bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative electron microscopic study on mitochondria of smooth muscle in piqixuzheng rat
Authors: Liu Hepei , Hou Yanming , Chang Xiaofeng , Wang Shulan
Published: June 2020

Johnson stated that man has always been interested in the structure of things in the world around him. In this regard, the electron microscopy, a modern technique, is used for research in traditional Chinese medicine. The terms and theories of TCM are quite different from those of Western medicine. The theory of TCM indicates that pi (spleen in TCM) has important roles in human health. According to the basic theory of TCM, one such function of the pi is nourishing the muscles. A significant decrease in the number of mitochondria with normal structure and a significant increase in the number of mitochondria with abnormal structure in skeletal muscle of piqixuzheng (spleen energy deficiency in TCM) rats have been reported. The present work is a quantitative electron microscopic study on mitochondria of smooth muscle in piqixuzheng rats.

Male Wistar rats were used in this study. The piqixuzheng rats were produced by treatment with houpo (Magnolia officinalis), Zhishi(Fructus aurantii immaturus), dahuang (Radix et rhizoma) and fasting day other day for six weeks.
A cell biological study of the micropylar apparatus in dipterans of economic importance
Authors: Zarani Flora E. , Margaritis Lukas H.
Published: June 2020

The micropylar apparatus is a specialized region of the eggshell, serving as a passage (micropylar canal) to the spermatozoon. The entire eggshell is secreted by the follicle cells during oogenesis, in successive layers, in the following order: vitelline membrane, wax layer, innermost chorionic layer, endochorion, exochorion. This study is an E.M. investigation of the structure and morphogenesis of the micropylar apparatus at the follicles of Dipterans of economic importance, such asCeratitis capitata,Dacus oleaeandRhagoletis cerasi. These insects affect the fruits of orange, olive and cherry trees respectively.

The micropylar apparatus of the DipteransC. capitata,D. oleaeandR. cerasi. forms a protrusion at the anterior part of the egg. This apparatus consists of the vitelline membrane (Vm), which is surrounded by the wax layer (wl); the innermost chorionic layer (icl); the endochorion (En); and the exochorionic “tuft”. The endochorion has many cavities which probably take part in the air storage, in order to facilitate the embryo’s respiratory needs (Figs. 1,3,5). The micropylar canal is covered by the characteristic tuft, resulting in the vitelline membrane, where it becomes thinner and forms the pocket.
Cuticular sensory structures on the legs of Orthetrum sabina (Odonata: Libellulidae) as revealed by SEM
Authors: Gupta Abhik , Dey Sudip , Gupta Suswita
Published: June 2020

Occurrence and distribution of various cuticular sensory structures such as Sensilla chaetica, S. trichoidea, S. basiconica, pore sensilla, hairplates, S. campanifonuia, microtrichial structures, as well as unique hemispherical protuberances and flattened branched setae are presented. Their morphology is described, and size ranges recorded.

As seen in figures 1 and 2, the coxa has two hairplate like structures, each comprising a group of trichoid sensilla with blunt and slightly bulbous tips. The femur has minute basiconic pegs, and pores as well, scattered on its surface(figure - 3 ). The tibial surface shows a profusion of unique flattened and branched setae (figure - 4 ). Similar setae are also present along the outer margin of the tarsus (figure - 5) which also bears a number of hemispherical protuberances(figure - 6 ).
Three-dimensional construction of the kinetosomal complex in a ciliate Blepharisma, revealed by isolation technique and serial sectioning
Authors: Shigenaka Yoshinobu , Ishida Masaki , Suzaki Toshinobu
Published: June 2020

In a heterotrichous ciliate, Blepharisma japonicum, pairs of basal bodies (kinetosomes) and their fibrous associates containing microtubular sheets and bundles are closely related to cell elongation in response to light stimulation. Up to date, however, three dimensional construction of them has not been clarified because of their own complexity and difficulty of establishment of their isolation technique. By applying the newly devised isolation medium, however, they were found to be isolated fruitfully from the cell bodies. The present isolation medium was composed of 10 mM MgSO4, 10 mM EGTA, 1 mM dithiothreitol, 32 mM sucrose, AH Triton X-100, 20% glycerol, 100 mM HEPES and 20 mM PIPES adjusted to pH 7.2 with KOH. The isolated complexes were mounted onto Formvar-coated grid, fixed briefly with 2% glutai-aldehyde (GA) buffered with 0.1 mM sodium cacodylate at pH 7.2, rinsed with deionized distilled water and finally stained with 1% aqueous uranyl acetate (UA). For thin sectioning, on the other hand, the organisms were fixed for 10 min with a mixture of 2.5% GA and 1% OsO4 in cacodylate buffer (pH 7.2) at room temperature.
A trial of 3-D observation on the ultrastructure of human enamel crystals
Authors: Ichijo Takashi , Yamashita Yasuo , Terashima tatsuo , Nagata Humio , Okamura Sadahiko , Tsuruta Chusei
Published: June 2020

