bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of differences in cancer cells and tumor growth sites on recruiting bone marrow‐derived endothelial cells and myofibroblasts in cancer‐induced stroma
Authors: Sangai Takafumi , Ishii Genichiro , Kodama Keiji , Miyamoto Shin'ichi , Aoyagi Yasuyuki , Ito Takashi , Magae Junji , Sasaki Hiroki , Nagashima Takeshi , Miyazaki Masaru , Ochiai Atsushi
Published: February 2005

Cancer‐stromal interaction is well known to play important roles during cancer progression. Recently we have demonstrated that bone marrow‐derived vascular endothelial cells (BMD‐VE) and myofibroblasts (BMD‐MF) are recruited into the human pancreatic cancer cell line Capan‐1 induced stroma. To assess the effect of the difference in cancer cell types on the recruitment of BMD‐VE and BMD‐MF, 10 kinds of human cancer cell line were implanted into the subctaneous tissue of the immunodeficient mice transplanted with bone marrow of double‐mutant mice (RAG‐1−/− β‐gal Tg or RAG‐1−/− GFP Tg). The recruitment frequency of BMD‐VE (%BMD‐VE) and BMD‐MF (%BMD‐MF), and tumor‐associated parameters [tumor volume (TV), microvessel density (MVD) and stromal proportion (%St)] were measured. The correlation among them was analyzed. Although %BMD‐VE and %BMD‐MF varied (from 0 to 21.6%, 0 to 29.6%, respectively), depending on the cancer cell line, both parameters were significantly correlated with %St (p < 0.005). Furthermore %BMD‐VE and %BMD‐MF also significantly correlated (p < 0.005). In order to assess the effect of tumor growth sites on the recruitment of the cells of interest, a human pancreatic cancer cell line, Capan‐1, was transplanted into 5 different sites: subcutaneous tissue, peritoneum, liver, spleen and lung. Tumors in the subcutaneous tissue and peritoneum induced desmoplastic stroma (%St = 22.7%, 19.5%, respectively) and contained BMD‐VE (%BMD‐VE = 21.6%, 16.5% respectively) and BMD‐MF (%BMD‐MF = 29.6%, 24.5%, respectively), but weak stromal induction without recruitment of BMD‐VE or ‐MF was observed in the tumors at of the liver, spleen and lung (%St = 9.7%, 9.1%, 5.4%, respectively). cDNA microarray analysis identified the 29 genes that expression was especially up‐ or down‐regulated in the cell line that induced an abundant stromal reaction. However they did not encoded the molecules that were directly involved in stromal cell recruitment (chemokines), differentiation (cytokines) or proliferation (growth factors). These results indicate that the recruitment of BMD‐VE and ‐MF is required for stromal formation during cancer progression and that the cancer microenvironment is important in stromal reaction and the recruitment of BMD‐VE and ‐MF. © 2005 Wiley‐Liss, Inc.
Fitting complex potential energy surfaces to simple model potentials: Application of the simplex‐annealing method
Authors: Bustos Marún Raúl A. , Coronado Eduardo A. , Ferrero Juan C.
Published: February 2005

A stochastic method of optimization, which combines simulated annealing with simplex, is implemented to fit the parameters of a simple model potential. The main characteristic of the method is that it explores the whole space of the parameters of the model potential, and therefore it is very efficient in locating the global minimum of the cost function, in addition to being independent of the initial guess of the parameters. The method is employed to fit the complex intermolecular potential energy surface of the dimer of water, using as a reference the spectroscopic quality anisotropic site–site potential of Feller et al. The simple model potential chosen for its reparameterization is the MCY model potential of Clementi et al. The quality of the fit is assessed by comparing the geometry of the minimum, the harmonic frequencies, and the second virial coefficients of the parameterized potential with the reference one. Finally, to prove more rigorously the robustness of this method, it is compared with standard nonstochastic methods of optimization. © 2005 Wiley Periodicals, Inc. J Comput Chem 26: 523–531, 2005
Electronic structure study of the initiation routes of the dimethyl sulfide oxidation by OH
Authors: González‐García Núria , González‐Lafont Àngels , Lluch José M.
Published: February 2005

In the present work the potential energy surface (PES) corresponding to the different initiation routes of the oxidation mechanism of DMS by hydroxyl radical in the absence of O2 has been studied, and connections among the different stationary points have been established. Single‐point high level electronic structure calculations at lower level optimized geometries have been shown to be necessary to assure convergence of energy barriers and reaction energies. Our results demonstrate that the oxidation of DMS by OH turns out to be initiated via three channels: a hydrogen abstraction channel that through a saddle point structure finally leads to CH3SCH2 + H2O, an addition‐elimination channel that firstly leads to an adduct complex (AD) and then via an elimination saddle point structure finally gives CH3SOH and CH3 products, and a third channel that through a concerted pathway leads to CH3OH and CH3S. The H‐abstraction and the addition‐elimination channels initiate by a common pathway that goes through the same reactant complex (RC). Our theoretical results agree quite well with the branching ratios experimentally assigned to the formation of the different products. Finally, the calculated equilibrium constants of the formation of the complex AD and the hexadeuterated complex AD from the corresponding reactants, as a function of the temperature, are in good accordance with the experimental values. © 2005 Wiley Periodicals, Inc. J Comput Chem 26: 569–583, 2005
Double proton transfer and one‐electron oxidation behavior in double H‐bonded glycinamide–glycine complex in the gas phase
Authors: Li Ping , Bu Yuxiang
Published: February 2005

