientation of nuclear substance takes on the appearance of 60 A thick sheets bent in a direction parallel to the sperm axis. Soon the sheets fill the entire nucleus. The nucleus then begins to twist along its axis so that it starts to take on the mature, flame-shaped form. At this time the flat sheets begin to disappear and in the mature sperm head they are no longer visible (see Text-fig. 2 B).     

Aggregation and proton release of purple and white membranes following cleavage of the carboxyl-terminal tail of bacteriorhodopsin

Our results indicate that the previously reported decrease in proton release by proteolyzed purple membrane sheets was due merely to the aggregation state of these preparations and not to the loss of the carboxyl-terminal tail. Changes in H+/M412 ratios obtained for purple and white membrane preparations correlate with the measured aggregation. White membrane preparations consistently exhibit H+/M412 ratios more than twice those measured for native purple membranes under the same conditions. Quasi-elastic light scattering was used to characterize the size of isolated purple and white membrane sheets before and after proteolysis. The results clearly show that native purple membrane preparations are larger in size than would be expected and that, following trypsin treatment, they are on average more than an order of magnitude larger. Negative staining electron microscopy showed that the purple membrane became aggregated in stacked arrays. Bleaching and reconstitution with retinal also affect aggregation, but iodination or nitration of purple membrane does not affect the measured size. The average size of white membranes is smaller; this is consistent with results of electron microscopy and the size increase is much less than that of purple membranes following trypsin treatment. No size change occurs with retinal reconstitution. In aggregated purple membrane preparations, protons and other cations are unable to exchange freely with the aqueous medium, explaining why proteolysis lowers the proton release from purple membrane sheets in suspension.     

Correlates of sprinting, jumping and parachuting performance in the butterfly lizard, Leiolepis belliani

Leiolepis belliani , a cursorial, beach-dwelling lizard, moves by running and jumping. The lizards' ability to flatten dorsoventrally, thereby increasing surface area and decreasing wing loading, may also confer parachuting ability. We measured locomotor performance of three ecologically relevant tasks: running, jumping and parachuting. In addition, we investigated whether, with the effect of size removed, locomotor performance capabilities are correlated, and whether they correlate with morphological features. Larger lizards fell and ran faster and jumped further. Lizards that were experimentally prevented from flattening fell faster than control lizards. When the effects of size were removed, limb length was uncorrelated with jumping and running performance; performance measures also were not correlated amongst themselves. The scant natural historical data available for this species suggests that lizards do not use their parachuting capability, and that dorsoventral flattening may have evolved for some other purpose. Leiolepis might serve as a useful model in understanding the evolution of gliding lizards (e.g. Draco ).     

Curvature of double-membrane organelles generated by changes in membrane size and composition

Transient double-membrane organelles are key players in cellular processes such as autophagy, reproduction, and viral infection. These organelles are formed by the bending and closure of flat, double-membrane sheets. Proteins are believed to be important in these morphological transitions but the underlying mechanism of curvature generation is poorly understood. Here, we describe a novel mechanism for this curvature generation which depends primarily on three membrane properties: the lateral size of the double-membrane sheets, the molecular composition of their highly curved rims, and a possible asymmetry between the two flat faces of the sheets. This mechanism is evolutionary advantageous since it does not require active processes and is readily available even when resources within the cell are restricted as during starvation, which can induce autophagy and sporulation. We identify pathways for protein-assisted regulation of curvature generation, organelle size, direction of bending, and morphology. Our theory also provides a mechanism for the stabilization of large double-membrane sheet-like structures found in the endoplasmic reticulum and in the Golgi cisternae.     

Factors promoting the establishment of primary cultures of liver cells fromXenopus larvae

Summary The requirements for establishment and survival of primary cultures of larval amphibian liver cells were investigated.Plating efficiency was found to be enhanced by a collagen substrate, by diluted conditioned medium from an adultXenopus kidney cell line and by high initial cell densities. Plating efficiency was highest at a tonicity of 165–220 mOsm/kg. In cultures with undiluted conditioned medium the increase in cell number was 50–60% greater than in controls, where it was about 2-fold between day 3 and 6 of culture. Conditioned medium from theXenopus kidney cell line is assumed to contain at least two components, which are effective at different concentrations and stimulate either plating efficiency and cell aggregation or cell proliferation.In cultures without collagen sheets, cell flattening is greatly reduced, indicating that cell shape is also dependent upon the substrate.