Recently it has become generally agreed that tooth and bone crystals consist principally of hydroxyapatite, Ca10(PO4)6(OH)2. Over several years, we have been pursuing observations on apatite crystals composing the teeth and bones at the level of their atomic configuration using transmission electron microscope for the purpose of examining their structural features of biological crystals. In this study, observations using the same approach were conducted a higher resolution than before. In this report, because of the highest resolution ever obtained that, crystals could be observed at a level of ⅙ lattice (0.136 nm intervals). Using this approach, subsequentry, the possibility of the three-dimensional observation on the configration composing hydroxyapatite structure in the unit cells was pursued. The crystals composing teeth and bone known to have slight curves and twists. The authors paid attention to this fact, and attempted to three-dimensionally observe the portion where the c axis of the crystal unit cells tilt slightly due to some proper curving of the crystals.
Ultrastructural study of cytoskeletal interactions with endosomes in macrophages by quickfreezing and deep-etching method
Authors: Baba Takeshi , Shiozawa Nobuki , Hotch Masao , Ohno Shinichi
Published: June 2020

Endosomes are vesicular or tubular organelles that play important roles in transports of receptors and receptor―bound ligands during receptor-mediated endocytosis. The mechanisms of endocytic transports from clathrin-coated pits to lysosomes have been studied by many investigators. However, few studies were reported about the interactions between endosomes and cytoskeletons. In this study, Fc-receptor-mediated endocytosis in macrophages are investigated by quick-freezing and deep-etching (QF-DE) method combined with gold-labeled immune complex and “replica scraping method”.
Contributions of electron microscopy towards the understanding of flavivirus replication process
Authors: Lee Ng Mah
Published: June 2020

The techniques of electron microscopy was extensively used in our study on flavivirus replication. The initial event of attachment and entry of the virus was visualised (Fig 1). It revealed that virus (Vi) particle attachment (double arrow) was most likely receptor-mediated. Entry was through clathrin-coated endocytotlc vesicles (Eve). Uncoating of the virus envelope (short arrows) seemed to occur within the endocytotic vesicles.

After entry and during replication, there are several virus-induced changes (Fig 2). Three most prominent features are the formation of vesicles (Ve), the microtubule paracrystals (Pc) and the proliferation of the endoplasmic reticulum (ER). These components are always in close vicinity to each other and could be the replication complex for the virus.
ElevatedCandida albicansscores obtained by microscopic examination of subgingival plaque from atypical periodontal disease sites in immunocompromised states (especially AIDS)
Authors: Hanker J. , Webster W.P. , Burkes E.J. , Greco G. , Hamamoto D. , Hopfer R.L. , Kutcher M. , Yates P. , Beck J. , Anderson E. , Giammara B.
Published: June 2020

Gingivitis and periodontitis have immunologic aspects that can influence or be influenced by the pathogenic microbiota present. In immunocompromised states, especially AIDS, an abnormal gingivitis (HIV-G) and periodontitis (HIV-P) are known. Although acute periodontal lesions generally respond to conventional therapy, in AIDS patients the atypical lesions may also be present which deteriorate further.Candida albicans(CA) lesions of the oral mucosa (not due to diabetes, cancer chemotherapy, radiation therapy or antibiotics) were present in almost all AIDS patients and were helpful in defining this disease. In 1988 it was shown that CA could be cultured from a greater percentage of the atypical than the conventional gingivitis or periodontitis sites in AIDS patients but this required many hours and could not show the relative number of CA present in the different sites.
Germination and multiplication of Bacillus thuringiensis var. kurstaki in the midgut of Pieris canidia larva: An Electron Microscopic study
Authors: Cheung Wilkin W.K. , Lam Yui F.
Published: June 2020

Presently Bacillus thuringiensis var. kurstaki (Btk, thuricide) is a popular microbial insecticide on market for controlling lepidopterous pests. The damage of Btk on lepidopterous larvae have been well documented. However, practically few references give sufficient ultrastructural details on the germination and multiplication of Bt spores and bacterial cells after infecting the larvae. The present study is aimed to fill the gap of the necessary information.

Pieris canidia L. larvae were immersed in 2.5% (w/v) thuricide (40,000 IU, Sandoz Co.) spore-crystal suspension for 5 sec. dip and were put onto petri dishes for observation in various time intervals after blotted dry with filter paper. Control larvae had not been treated with Btk.
Use of a computerized image analysis program to statistically characterize the ultrastructural features of chloroplasts
Authors: Wergin William P. , Pooley Christopher
Published: June 2020

During normal plant growth, the development and structure of the chloroplast is largely influenced by genetics. However, environmental and biological conditions such as light (intensity and quality), temperature, draught, herbicides, nutrition, pollutants and pathogens also affect the ultrastructure of this organelle. As a result, fine-structural investigations of higher plants frequently describe the effects of experimental treatments on the relative size of the chloroplast, as well as on the occurrence, size and number of its internal structures, which include the starch and osmiophilic bodies and the grana and stroma lamellae. These descriptions require either a tedious examination of numerousorganelles in an effort to gain a subjective impression of the changes that have occurred or a painstaking effort of counting, measuring and averaging the various structural features to provide a statistical representation of the alterations. In an effort to gain objective data in a more efficient manner, a software program was written in Quips, a Pascal based programming language. This program, whichis used on a Cambridge Quantimet 970 image analysis system, scans the electron micrograph and assigns 256 grey levels to the structural elements. This process enables one to isolate and to measure the structural features of the chloroplast.
Correlation of ultrastructure with the mechanism of wax filament production in cork cells in Johnsongrass [Sorghum halepense (L.) Pers]
Authors: Paul R. N. , McWhorter C. G.
Published: June 2020

Wax filament production has been previously reported to be a secretory function of costal and intercostal cork cells in johnsongrass leaf and sheath epidermis. The “waxy bloom” observed on the sheath of johnsongrass during certain stages of plant development is the result of massive quantities of long wax filaments (>200 um) deposited on the sheath cuticle. SEM investigations of intact and dewaxed sheath tissue have indicated that wax filaments are the result of extension of material through cuticular pores. These pores are observed in the cork cell cuticle in dewaxed tissue. The purpose of this investigation is to correlate cork cell ultrastructure with the process of wax filament production through the use of a procedure maximizing lipid retention in epoxy-resin embedded materials.