The behaviors of double proton transfer (DPT) occurring in a representative glycinamide–glycine complex have been investigated employing the B3LYP/6‐311++G** level of theory. Thermodynamic and especially kinetic parameters, such as tautomerization energy, equilibrium constant, and barrier heights have been discussed, respectively. The relevant quantities involved in the DPT process including geometrical changes, interaction energies, and deformation energies have also been studied. Analogous to that of tautomeric process assisted with a formic acid molecule, the participation of a glycine molecule favors the proceeding of the proton transfer (PT) for glycinamide compared with that without mediator‐assisted case. The DPT process proceeds with a concerted mechanism rather than a stepwise one because no zwitterionic complexes have been located during the DPT process. The barrier heights are 12.14 and 0.83 kcal/mol for the forward and reverse directions, respectively. However, both of them have been reduced by 3.10 and 2.66 kcal/mol to 9.04 and −1.83 kcal/mol with further inclusion of zero‐point vibrational energy (ZPVE) corrections, where the disappearance of the reverse barrier height implies that the reverse reaction should proceed with barrierless spontaneously, analogous to those of DPTs occurring between glycinamide and formic acid (or formamide). Additionally, the oxidation process for the double H‐bonded glycinamide–glycine complex has also been investigated. The oxidated product is characterized by a distonic radical cation due to the fact that one‐electron oxidation takes place on glycine fragment and a proton has been transferred from glycine to glycinamide fragment spontaneously. As a result, the vertical and adiabatic ionization potentials for the neutral complex have been determined to be about 8.71 and 7.85 eV, respectively, where both of them have been reduced by about 0.54 (1.11) and 0.75 (1.13) eV relative to those of isolated glycinamide (glycine) due to the formation of the intermolecular H‐bond. © 2005 Wiley Periodicals, Inc. J Comput Chem 26: 552–560, 2005
Computational methods for the study of enzymic reaction mechanisms III: A perturbation plus QM/MM approach for calculating relative free energies of protonation
Authors: Cummins Peter L. , Gready Jill E.
Published: February 2005

We describe a coupling parameter, that is, perturbation, approach to effectively create and annihilate atoms in the quantum mechanical Hamiltonian within the closed shell restricted Hartree–Fock formalism. This perturbed quantum mechanical atom (PQA) method is combined with molecular mechanics (MM) methods (PQA/MM) within a molecular dynamics simulation, to model the protein environment (MM region) effects that also make a contribution to the overall free energy change. Using the semiempirical PM3 method to model the QM region, the application of this PQA/MM method is illustrated by calculation of the relative protonation free energy of the conserved OD2 (Asp27) and the N5 (dihydrofolate) proton acceptor sites in the active site of Escherichia coli dihydrofolate reductase (DHFR) with the bound nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. For a number of choices for the QM region, the relative protonation free energy was calculated as the sum of contributions from the QM region and the interaction between the QM and MM regions via the thermodynamic integration (TI) method. The results demonstrate the importance of including the whole substrate molecule in the QM region, and the overall protein (MM) environment in determining the relative stabilities of protonation sites in the enzyme active site. The PQA/MM free energies obtained by TI were also compared with those estimated by a less computationally demanding nonperturbative method based on the linear response approximation (LRA). For some choices of QM region, the total free energies calculated using the LRA method were in very close agreement with the PQA/MM values. However, the QM and QM/MM component free energies were found to differ significantly between the two methods. © 2005 Wiley Periodicals, Inc. J Comput Chem 26: 561–568, 2005
Multiple acute effects on the membrane potential of PC12 cells produced by nerve growth factor (NGF)
Authors: Shimazu Kazuhiro , Takeda Kazuyo , Yu Zu‐Xi , Jiang Hao , Liu Xu‐Wen , Nelson Phillip G. , Guroff Gordon
Published: February 2005

We studied whether nerve growth factor (NGF) can affect the membrane potential and conductance of PC12 cells. We demonstrate that NGF depolarizes the membrane of PC12 cells within a minute and by using transfected NIH 3T3‐Trk and ‐p75 cells we show that both the high affinity NGF receptor p140trk and the low affinity NGF receptor or p75NGF may be involved in the depolarization. Tyrosine kinase inhibitor, K252a, partially inhibited the depolarization, but two agents affecting intracellular calcium movements, Xestospongin C (XeC) and thapsigargin, did not. The early depolarization was eliminated in Na+ free solutions and under this condition, a ‘prolonged’ (>2 min) hyperpolarization was observed in PC12 cells in response to NGF. This hyperpolarization was also induced in PC12 cells by epidermal growth factor (EGF). Voltage clamp experiments showed that NGF produced a late (>2 min) increase in membrane conductance. The Ca2+‐dependent BK‐type channel blocker, iberiotoxin, and the general Ca2+‐dependent K+ channel blocker, TEA, attenuated or eliminated the hyperpolarization produced by NGF in sodium free media. Under pretreatment with the non‐selective cation channel blockers La3+ and Gd3+, NGF hyperpolarized the membrane of PC12 cells. These results suggest that three different currents are implicated in rapid NGF‐induced membrane voltage changes, namely an acutely activated Na+ current, Ca2+‐dependent potassium currents and non‐selective cation currents. Published 2005 Wiley‐Liss, Inc.
The blood‐testis barrier and sertoli cell junctions: Structural considerations
Authors: Pelletier R.‐Marc , Byers Stephen W.
Published: February 2005