Johnsongrass tissue used in this study was taken from greenhouse-grown plants ranging from 10 cm to 20 cm in height. Sheath tissue subtending the youngest leaves was used.
Portein-gold labelling of ultrathin sections of wheat spot mosaic-affected wheat plants
Authors: Zaychuk K. S. , Chen M. H. , Hiruki C.
Published: June 2020

Wheat spot mosaic (WSpM), which frequently occurs with wheat streak mosaic virus was first reported in 1956 from Alberta. Singly isolated, WSpM causes chlorotic spots, chlorosis, stunting, and sometimes death of the wheat plants. The vector responsible for transmission is the eriophyid mite,Eriophyes tulipaeKiefer. The examination of leaf ultrastructure by electron microscopy has revealed double membrane bound bodies (DMBB’s) 0.1-0.2 μm in diameter. Dispersed fibrils within these bodies suggested the presence of nucleic acid. However, neither ribosomes characteristic of bacteria, mycoplasma and the psittacosis group of organisms nor an electron dense core characteristic of many viruses was commonly evident.

In an attempt to determine if the DMBB’s contain nucleic acids, RNase A, DNase I, and lactoferrin protein were conjugated with 10 nm colloidal gold as previously described. Young root and leaf tissues from WSpM-affected wheat plants were fixed in glutaraldehyde, postfixed in osmium tetroxide,and embedded in Spurr’s resin.
Cytochemical and immunocytochemical localization of the endosperm cells of ginseng
Authors: Kim Eun-Soo , Yu Seong-Cheol , Kim Woo-Kap
Published: June 2020

Storage material of endosperm cells digested by various enzymes is transported to the embryo. At this time, the cellulose of the endosperm cell wall is guessed to be hydrolyzed by the cellulase in order to help the storage material to move from the endosperm cell to the embryo. Therefore, this study has been carried out to investigate the localization of the cellulase activity on the endosperm cells of Panax ginseng C.A. Meyer with the cytochemical and immunocytochemical techniques during germination.

Ripened red fruits of 4-year-old ginseng were collected at the cultivated field in Korea. After the sarcocarps were removed, the seeds were mixed with sands at the ratio of 1 to 5 to split the seed coat along the raphe. They were sown on moist filter paper in petri dishs and kept in the dark at 4 C. For the cytochemistry, tissues were fixed in Karnovsky fixative at 0 C for 1 hr, rinsed in 0.1 M phosphate buffer, pH 7.2, and stored in this buffer at 0 C for 16 hr.
Cytoskeleton in tobacco plant cells after propane jet freezing and freeze substitution
Authors: Ding Biao , Turgeon Robert , Parthasarathy M.V.
Published: June 2020

Microfilaments in plant cells are very labile and in some cells difficult to preserve by chemical fixation. Single microfilaments are even more labile and are rarely preserved. Cryofixation is so far the best means to preserve these labile cellular components for electron microscopy. However, previous studies only dealt with single plant cells because of the shallow depth of good freezing limited by the technique (plunge freezing). Here we report our results of preserving cytoskeleton in plant leaf and root cells using propane jet freezing and freeze substitution.

Tobacco (Nicotiana tabacum L. var. Maryland Mammoth) plants were grown under greenhouse conditions. A developing leaf was cut from a two-month old plant and immersed in a 0.2 M sucrose solution containing 20 mM MES, 20 mM CaCl2 and 20 mM KCl. Samples of 1 mm2 were cut out of the leaf and incubated for 10 to 30 minutes. The leaf samples were then frozen in a commercial propane jet-freezer (Model MF7200, KMC, Tucson, Arizona).
Cathorops spixii (Pisces) gill study through Scanning Electron Microscopy
Authors: Eiras-Stofella D. R. , Fanta E. , Santos V. L. P.
Published: June 2020

The scanning electron microscope is very useful for the detail three-dimensional external understanding of structures, such as fish gills, that present varied aspects according to systematic position or to adaptations to the environment. Cathorops spixii lives near to muddy bottom, in brackish water, and was collected from the Paranaguá bay in the south of Brazil. The 4 gill arches, with 2 hemibranchiae each, have primary gill filaments covered with a corrugated epithelium (Figs. 2 and 4). The epithelial cells are also covered with clear parallel ridges. The secondary or respiratory lamellae are well-developed leaflike structures, which become flatter the father they are from the filament tip. Compared to the primary lamella they are formed by a smooth lininig of epithelial cells, each with concentric ridges. The border between the polygonal cells is clearly visible. Some apical openings of chloride cells can be seen between the cells. All these structures are probably adaptations to the environment where the salinity levels may change, and to the needs of a mucous lining for protection against the environment on the one hand, and an increase of the contact surface of the water and the epithelium on the other.
Directional observation on lipid of male nectary of Lindera megaphylla hemsl. by the rapid embedding method with polyethylene glycol (PEG)
Authors: Dalin Dai , Jianmin Guo
Published: June 2020