In this review, a few well‐established axioms have been challenged while others were viewed from a new perspective. The extensive literature on the blood‐testis barrier has been scrutinized to help probe its mechanics and hopefully to promote understanding of the constant adaptation of the barrier function to germ cell development. Our principal conclusions are as follows: (1) Although the barrier zonule is topographically located at the base of the seminiferous epithelium it actually encircles the apex of the Sertoli cell. Consequently the long irregular processes specialized in holding and shaping the developing germ cells should be considered as apical appendages analogous to microvilli. (2) The development of the barrier zonule does not coincide with the appearance of a particular class of germ cells. (3) The barrier compartmentalizes the epithelium into only two cellular compartments: basal and lumenal. (4) Although the blood‐testis barrier does sequester germ cells usually considered antigenic, immunoregulator factors other than the physical barrier seem to be involved in preventing autoimmune orchitis. (5) Structurally, a Sertoli cell junctional complex is composed of occluding, gap, close, and adhering junctions. The Sertoli cell membrane segments facing germ cells are presumably included in the continuum of the Sertoli cell junctional complex that extends all over the lateral and apical Sertoli cell membranes. (6) The modulation (i.e., formation and dismantling) of the junctions in a baso‐apical direction is characteristic of the seminiferous epithelium and may be dictated by germ cell differentiation. The formation of tubulobulbar complexes and the following internalization of junction vesicles conceivably represent sequential steps of a single intricate junction elimination process that involves junction membrane segments from different cell types as part of a continual cell membrane recycling system. (7) The preferential association of junctional particles with one or the other fracture‐face reflect a response to various stimuli including seasonal breeding. Changes in the affinity of the particles are generally coincidental with cytoskeletal changes. However, changes in the cytoskeleton are not necessarily accompanied by permeability changes. The number of strands seems to reflect neither the junctional permeability nor the transepithelial resistance. The diverse orientation of the strands seems to be related to the plasticity of the Sertoli cell occluding zonule. (8) Cooperation between all constituents (Sertoli cells, myoid cells, cell substratum, and germ cells) of the epithelium seems essential for the barrier zonule to function in synchrony with the germ cell differentiation. This cooperation ensures that the Sertoli cell barrier zonule is able to continually adapt to the changing requirements of spermatogenesis.
Nickel nitrate: A new junction permeability tracer for the study of the blood‐testis barrier
Authors: Cavicchia Juan C. , Sacerdote Fabio L. , Gutierrez Luis A.
Published: February 2005

With the purpose of evaluating a new intercellular tracer, nickel‐K ferrocyanide, we compared results yielded by lanthanum with information provided by nickel. This was done in the seminiferous epithelium of Holtzman rats of several postnatal ages and in a wild local seasonal breeder Galea musteloides. Tissues were studied with transmission electron microscopy and freezefracture replications. Nickel tracing proved to delineate cell contours more intensely and less interruptedly than lanthanum. With regard to seasonal variations in adult galea, the limits of the barrier were similar to those described in other mammals: spermatogonia, preleptotene, and leptotene spermatocytes were surrounded by the tracer in the basal compartment. The zygotenepachytenes were contained in the lumenal compartment and tracers were stopped at the inter—Sertoli cell tight junctions. During the inactive spermatogenic phase in winter, the seminiferous epithelium contained Sertoli cells and occasional germ cells, never beyond the spermatocyte stage. The tracer filled intercellular spaces, indicating that the barrier was incompetent. Some resting germ cells showed nuclear hyperchromasia, karyolysis, organelle loss, cell shrinkage, and cell fusion leading to a multinucleated cells. The inter‐Sertoli tight junctions were scanty and had randomly oriented and discontinuous junctional strands. Moreover, inter—Sertoli cell gap junctions proliferated. During the active spermatogenic phase in summer, junctions were numerous. Their junctional strands were parallel to each other, and continuous.
Ultrastructural diagnosis of human pituitary adenomas
Authors: Horvath Eva , Kovacs Kalman
Published: February 2005

Electron microscopy, which has been instrumental in the characterization of normal pituitary cell types, has also played a crucial role in the mirphologic classification pituitary adenomas arising in the presently known 5 cell types, and in the recognition of 3 adenoma types with yet undisclosed cell derivation. This review deals with the application of electron microscopy for study of pituitary adenomas in order to provide specific pathological diagnosis and aid the clinician in selecting appropriate postoperative treatment. In addition to the ultrastructural appearance and diagnostic features of 15 adenoma types, the morphology of hyperplastic proliferations and that of known normal counterparts of various adenoma types are also discussed. Specific morphologic diagnosis of pituitary lesions is important not only for adequate postoperative management of patient, but is also a prerequisite for study of the natural history and biological behaviour of various adenoma types.
Use of ultrastructural immunohistochemistry in human pituitary pathology
Authors: Sano Toshiaki
Published: February 2005