Lipid cytochemistry has not yet advanced far at the EM level. A major problem has been the loss of lipid during dehydration and embedding. Although the adoption of glutaraldehyde and osmium tetroxide accelerate the chemical reaction of lipid and osmium tetroxide can react on the double bouds of unsaturated lipid to from the osmium black, osmium tetroxide can be reduced in saturated lipid and subsequently some of unsaturated lipid are lost during dehydration. In order to reduce the loss of lipid by traditional method, some researchers adopted a few new methods, such as the change of embedding procedure and the adoption of new embedding media, to solve the problem. In a sense, these new methods are effective. They, however, usually require a long period of preparation. In this paper, we do research on the fiora nectary strucure of lauraceae by the rapid-embedding method wwith PEG under electron microscope and attempt to find a better method to solve the problem mentioned above.
A rapid silver stain for the DNA of microorganisms cultured from AIDS and other immunocompromised patients
Authors: Giammara B.L , Hopfer R.L. , Yates P.E. , Hanker J.S.
Published: June 2020

Although a number of cytochemical methods have been introduced for nuclei at the ultrastructural level, many of these are topographic, i.e. identify nuclei rather than nuclear chromatin and DNA. An improved silver method for the Feulgen reaction for the light and electron microscopy of DNA was introduced in 1984 (FETS reaction). It required application of the silver methenamine for 15 min. under UV-irradiation and subsequent development of the stain overnight in glycerine. This type of methodology has been considerably accelerated by the application of silver solutions, especially silver methenamine solutions prepared from the solid, under microwave(MW) irradiation. The current procedure employs 1 min. formalin fixation of a smear of the sample on a slide or coverslip. The fixed smear is then rinsed 1 hr. in buffer. Aldehyde or pseudoaldehyde groups of apurinic acids are generated by the action of 2.5N HCl for 10 min. on the DNA. These groups are then condensed with thiocarbohydrazide and the resulting product reacted with silver methenamine solution under MW irradiation for 4 min.
Flat embeddment of monolayer cells, tissue sections, and other cellular materials attached onto glass substrate
Authors: Chang Jeffrey P. , Wang Jaang J.
Published: June 2020

Flat embeddment of certain specimens for electron microscopy is necessary for three classes of biological materials: namely monolayer cells, tissue sections of paraffin or plastics, as well as cell concentrations, exfoliated cells, and cell smears. The present report concerns a flat-embedding technique which can be applied to all these three classes of materials and which is a modified and improved version of Chang's original methodology.

Preparation of coverglasses and microslides. Chemically cleaned coverglasses, 11 × 22 mm or other sizes, are laid in rows on black paper. Ink-mark one coner for identifying the spray-side of the glass for growing cells. Lightly spray with Teflon monomer (Heddy/Contact Inductries, Paterson, NO 07524, U.S.A.) from a pressurized can. Bake the sprayed glasses at 500°F for 45 min on Cover-Glass Ceramic Racks (A. Thomas Co. Philadelphia), for Teflon to polymerize.

Monolayer Cells. After sterilization, the Teflon-treated coverglasses, with cells attached, are treated or fixed in situ in Columbia staining dishes (A. Thomas Co., Philadelphia) for subsequent processing.
Analysis of rat adrenocortical cells by analytical color fluorescence EM
Authors: Nakano Tohru , Ning Gang , Fujimoto Toyoshi , Koike Hirotami , Ogawa Kazuo
Published: June 2020

Distribution of various steroids in adrenocortical cells has not been unequivocally demonstrated, because specific histochemical techniques were not available. In recent years, we have developed an analytical color fluorescence electron microscope (ACFEM) which was assembled on the basis of a high resolution SEM (AKASHI : DS-130). The ACFEM enabled us to detect and analyze cathodoluminescence (CL) which is a very weak light emission under electron beam bombardments, and color CL images could be obtained simultaneously with secondary electron images of the same field. We have previously reported that rat adrenocortical cells emit strong blue-green CL, and intermediary metabolites of the steroid hormones can be separated by CL spectroscopy with the ACFEM. In the present study, effects of hypocholesteremic drugs and adrenocorticotrophic hormone (ACTH) on the lipid droplets of adrenocortical cells were examined by employing the ACFEM.

Adult male Wistar rats weighing 250-300g were used, and divided into four groups :
Study on EPM to microvascular cast of human brain
Authors: Zhou Shanmin
Published: June 2020

While directing the microvascular cast of a sample of fresh brain under binocular microscope with methyl methacrylate, the specimen surface vacuum evaporation treating unit has been sprayed 100aA golden membrane for microvascular cast, on the condition of accelerating voltage 20kV and probe current 0.02nA, with the scanning of EPM Shimadzu Corporation JAPAN. You will find that the corterartery of brain substance artery has various types of the branches, the blood capillary circulation tallies one with another to form well developed blood capillary network and there are cobble-like marks formed by internal cells on the surface of arteriole blood vessels.