Recent advances in ultrastructural immunohistochemistry have provided insight into not only the subcellular localization of single antigens but also the colocalization of two distinct antigens in the same cellular constituent. In the field of pituitary pathology, precise identification of cell types, mechanism of processing, and dynamic intracellular transportation of hormones, as well as produciton of multiple hormones in the same cells of nontumorous and neoplastic adenohypophyses, have been documented by use of these techniques. The present review deals with the use of major methods for ultrastructural immunohistochemistry including pre‐, post‐, and non‐embedding methods, paricularly focusing on their application to human pituitary pathology. Problems of tissue processing and a protocol for double labeling technique using the protein A–gold complex are also described.
Effect of drugs on pituitary ultrastructure
Authors: Sager W.
Published: February 2005

Various drugs and hormones influence the light microscopic and especially the electron microscopic structure of the anterior pituitary and its tumors. Many structural effects are known only from animal experiment since specimens from human pituitaries are mostly not available. The structure of growth hormone (GH) Cells is relatively stable. A massive GH cell hyperplasia is known only in rare cases with growth hormone releasing factor (GRF) excess from tumors. Prolactin cells can be stimulated by drugs, neurotransmitters, and hormones which decrease the dopamine inhibition. Adrenocorticotropic hormone (ACTH) cells are stimulated by stress, some hormones, loss of adrenals, and drugs which activate the α1‐ and β‐redceptors or inhibit the α2‐receptors. They are suppressed and changed into Crook's cells by treatment with glucocorticoids. Thyroid‐stimulating hormone (TSH) cells increase in number and size in states of over‐stimulation especially by thyrotropin releasing hormone (TRH). A decrease results from hyperthyroidism and possibly from somatostatin, L‐dopa, and dopamine. Gonadotroph cells transform into castration cells in strongly hyperactive states (gonadectomy, antiandrogens, gonadotropin releasing hormone [Gn‐RH]agonists, aminoglutethimide). Special types of pituitary adenomas can be treated with drugs which suppress hormone production and proliferation. Dopamine agonists and somatostatin reduce the tumor size of varying proportions of GH secreting adenomas in acromegaly. Ultrastructurally, a decrease of cytoplasmic and nuclear volume and an increase of lysosomes are found. Bromocriptine and other dopamine agonists are establiched in the treatment of prolactin secreting adenomas. They induce a shrinkage in many cases. Ultrastructurally, a reduction of cellular and nuclear size, an increase in number of secretory granules and of lysosomes, and a reduction of rough endoplasmic reticulum can be demonstrated.
Improved structural detail in freeze‐fracture replicas: High‐angle shadowing of gap junctions cooled below −170°C and protected by liquid nitrogen—cooled shrouds
Authors: Rash John E. , Yasumura Thomas
Published: February 2005

In conventional freeze‐fracture replicas, precise complementarity of membrane faces is seldom achieved. In a model system frequently used to evaluate replica quality, vertebrate gap junctions are usually visualized as patches of 8–10 nm P‐face intramembrane particles separated by 1–2 nm spaces, while E‐face images are represented by 4–6 nm conical pits separated by 5–7 nm wide membrane ridges. However, that disparity in sizes of particles versus pits, as well as the disparity in the widths of the spaces separating particles versus pits, suggests that a significant reduction in complementarity of membrane faces has occurred. In this investigation, a JEOL JFD‐9000 freeze‐etch machine was modified so that fracturing and replication could be performed at temperatures much colder than commonly employed. With the addition of cryopumps to improve overall vacuum and the installation of optically tight LN2‐cooled shrouds surrounding the specimen and the knife, water vapor contamination arising from all sources within the vacuum chamber was reduced substantially, allowing replicas to be made at temperatures down to −185°C. With the specimen at these much colder temperatures, water vapor released by the heat of cleaving was also reduced significantly, providing additional improvement in replica quality. In addition, with higher shadowing angles (< 60°) and with the specimen at a much lower temperature, the grain size of the platinum film was noticeably reduced, thereby improving resolution at the molecular level. Under these improved conditions, replicas of rat liver gap junctions revealed that many of the P‐face IMPs were tubes 6–7 nm in diameter, but that other IMPs had been stretched and distorted by the fracturing process. More important, however, these high resolution replicas revealed that the replicas of the E‐face pits represented three‐dimensional molecular casts of the transmembrane proteins comprising the connexon hexamer. This means that before they were replicated, the E‐face pits faithfully maintained the shape that the IMPs had before fracturing. These more detailed images revealed a new structure in the center of each E‐face pit: a 2–3 nm “peg” that may represent the frozen aqueous matrix of the connexon ion channel that remained after elastic extraction of the surrounding six connexin molecules. Thus, high‐angle shadowing at very low specimen temperature under virtually non‐contaminating conditions has revealed a new level of detail for membrane structure in freeze‐fracture replicas.
Effect of a 2‐month light cycle regimen on testicular parameters of adult Ile‐de‐France rams
Authors: Hochereau‐de Reviers M. T. , Perreau C. , Pisselet C. , Pelletier J.
Published: February 2005

This experiment was conducted in Ile‐de‐France adult rams to examine the target point of a 2‐month light cycle regimen on seminiferous tubule functions, on intertubular compartment and on Leydig cell parameters. Eight rams were subjected to a 2‐month light cycle regimen and were compared to sexually active or inactive rams. In light‐treated rams, testis weight was maintained equal or was higher than that of sexually active rams. Both tubular and intertubular tissues were found significantly higher in light‐treated than in sexually active rams. The mean ratio of basement membrane area of the seminiferous tubules per Sertoli cells and the daily productions of A1 spermatogonia and of leptotene primary spermatocytes were significantly increased in light‐treated rams as compared with sexually active or inactive rams. Meanwhile, the dairy productions of diplotene primary spermatocytes, of round spermatids, of spermatozoa and of the rete testis fluid were not significantly increased in light‐treated as compared with sexually active rams but significantly greater than those of sexually inactive rams. Total volume, total numbers, and individual volumes of Leydig cells were at least equal or higher in light‐treated than in sexually active rams.
Medium temperature epoxy resin for immunocytochemistry: Quetol 651 with water
Authors: Abad André R.
Published: February 2005

The addition of 1% water to the epoxy resin Quetol increased the labeling intensity of the sample. The significant decrease of the curing temperature of the epoxy resin may assist in preservation of antigens. Water may also reduce the cross‐linkage of the resin allowing more antigen to be available to the antibodies. The modified Quetol resin is an option for use in immunocytochemistry studies.
Ultrastructural distribution of the M form of creatine phosphokinase in human muscle by immunogold labeling
Authors: Dankert John R. , Papadi George P. , Shields Robert P.
Published: February 2005

Creatine phosphokinase regenerates ATP from ADP using creatine phosphate. Isoenzymes of creatine phosphokinase are bound to certain cellular structures or are compartmentalized in areas of the cell, and this has been used as a basis for defining the role of these isoenzymes in energy metabolism. The M isoenzyme of creatine phosphokinase has been morphologically associated with the M‐line of striated muscle in many species. In this present study the ultrastructural distribution and the relative concentration of the M form of creatine phosphokinase in human muscle tissue was determined using immunogold and electron microscopy. The M‐line of the sarcomere, comprising only 3—4% of the sarcomere area, was found to contain over 20% of the total M isoenzyme signal of the entire sarcomere. This technique represents a quantitative, ultrastructural method to study the subcellular distribution of this isoenzyme. These data suggest that localized concentrations of M‐CPK may be important for normal energy metabolism, and may also serve as a foundation for a better understanding of the relationship between abnormal creatine metabolism and the pathogenesis of neuromuscular disease.
Atypical cells in the normal guinea pig organ of Corti as revealed by micromanipuiation in SEM
Authors: Reiss Gebhard
Published: February 2005

Inner ear tissue of the normal guinea pig was conductively stained (OTOTO‐method) for SEM investigations. The Hensen's cells of the organ of Corti were removed using a micromanipulator inside the SEM. By this method atypical bodies of sensory and supporting cells were revealed in the apical turns of the cochlea. Atypical sensory cells showed great variations in size and shape. Several had no contact to Deiter's cells and no or only one nerve supply at their basal end. Atypical Deiter's cells showed alterations in shape and in the form of their phalangeal processes.

Additionally altered parts of the organ of Corti were isolated by micromanipulation and embedded for correlative TEM‐investigations.
Intercellular junctions in embryonic chick cardiac muscle revealed by rapid freezing and freeze‐substitution
Authors: Shiozaki Masayuki , Shimada Yutaka
Published: February 2005

Using the method of rapid freezing and freeze‐substitution, the embryonic chick cardiac muscle was investigated by transmission electron microscopy. Initially, the intercellular junctional complexes (fasciae adherentes and desmosomes) were formed in close proximity to each other along a nearly straight line. Subsequently, the separation of fasciae from desmosomes took place to form intercalated discs. The cell membranes of fasciae adherentes were reinforced with highly interwoven fine fibrils at which myofibrils terminated. The intercellular space of fasciae was bridged with fine fibrillar structures seemingly connected by a thin line at their middle portions. In the intercellular space of desmosomes, central lamina and traversing filaments were clearly observed. The outer and inner leaflets of the desmosomal plasmalemma were asymmetrically differentiated; the outer leaflet was thinner than the inner leaflet. On the inner side of the cell membrane, an electron‐lucent layer and a dense desmosomal plaque were observed. The latter structure had protrusions with less electron density towards the cytoplasmic side. Further inside, a meshwork of fine fibrils was seen along and toward which bundles of intermediate filaments ran. The results obtained with freeze‐substitution appeared to provide more information than those with thin sections after conventional fixation or with replicas of chemically fixed/glycerinated or physically fixed/deep‐etched materials.
Bloch wave treatment of symmetry and multiple beam cases in reflection high energy electron diffraction and reflection electron microscopy
Authors: Peng L.‐M. , Gjønnes K. , Gjønnes J.
Published: February 2005

Bloch wave equations for the multiple beam cases in reflection high energy electron diffraction (RHEED) are derived from the integral equation by forward‐ and back‐scattering Green function operators. A linearization is achieved through separation in a forward‐ and a back‐scattering component for each beam. This leads to a set of fundamental equations similar to the transmission case, but with a non‐Hermitian matrix, and the beams may be entered as either forward‐, back‐scattered, or both. The number of beams needed to be included in RHEED calculations is thus reduced, and so are the computing time and computer space required. The systematic row case, corresponding to reflections from planes parallel to the crystal surface, is treated in detail and illustrated by calculations of dispersion surfaces and rocking curves for Au(001). Symmetry relations for the systematic row and between reciprocal rows are discussed and illustrated.
Developments in the dynamical theory of high energy electron reflection
Authors: Ma Y. , Marks L. D.
Published: February 2005

High energy electron reflection (HEER) is an important technique in surface science and uses the information carried by high energy electrons reflected from surfaces to study surface structures and surface electronic states. With the development of reflection high energy electron diffraction (RHEED), high energy electron microscopy (REM), and high energy electron energy loss spectroscopy (EEL) in surface science, the usefulness of HEER has been widely recognized and demonstrated. However, a stationary dynamical solution for an arbitrary surface for HEER has not been obtained yet.