Mixing nine methyl methacrylate with one methyl acrylate and adding into it 6% benoyl peroxide of the liquids above, putting this mixture into a cone-shaped bottie , and heating the liquid to 80-P5 C, when the solution is gradually getting dense set and is ready to gettogether.
Effect of verapamil on myocardial changes in experimental hypertensive rats
Authors: Yilmazer Selma , Seçkin Ismail , Tasyürekli Mustafa , Arda Oktay , Satiroglu Güngör
Published: June 2020

Recent experimental and clinical studies have reported on the effectiveness of calcium antagonists in the treatment of hypertension. The influence of such therapy on the morphologic features of heart, however has not been systematically studied. In the present study we have investigated the effect of verapamil, a calcium antagonist, on blood pressure and on ultrastructural features of myocardium in rats made hypertensive with DOCA + salt.

Experiments were carried out with 24 female Wistar rats, 4 weeks of age. The animals were divided into 3, 8 animals per group. Group I was considered as the control group. Group II was injected intraperitoneally with 3 mg/kg daily of DOCA for 5 weeks and 154 NaCl was added in their drinking water. Group III received 8mg/kg daily of verapamil hydrochloride (Isoptin, Knoll Pharmaceutical Co.) orally, in additon to DOCA and salt. Five weeks after the start of the experiment systolic blood pressure was recorded and animals were killed. Left ventricle was dissected and prepared for electron microscopy.
Morphologic alterations in porcine endothelial cells by the plant toxin metabolite, monocrotaline pyrrole
Authors: Hogan M.E. , Wagner J.G. , Klomparens K.L. , Roth R.A.
Published: June 2020

Animals treated with the pyrrolizidine alkaloid metabolite, monocrotaline pyrrole (MCTP), show a progressive pulmonary hypertension associated with interstitial edema and smooth muscle thickening of muscular vessels in the lung. To determine what effect the blood borne toxin has on the endothelium, primary tissue cultures were treated with MCTP and were examined using the scanning electron microscope.

Porcine endothelial cells were isolated, cultured, then plated on tissue culture slides and Millipore filters. The effect of cell density was examined by plating at three different concentrations, the most dense (control cells) being confluent at time of fixation. Cells received no treatment, the vehicle (N,Ndimethylformamide), 5μg MCTP/ml or 50μg MCTP/ml 24 hours after plating and were allowed to grow for an additional four days.

The most striking changes were cell hypertrophy and an alteration in the cell's plating character at a dose of 50μg MCTP/ml. Hypertrophy of the endothelial cells was evident at the 50μg MCTP/ml dose, at which the treated cells approached 4 to 5 times the size of the untreated cells.
Ultrastructural observations of the aorta of calves implanted with a mechanical left ventricular assist device
Authors: Cassella J P , Salih V , Graham T R
Published: June 2020

Left ventricular assist systems are being developed for eventual long term or permanent implantation as an alternative to heart transplantation in patients unsuitable for or denied the transplant option. Evaluation of the effects of these devices upon normal physiology is required. A preliminary study was conducted to evaluate the morphology of aortic tissue from calves implanted with a pneumatic Left Ventricular Assist device-LVAD. Two 3 month old heifer calves (calf 1 and calf 2) were electively explanted after 128 days and 47 days respectively. Descending thoracic aortic tissue from both animals was removed immediately post mortem and placed into karnovsky’s fixative. The tissue was subsequently processed for transmission electron microscopy (TEM). Some aortic tissue was fixed in neutral buffered formalin and processed for routine light microscopy.
Elemental silicone analysis in human breast and capsular tissue surrounding prostheses and expanders
Authors: Jennings Debby , Morykwas Michael , DeFranzo Anthony , Argenta Louis
Published: June 2020

polydimethylsiloxane is the most frequently used polymer for manufacture of breast prostheses and soft tissue expanders. The diffusion of lower molecular weight molecules and there lease of silicafiller from the outer shell have been speculated to cause capsular contraction (capsule formation is normal, capsular contraction is pathologic). Previous research has identified silicone in the tissue surrounding gel-filled implants. This preliminary project analyzed human breast tissue and capsular tissue from around expanders and implants for the presence of silicone by electron dispersive x-ray analysis (EDXA).

Tissue samples were divided into four categories based on device: single lumen, standard bleed gel filled implant (n=2); single lumen, low bleed gel filled implant (n=2); double lumen (saline outer lumen) gel filled implant (n=3); and expander or inflatable prostheses (nogel) (n=4). Breast and subcutaneous tissue external to the capsules were used as controls (n=4).
Orthopedic prophylaxis: Bacterial adherence to antibiotic-impregnated polymethylmethacrylate (bone cement)
Authors: Edmiston Charles E. , Siverhus David J. , Goheen Michael P. , Stiehl James B.
Published: June 2020

While the overall incidence of infection following joint and hip replacement is low, infection when it occurs is often catastrophic and requires replacement of the contaminated prosthetic. The addition of antibiotics to polymethylmethacrylate (PMM) in total joint and hip replacements has been suggested to reduce the risk of post-surgical infection. However, no studies have demonstrated the effect of antibiotic impregnation on microbial adherence and colonization of the resin surface. In the present study cefazolin, tobramycin, or ciprofloxacin were added to the polymethylmethacrylate prior to polymerization and exposed to one of four microbial strains: Staphylococcus aureus (LDE26), Staphylococcus epidermidis (RP62 mucin + or SP2 mucin -) and Escherichia coli (PB103) for a period of 48 hours in PBS with 0.25% dextrose. All bacteria were sensitive to the three test drugs. At 2, 8, 24, and 48 hours a resin block was removed from the test solution, rinsed (3x) and ultrasonically oscillated to remove adherent bacteria. The sonicate was serially diluted, plated to antibiotic free media and microbial quantitation expressed as the log10 colony forming units (cfu) per mg of bone cement. Antibiotic free resin blocks were run in parallel with impregnated samples.
Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation
Authors: Shioda Seiji , Nakai Yasumitsu , Ichikawa Atsushi , Ochiai Hidehiko , Naito Nobuko
Published: June 2020

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.

Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.
Membranous diffusion of Dil (a phospholipid-like fluorescent probe) in retrogradely labeled neurons: Analysis with the EM
Authors: Cunningham D.E. , von Bartheld C.S. , Rubel E.W
Published: June 2020

The compound 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (Dil) is a fluorescent molecule with diffusion properties similar to those of phospholipids. Intramembranous (“lateral”) diffusion of Dil is preserved in aldehyde-fixed tissues and has been used to label neuronal pathways. We found that Dil-label, when illuminated (“photo-converted”) in the presence of diaminobenzidine (DAB), forms an electron-dense reaction product that is suitable for EM analysis. We show that the Dil molecule has a surprising diffusion ability and differential affinitiy to internal membranes after retrograde diffusion along neurites in fixed tissue. This finding may have implications for normal diffusion of phospholipids along intracellular pathways in vivo.

Two 20-day old White Leghorn chick embryos were anesthetized and perfused intracardially with 0.1M phosphate buffer (PB, pH 7.4) followed by perfusion with 2% paraformaldehyde and 1% glutaraldehyde in PB.
Horseradish peroxidase as a cytochemical marker for altered cell membrane permeability in cultured myocytes after oxidative stress
Authors: Ashraf M. , Aida K. , Oguro T.
Published: June 2020

The development of a selective staining technique is important for the study of cell membrane permeability altered under different experimental conditions. This is particularly useful if the same technique or procedure is used for both light and electron microscopy examination. In this study, the effect of oxidative stress in the cultured myocytes using lanthanum (La+++), ruthenium red (RR) and horseradish peroxidase (HRP) as cytochemical markers for leaky myocytes was studied. Myocytes from adult rat hearts were isolated and cultured in modified MEM (Sigma Chemical Co.) at 37°C. Myocytes were exposed to 300 μM H2O2 for 15, 30 and 60 minutes. The cells were then rinsed, incubated in 1% HRP, 5 mM La+++ or 0.3. RR, and were fixed in 2.5% buffered glutaraldehyde for 1 hour. The HRP-treated cells were incubated in diaminobenzidine as described.

After treatment with H2O2 for 15 minutes, all myocytes remained rod-shaped, and their number was decreased to 80% after 60 minutes. The staining of cells with La+++ could not be visualized under the light microscope even in the round cells, and with RR, only heavily damaged cells were distinguishable from normal cells. HRP staining yielded excellent results.
Immunogold labeling of the calcium binding protein synexin in isolated adrenal chromaffin granules and chromaffin cells
Authors: Kuijpers Gemma A.J. , Pollard Harvey B.
Published: June 2020

Exocytotic fusion of granules in the adrenal medulla chromaffin cell is triggered by a rise in the concentration of cytosolic Ca2+upon cell activation. The protein synexin, annexin VII, was originally found in the adrenal medulla and has been shown to cause aggregation and to support fusion of chromaffin granules in a Ca2+-dependent manner. We have previously suggested that synexin may there fore play a role in the exocytotic fusion process. In order to obtain more structural information on synexin, we performed immuno-electron microscopy on frozen ultrathin sections of both isolated chromaffin granules and chromaffin cells.

Chromaffin granules were isolated from bovine adrenal medulla, and synexin was isolated from bovine lung. Granules were incubated in the presence or absence of synexin (24 μg per mg granule protein) and Ca2+(1 mM), which induces maximal granule aggregation, in 0.3M sucrose-40m MMES buffer(pH 6.0). Granules were pelleted, washed twice in buffer without synexin and fixed with 2% glutaraldehyde- 2% para formaldehyde in 0.1 M phosphate buffer (GA/PFA) for 30 min. Chromaffin cells were isolated and cultured for 3-5 days, and washed and incubated in Krebs solution with or without 20 uM nicotine. Cells were fixed 90 sec after on set of stimulation with GA/PFA for 30 min. Fixed granule or cell pellets were washed, infiltrated with 2.3 M sucrose in PBS, mounted and frozen in liquid N2.
STEM analysis of Janssen AuroProbe One
Authors: Hainfeld James F.
Published: June 2020

The announcement of a smaller (1.0 nm) colloidal gold immunolabel by Janssen Life Sciences recently has heightened the hopes of many researchers to be able to label at higher resolution or have a smaller probe that could better access secluded antigens that are frequently accessible to fluorescent labeled antibodies or Fab fragments but not to the usual 3.0 to 5.0 nm colloidal gold immunolabels. Because the 1.0 nm gold is difficult to visualize directly in the TEM, it must usually be enhanced by a silver developer that nucleates on the gold and grows to grains that are of a useful size.

While AuroProbe One has met with some success, some shortcomings have been found. One is that the silver developer can self nucleate, giving some false spots. Also, the grains do not grow uniformly and yield an irregular staining.