In this paper, some developments in understanding the dynamical theory of HEER, particularly in recent years, are reviewed:

The introduction of the concept of current flow for a semi‐infinite crystal model has removed the confusion around the wave points in the “band gap”.

The consistency between the Bloch wave and multislice in the Bragg case has verified the validity of the argument of current flow and led to the emergence of the BMCR method (Bloch wave + Multislice Combined for Reflection).

The failure of the Bloch Wave‐Only solution (the BWO solution) on Au (110) surfaces in the Bragg case revealed by the BMCR method implies that previous BWO calculations in the Bragg case might be at fault.

The 2‐D dependence of the electron wave fields and Picard iteration‐like character of multislice calculation in the Bragg case has led to the emergence of an Edge Patching method in Multislice‐mode‐Only (the EPMO method). The new method yields an infinitely convergent stationary dynamical solution for an arbitrary surface.
Characterization of the annealed (0001) surface of sapphire (α‐al2o3) and interaction with silver by reflection electron microscopy and scanning reflection electron microscopy
Authors: Ndubuisi Godfrey C. , Liu J. , Cowley J. M.
Published: February 2005

Annealed (0001) surfaces of single‐crystal sapphire (α‐Al2O3) rod have been studied in the electron microscope using reflection electron microscopy (REM), scanning reflection electron microscopy (SREM), and reflection high energy electron diffraction (RHEED). Annealed surfaces of (0001) sapphire are vicinal and characterized by close‐packed (0001)‐oriented terraces separated by faceted multiple‐height steps, with edges parallel to energetically preferred low‐index directions (<101―0> and <112―0>). These structural features are not seen on cleaved surfaces or polished surfaces treated at temperatures < 1,250°C. Oxygen‐annealing produces clean surfaces which prove useful for investigating the interaction of deposited metals with the (0001) sapphire. Both REM and SREM (with microdiffraction spots) techniques have been used to observe fine structure of flat Ag islands on the scale of 1—100 nm on the (0001)‐oriented terraces as well as aggregations at the steps. A preliminary result on interaction with Cu is also included.
Flat embedding and immunolabelling of SW 1116 colon carcinoma cells in LR white: An improved technique in light and electron microscopy
Authors: Goping Gertrud , Yedgar Saul , Pollard Harvey B. , Kuijpers Gemma A. J.
Published: February 2005

Human SW 1116 colon carcinoma cells were grown on matrix‐covered coverslips and flat embedded in specially prepared gelatin capsules in the hydrophylic resin LR White. Dehydration and polymerization were carried out so as to maximize preservation of antigenicity. Sections were cut perpendicular to the substratum. To visualize mucin, semithin sections of SW 1116 cells were stained with periodic acid Schiff (PAS) reagent for light microscopy, and ultrathin sections were labelled with a monoclonal mucin antibody (Mab 19‐9) and immunogold for electron microscopy. Immunofluorescence was carried out on whole cultured cells using Mab 19‐9. The morphological preservation of SW 1116 cells embedded in LR White was comparable to that of Epon‐embedded cells. Mucin was localized on the microvillar surface of the apical plasma membrane and occasionally in intercellular spaces between adjacent cells. Mucin was also present in vesicles in the apical and lateral part, and to a lesser extent in the basal part of the cells. We conclude that this new technology significantly improves the morphological preservation of cells and tissues in LR White, while also serving to sustain the antigenicity of cellular antigens.
Stepped surfaces of sapphire (α‐Al2O3) with low miller indices
Authors: Ndubuisi Godfrey C. , Liu J. , Cowley J. M.
Published: February 2005

Oxygen‐annealed surfaces of sapphire with low Miller indices ((0001), {1010}, {1120}, {1011}) have been studied in both transmission electron microscopy (TEM) and reflection electron microscopy (REM) configurations. The significance of REM diffraction conditions for the determination of the nature of the step heights is discussed. The relationship between the TEM and REM images is explained. The structural features are those that might be expected from considerations of the atom arrangement in the low Miller index planes. The structural features on the surfaces varied with respect to annealing temperature and surface condition. Thermally stable structures that might appear from consideration of the equilibrium‐annealing temperature are proposed.
Acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: Comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine
Authors: Edwards Harold H. , Yeh Yu‐Yan , Tarnowski Betty I. , Schonbaum Gregory R.
Published: February 2005

Tissue processing for transmission electron microscopy (TEM) is commonly accomplished using ethanol (EtOH) as a dehydrating solvent and propylene oxide (PO) as a transition fluid. Both solvents have some undesirable properties: EtOH solubilizes lipids; PO is highly flammable, volatile, toxic, and potentially carcinogenic. Their replacement by a compound devoid of these characteristics is therefore desirable. Acetonitrile (AN) appears to be such a solvent. It is freely miscible with water, alcohols, acetone, and epoxy resins; it does not interfere with epoxy polymerization; and the resulting cured resins have excellent cutting quality and beam stability. AN is also an excellent dehydrating agent whose use does not necessitate modification of current techniques. Most importantly, the low solubility of phospholipids (PL) in AN limits the loss of membrane lipids and, hence, leads to a better preservation of tissue features.
Observation by transmission electron microscopy of etch figures obtained on an organic molecular crystal
Authors: Mercier Michel , Raimi Mohamed Karim , Bonpunt Louis
Published: February 2005

It is interesting to apply the method of etch figures to the study of organic molecular crystal defects, by observing the etch pits as soon as they are produced. We have set up a method to determine the geometrical forms of such small etch pits, observed on pre‐shadowed replicas of naphthalene crystal surfaces. The described experimental procedure was designed to avoid artefacts due to vacuum sublimation and moisture traces on the replicated surface. Stereoscopic observation makes interpretation possible. The 3‐D morphology and size of etch figures smaller than 1 μm can be determined.
Pinealocyte synaptic ribbons and neuroendocrine function
Authors: McNulty John A. , Fox Linda M.
Published: February 2005

A comparative study of pinealocyte synaptic ribbons (SR) revealed two predominant populations exhibiting either a rod/ribbon shape (SRr) or a spherical/punctate shape (SRsp). Species‐specific differences were found in the abundance of SR, the ratio of SRr/SRsp, and the occurrence of SR in ribbon fields. The close topographical relationship of SR to the plasma membrane and the numerical changes that occurred with changes in metabolism of the pinealocytes suggest that SR have important vesicle‐mediated interactions with the cell membrane. Experiments designed to clarify the relationship between SR and pineal neuroendocrine function revealed a positive correlation between SR numbers and indole intermediates during pineal development in the rat, and increased SR frequency after denervation of the rat pineal gland or administration of the β‐adrenergic agonist, isoproterenol. These data are consistent with the hypothesis that SR function is linked to receptor mechanisms regulating indoleamine production in the pineal gland. © 1992 Wiley‐Liss, Inc.
Third ventricular pineal gland grafts in the gerbil: An electron microscopical and immunohistochemical investigation
Authors: Welsh Marcia G. , Li Kang
Published: February 2005

The superficial pineal gland was grafted into the third ventricle of adult mongolian gerbils. Donor pineal glands from both neonatal and 3–4 week old gerbils were able to survive for at least 6 months. The pinealocytes of the grafted superficial pineal glands maintained the morphology and the S‐antigen immunoreactivity of the in situ pineal complex. Synaptic ribbons and spherules were present but rare. Unlike the in situ pinealocytes, glycogen accumulations were common in the graft pinealocytes. Site specific modulation of structure was indicated as the ventricular surface of the grafts became covered with cerebrospinal fluid (CSF)‐contacting pinealocytes typical of those seen in the deep pineal. The CSF‐contacting pinealocytes of the graft had numerous processes that extended along the ventricular surface of the graft. The blood vessels of the grafts had non‐fenestrated endothelium and wide perivascular areas typical of those seen in the in situ pineal complex. Tyrosine hydroxylase‐immunopositive nerve fibers were present in the grafted tissue indicating reinnervation of the graft. The source of the fibers was not determined. The nerve fibers were present both within the perivascular area and within the parenchyma where they were associated with pinealocytes. The results demonstrate that the cerebral ventricles are an ideal location for the survival of superficial pineal gland grafts. It is suggested that pineal grafts may be a means to further study pineal development and innervation. © 1992 Wiley‐Liss, Inc.
Quantitative perimeter and area measurements of digital images
Authors: Barba Joseph , Chan Kin S. , Gil Joan
Published: February 2005

Quantitative estimations of perimeter and area from digitized video images, and the application of these features in morphometry, are discussed. Estimations from manual tracings via interactive peripherals and from chain codes are addressed. Topics discussed are calibration, determination of vertical and horizontal pixel resolution, effects of tracing jitter, and for chain codes, the spatial quantization scheme representation of the digital contour. Finally, new perimeter estimators for 4‐connected and 8‐connected chain codes for non‐unity pixel aspect ratio are presented with simulation results.
Quantitative morphology for biologists and computer scientists: I. Computer‐aided tutorial for biological stereology (version 1.0)
Authors: Bolender Robert P.
Published: February 2005

This paper describes a computer‐aided tutorial for biological stereology. Stereology, a type of quantitative morphology, includes a collection of statistical methods that quantify the structural compartments that can be viewed in sections with light and electron microscopy. These methods provide volume, surface, length, shape, and number data, and help define the quantitative relationships among the structural compartments of biological hierarchies. Hierarchies, which connect structural data ranging in size from molecules to organs, serve as a central core to which the data of biological databases can be linked. The tutorial focuses on two objectives. It provides the user primarily interested in using quantitative morphology databases with background information, and offers a set of state‐of‐the‐art tools to researchers wishing to use these methods in the laboratory. The main topics of the tutorial include: introduction to quantitative morphology, symbols/terms, data types, sampling, hierarchies, data interpretation, and utilities. The tutorial runs under the MS‐DOS operating system and requires at least an IBM PC AT (or compatible), a color monitor (EGA, VGA), 540 KB of RAM, and 3 MB of hard disk space. © 1992 Wiley‐Liss, Inc.
Deployment of extracellular matrix proteins in sea urchin embryogenesis
Authors: Alliegro Mark C. , Black Steven D. , McClay David R.
Published: February 2005