This report, however, takes a look at the AuroProbe One directly using the high (0.25 nm) resolution STEM in darkfield where 1.0 nm particles are easily seen.
Visualization of heavy metals in cultured macrophages and hepatocytes
Authors: Brunk Ulf T , Brunmark Anders , Zdolsek Johann M.
Published: June 2020

The objective of this study was to develop a cytochemical method, based on a modification of the Timm method, or the silver enhancement technique, whereby iron, which is not bound within very stable metallo-organic complexes can be visualized in glutaraldehyde-fixed cultured cells with preserved ultrastructure. This has previously not been possible due to a severe damage to the fine structure caused by exposure to high pH during the sulfidation process. However, if cells are wellstabilized by glutaraldehyde before sulfidation at pH-9, they withstand the alkaline environment needed to produce a level of sulfide ions adequate to generate sufficient amounts of heavy metal sulfides before physical development. We have compared this “high pH, high S2-” variety of the silver enhancement technique, at the light and electron microscopic level, with other EM-modifications of the technique used for the detection of easily accessible heavy metals, such as zinc.
Organs of a Baikal seal affected by a morbillivirus
Authors: Belykh O.I. , Likhoshway Ye.V. , Solodun Yu.V. , Goldberg O.A. , Kumarev V.P.
Published: June 2020

The population of Baikal seals Phoca sibirica has been plagued in 1987-88 by an unknown disease. Oligonucleotide probing of nucleic acids isolated from tissues of ill and dead animals, as well as immunological evidence and clinical data suggested that seals were infected by a morbillivirus. Morbillivirus antigen has been vizualized in dead seal tissues by immunoelectron microscopy (preembedding technique).

The present report gives outline of electron microscopic studies of the tissues of infected Baikal seals. Morbillivirus antigens were vizualized as clusters of gold spheres by postembedding technique with monoclonal antibodies against measles virus and protein A-colloid gold conjugates in nuclei and cytoplasm of liver and kidney cells. Some clusters were associated with virus-like particles having a diameter of 80-100 nm. Electron microscopy of ultrathin sections stained with uranyl acetate revealed nucleocapsides having length of up to 1400 nm, and a diameter of 13-17 nm, morphologically similar to measles and seals distemper virus.
Visualization of DNA and DNA-protein complexes by TEM
Authors: Griffith Jack D.
Published: June 2020

A large class of DNA and DNA-protein complexes of great interest to modern molecular biologists lie in a realm of size and complexity that it is too large for structural approaches using X ray diffraction or NMR techniques. Such complexes are usually highly irregular so that EM methods employing the formation of 2 dimensional crystals or dense-packing followed by image averaging are not usable. Examples of such complexes include topoisomerase II heterodimers bound to supercoiled DNA, plasmid DNAs being replicated by the battery of nearly 20 different proteins that initiate and carry out replication in E. coli, complexes of reverse transcriptase enzyme degrading the RNA strand of an RNA/DNA hybrid duplex, and many of the recombinational intermediates of DNA strand exchanges. In the latter complexes large protein scaffolds built of hundreds of RecA protein monomers form filaments with in which the events of DNA strand exchange occur.

To visualize such large and complex structures the demands of the biochemical reactions must be given first prior ity: is ATP, glycerol, or salt required for the on going reactions? Do the reactions occur at room temperature or only at 37 degrees? Efforts in this laboratory have been focused on determining which of the various routes for visualizing macromolecules provides the best general approach to obtaining useful structural information that can be directly related to the biochemical processes.
Analysis of Myofibrillar Organization and Degeneration by Fluorescence Confocal Microscopy
Authors: Sussman Mark A.
Published: June 2020

Myofibrillar degeneration is an important pathological process in progressive cardiomyopathy leading to heart failure. Loss of myofibrils in vivo has been observed in both adaptive cardiac responses (i.e. hypertrophy) as well as in chemotherapeutic use of antitumor drugs with cardiotoxic side effects (i.e. doxorubicin). The molecular mechanism(s) of myofibrillar degeneration are poorly understood in comparison with the sequence of events involved in myofibrillar assembly and organization. Maintenance of myofibril integrity is dependent upon a variety of factors, including contractile protein stoichiometry and protein kinase activity.

The repeating sarcomeric architecture of myofibrils is well suited to structural analysis, since disruption of normal organization is easily visualized by fluorescence microscopy. Many antibodies are available for use in observation of myofibril structure (see Table at right). Confocal microscopy provides advantages in studies of myofilament organization by allowing for direct and accurate measurement of distances to within 0.2 βm and the ability to perform vertical sectioning through individual cells. This vertical (Z-axis) sectioning can be used to select the focal plane within the cell for observation, resulting in higher resolution images by reducing fluorescent signals from above and below the plane. Image analysis software enables the user to create projections of optically sectioned cardiomyocytes which can be rotated to reveal interior structural relationships previously unobserved with single sections or conventional epifluorescence microscopy. Examples of image analysis will highlight useful features such as distance measurement, periodicity, pixel intensity, colocalization of dual labels, and three dimensional reconstruction.
Fluorescence, Confocal, and Intermediate Voltage Electron Microscopy of Centractin Localization in Transfected PtK2 Cells
Authors: Holleran Elizabeth A. , Gray-Board Gladys , Holzbaur Erika L.F. , Peachey Lee D.
Published: June 2020

Centractin (Arp 1) is an actin-related protein that shares 53% sequence identity with conventional actin and is predicted to have a similar core structure for nucleotide binding. Biochemical studies of centractin, named for its apparent localization to the centrosome, reveal that it is a member of a stable macromolecular complex, dynactin. The dynactin complex consists of at least 9 polypeptides and has been implicated in activating dynein mediated vesicle transport along microtubules. Ultra-structural analysis, by rotary shadowing electron microscopy with antibody decoration, reveals that within the context of the dynactin complex, centractin forms a 37 nm filament resembling the structure of an actin filament with capping protein and p62 localizing to opposite ends.