The apical extracellular matrix of the sea urchin embryo, known as the hyaline layer (HL), is a multi‐laminate organelle composed of at least 10 polypeptides. Although integrated into one ECM, HL proteins exhibit individual temporal and spatial dynamics throughout development. These molecules are stockpiled in the oocyte during vitellogenesis in at least four distinct vesicle populations. They are released onto the cell surface at fertilization in a specific order, and interact differentially with embryonic cells as development proceeds. Many experiments have suggested that the HL is vital for embryogenesis, but relatively little is known about the functions and interactions of its constituent molecules. The purpose of the present review has been to gather information on the basic characteristics of the known HL proteins together with data on their expression in the embryo, and where possible, their biological activities. Compiled, these observations may provide some insight into the workings of a uniquely embryonic organelle. © 1992 Wiley‐Liss, Inc.
Cell lineage in molluscan development
Authors: Dohmen R. M.
Published: February 2005

Cell lineage specification in molluscs is brought about by two mechanisms: the segregation of morphogenetic plasms and inductive cell interactions. The evidence for the existence of morphogenetic plasms is largely circumstantial, but in one species, Bithynia, such a plasm has been identified in the polar lobe that forms at first cleavage. Inductive cell interactions are thought to be a prerequisite for the development of a large number of tissues and organs. The most extensively studied example is the specification of the mesodermal stem cell in Lymnaea and Patella, which occurs between 5th and 6th cleavage through an interaction between one macromere and a large number of micromeres.

Both segregation and induction are tuned to the animal‐vegetal polarity of the egg, at least during early development. This polarity probably arises during oogenesis and is manifest in regional differentiations of the surface architecture of the egg, in the distribution of inner membrane particles in the plasma membrane, in membrane fluidity characteristics, in ionic conductance properties of the plasma membrane, etc. All these phenomena have in common that they represent properties of the egg surface, suggesting that the polarity of the egg is somehow imprinted into the plasma membrane and the cortex of the egg during oogenesis. © 1992 Wiley‐Liss, Inc.
Role of ultrastructural studies in the analysis of cell lineage in the mammalian pre‐implantation embryo
Authors: Cruz P. Y.
Published: February 2005

Ultrastructural studies have contributed significantly to our understanding of cell lineage differentiation in the mammalian pre‐implantation embryo. Such studies have documented, and continue to document, morphological, biochemical, and physiological characteristics of the cell lineages established during the pre‐implantation period in eutherian embryos, principally that of the mouse. This review evaluates these contributions and identifies areas of study in which ultrastructural analysis is most likely to have an important role in the future. © 1992 Wiley‐Liss, Inc.
Chemical extraction of the cytosol using osmium tetroxide for high resolution scanning electron microscopy
Authors: Lea P. J. , Hollenberg M. J. , Temkin R. J. , Khan P. A.
Published: February 2005

Detailed examination of subcellular structures in three dimensions (3D) by high resolution scanning electron microscopy (HRSEM) is now possible due to improvements in the design of the scanning electron microscope and the introduction of methods of specimen preparation using chemical removal of the cytosol and cytoskeleton by dilute osmium tetroxide. Cells which have been fixed, frozen, cleaved, thawed, and subjected to cytosol extraction display intact intracellular structures in 3D including nuclear chromatin, endoplasmic reticulum, mitochondria, and the Golgi complex at a resolution close to that of conventional biological transmission electron microscopy (TEM). Small changes in the 3D structure of subcellular components can be conveniently examined in this way in development, in a variety of physiological processes and in disease. Broad areas of the specimen can be quickly surveyed by HRSEM since sectioning is not required and specimens of comparatively large size (up to 5 mm3) can be placed in the microscope. Extraction of the cytosol with dilute osmium tetroxide (OsO4) exposes subcellular structures in relief, permitting their examination in 3D from several aspects. However, the OsO4 extraction technique is limited, since significant intracellular structures, such as the cytoskeleton, vesicles, and antibody binding sites can be removed or inactivated during the cytosol removal steps. © 1992 Wiley‐Liss, Inc.
Determination of conditions for optimum resolution of a high angle thin window energy dispersive spectrometer
Authors: Blackford Mark G. , Smith Katherine L.
Published: February 2005

The energy resolution of an energy dispersive spectrometer (EDS) equipped with an ultrathin window (UTW) and mounted at a high take‐off angle (72°) on a transmission electron microscope has been studied under a variety of operating conditions. The spectrometer resolution is close to that specified by the manufacturer, up to count rates of 400 cps. Above 400 cps the resolution deteriorates rapidly, and the MCA dead time and zero width increase. Above 10 ke V, the height of the background is much greater than expected for bremsstrahlung and shows the shape which has previously been attributed to backscattered electron flux into the detector. It is postulated that the deterioration in resolution with count rate is caused by backscattered electrons reaching the detector through the UTW. © 1992 Wiley‐Liss, Inc.