We have used transient transfection assays to investigate the effects of overexpression of centractin in cells. Mammalian PtK2 cells were transiently transfected with centractin cloned in the pcDNA3 vector under the control of a CMV promoter. The cells were transfected for 24 hours using Ca2+/DNA co-precipitates. The transfection efficiency was as high as 41 % as determined by direct counting of immunostained cells. Cells were fixed at time points ranging from 18 to 24 hours post washing using 1% formaldehyde plus 1% Triton X-100 (5 min), 0.05% glutaraldehyde (5 min), 1 % formaldehyde (5 min), and 0.05% sodium borohydride to block free aldehyde groups (3×5 min). Immunofluorescence employed an affinity-purified, rabbit polyclonal antibody to centractin followed by a Texas Red-labeled secondary antibody. Epifluorescence microscopy revealed that all of the cells overexpressing centractin contained centractin structures with novel morphology. Two distinct patterns were observed consistently: filamentous centractin localized throughout the cytoplasm (Figure la) and perinuclear centractin accumulations (Figure lb). The perinuclear accumulations appear to be highly three dimensional and curved in structure (confirmed by confocal microscopy), and often have associated filamentous structures (Figure lb). The addition of 33 micromolar nocodozole (30 min. at 4°C) to the transfected cells results in thinner centractin filaments (Figure lc).
The structure of frozen-hydrated membrane skeletons of human red blood cells
Authors: Yang Li , Josephs Robert
Published: June 2020

The red cell membrane skeleton is a two-dimensional network of structural proteins attached to the cytoplasmic surface of the erythrocyte membrane. The major components of the skeletal network are spectrin, actin and band 4.1. Reversible shape changes in the red cell are believed to be mediated by changes in the conformation of spectrin. In this study cryo electron microscopy was used to examine membrane skeletons that were embedded in a thin layer of vitreous ice supported by a fenestrated carbon film. This avoids drying and flattening effects and the attendant changes in salt concentrations and pH associated with negative staining.

Ghosts derived from normal human red cells were incubated with 4 volumes of 2.5% Triton X-100 in 5 mM sodium phosphate (pH 7.6) for 1 hour at 4°C. Membrane skeletons were isolated on a discontinuous density gradient of 10% and 60% sucrose and dialyzed against 1 mM sodium phosphate buffer (pH 8) containing 0.1 mM DTT for 4-6 hours. Skeletons were applied to carboncoated fenestrated films that had been treated with 1% Alcian blue and rinsed thoroughly with distilled H2O and frozen in liquid ethane. The specimens were examined at -172°C in a Philips CM10 microscope.
Comparison of Polymerization of Deoxy-Sickle Cell Hemoglobin in High and Low Concentrated Phosphate Buffers
Authors: Wang Zhiping , Chen Yimei , Josephs Robert
Published: June 2020

Substitution of Val for Glu-β6 in the β-globin chain of hemoglobin (Hb) results in deoxy-sickle hemoglobin (HbS) forming fibers which cause sickle cell anemia. We have previously determined the three-dimensional structure of the HbS fiber from electron micrographs and synthesized a model depicting the contacts stabilizing the fiber. In order to test the contacts predicated by the model we are preparing site directed mutants. However high concentrations of hemoglobin (ca. 160 mg/ml) are required for polymers to form. Quantities of the order of 10-20 mg are available from most site directed mutant preparations. Increasing the phosphate buffer concentration to 1.5 M can reduce the concentration of HbS required for polymerization. However the use of high concentrations of phosphate buffer to induce polymerization has been criticized because there is little data relating the structure of fibers formed in high phosphate to the structure of fibers formed under physiological conditions.
Localisation of hyperaccumulated nickel in Stackhousia tryonii using Electron-probe microanalysis
Authors: Noell I. , Morris D.
Published: June 2020

Proton microprobe and electron probe X-ray microanalysis (EPXMA) simultaneously measure and map elemental content, and hence are excellent tools for investigating the distribution and function of elevated Ni levels in hyperaccumulating plants (Ni concentration >1000 μg g−1 dry weight). Five major hypotheses have been proposed for the function of Ni hyperaccumulation. Our research focuses on the hypothesis that Ni defends against herbivore or pathogen attack and examines the movement of Ni from soil through plant to herbivore in Stackhousia tryonii, the only known hyperaccumulator in eastern Australia. Using a JEOL JXA-840-A electron probe microanalyzer with Moran Scientific Analysis software, we located features of high mean atomic number in whole leaves and cross-sections through backscattered-electron imaging (BEI), then we used EPXMA to identify the elements present and to prepare semi-quantitative x-ray maps of seven key elements.