Electron Microscopic Appearance Research Articles

A eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis B virus capsid, a multimeric particle.

We have established a system for assembly of hepatitis B virus capsid, a homomultimer of the viral core polypeptide, using cell-free transcription-linked translation. The mature particles that are produced are indistinguishable from authentic viral capsids by four criteria: velocity sedimentation, buoyant density, protease resistance, and electron microscopic appearance. Production of unassembled core polypeptides can be uncoupled from production of capsid particles by decreasing core mRNA concentration. Addition of excess unlabeled core polypeptides allows the chase of the unassembled polypeptides into mature capsids. Using this cell-free system, we demonstrate that assembly of capsids proceeds by way of a novel high molecular weight intermediate. Upon isolation, the high molecular weight intermediate is productive of mature capsids when energy substrates are manipulated. A 60-kD protein related to the chaperonin t-complex polypeptide 1 (TCP-1) is found in association with core polypeptides in two different assembly intermediates, but is not associated with either the initial unassembled polypeptides or with the final mature capsid product. These findings implicate TCP-1 or a related chaperonin in viral assembly and raise the possibility that eukaryotic cytosolic chaperonins may play a distinctive role in multimer assembly apart from their involvement in assisting monomer folding.

Elemental analysis of non-living particles in picoplankton fractions from oligotrophic lake water

Electron microscopic (TEM) study of bacterial-sized planktonic fractions recovered from the water column samples of a small oligotrophic lake, consistently showed the presence of non-living organic and inorganic particles as prominent parts of the micro habitat. An all important objective of this work was to determine the composition and possible origins of the minute particles. They were of several morphological types, described by transmission electron microscopic (TEM) appearance as: spherical, oblong, cuboidal, plates and fibrillar. The TEM used in the scanning transmission mode (STEM) in conjunction with the Princeton Gamma Tech (PGT) System 4 energy dispersive X-ray spectrometer (EDX) allowed the determination of the elemental composition of individual particles. The particles referred to as spherical were electron dense and were less than 1 μm in diameter. They were mainly composed of either Fe or Ti but not both. Cuboidal iron particles 37–75 nm in diameter were found in about equal abundance, but they were arranged in chains of individuals. Oblong particles were from 0.18 to 0.2 nm in width to 0.2 to 0.45 μm in length. Most were composed mainly of Ca, S and O but other elemental compositions were observed also. Plates with jagged edges composed of Al and Si were somewhat electron transparent. Both were less than 1 μm in largest dimension. Fibrillar particles were most abundant and were composed of Fe, Si, Cl, K, Na, O and C. They ranged from 90 Å to 37 nm in thickness and 1 μm to several μm in length. Particles of planktonic origin were observed with regularity including fragments of diatom cell walls and scales. These particles were composed of Si and O. Hexagonally shaped virus-like particles of several sizes were observed also. The relationship between elements in micro particles and their possible origins are discussed.

Dimeric Assembly of Enterocyte Brush Border Enzymes

The noncovalent, dimeric assembly of small intestinal brush border enzymes was studied by sedimentation analysis in density gradients of extracts of pulse-labeled pig jejunal mucosal explants. Like aminopeptidase N (EC 3.4.11.2), sucrase-isomaltase (EC 3.2.1.48-10), aminopeptidase A (EC 3.4.11.7), and dipeptidyl peptidase IV (EC 3.4.14.5) were all observed to dimerize predominantly prior to the Golgi-associated complex glycosylation, i.e., in the endoplasmic reticulum or in an intermediate compartment between this organelle and the Golgi complex. However, small amounts of monomeric complex-glycosylated forms, in particular of sucrase-isomaltase, were detectable. This indicates that homodimerization cannot be an absolute requirement for transport to, and through, the Golgi complex although our data suggest that dimeric assembly may increase the rate of intracellular transport. Culture at low temperature (20 degrees C) reduced the rate of, but did not prevent, dimerization. Maltase-glucoamylase (EC 3.2.1.20) only appeared as a dimer when extracted and analyzed under low salt conditions, suggesting a weak association between the two subunits. This finding is consistent with the electronmicroscopic appearance of the liposome-reconstituted enzyme [Norén et al. (1986) J. Biol. Chem. 261, 12306-12309], showing only the inner, membrane-anchored domains of the monomers to be in close contact with one another while the outer domains are far apart. In contrast to the other brush border enzymes studied, lactase-phlorizin hydrolase (EC 3.2.1.23-62) was found to occur predominantly as a monomer in its transient, high mannose-glycosylated state.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement and neutrophil activation during cardiopulmonary bypass: A study in the complement-deficient dog

Cardiopulmonary bypass (CPB) is known to cause complement and neutrophil activation, but the relative importance and interaction of these components in CPB-induced inflammation is unknown. In this study, a strain of dogs genetically deficient in the third component of complement (C3) was used to determine the contribution of C3 to neutropnil activation and pulmonary injury after CPB. Eleven dogs (5 C3-deficient and 6 controls) underwent 150 minutes of hypothermic CPB (28 °C) followed by 2 hours of observation. Before CPB, C3 levels were normal in controls and less than 1% of normal in C3-deficient dogs. In control dogs, functional activity of C3 decreased to 53.2% of baseline after 1 hour of CPB and there was immunohistochemical evidence of C3 deposition in lung after CPB; C3-deficient dogs had no C3 deposition in lung. Although similar degrees of neutropenia occurred during CPB in the two groups, expression of neutrophil adhesion molecule subunit CD18 was significantly lower in C3-deficient dogs than controls after 1 hour of CPB (45.9 ± 3.7 versus 82.9 ± 10.0 mean fluorescence units; p < 0.02). Postbypass lung tissue myeloperoxidase content was also less in C3-deficient dogs (43.8 ± 4.6 versus 71.1 ± 8.6 μmol · 10 mg −1· min −1; p < 0.03). Cardiopulmonary bypass-associated lung injury (assessed by alveolar-arterial oxygen gradient, pulmonary vascular resistance, percent lung water, and light and electron microscopic appearance) was similar between groups. These results demonstrate that (1) C3 is deposited on pulmonary vascular endothelium during CPB and 12) C3 mediates increased expression of neutrophil CD18 and neutrophil sequestration in lung after CPB. Furthermore, these results suggest that modulation of complement activation is a potential means to reduce neutrophil activation during CPB, but that prevention of much of the organ injury associated with CPB will require inhibition of multiple arms of the inflammatory cascade.

Ultrastructural evidence for bladder MAST cell degranulation and their imminent association with nerve fibers in patients with interstitial cystitis

The ultrastructure of human mast cells (HMCs) in various diseases has been well documented; however, detailed morphological description of human bladder mast cells (HBMCs) in patients with interstitial cystitis (IC) is incomplete. The present study was undertaken to reveal any morphological modifications of human bladder mast cells (HBMCs) from the IC patients and to investigate the spatial relationships between nerve fibers and these mast cells at the ultrastructural level.Fresh-fixed surgical biopsy specimens from IC patients as well as paraffin blocks of IC retrieved through our medical record archival system were processed with routine and deparaffinization methods for transmission electron microscopy (1). No differences in mast cell morphology were noticed between the fresh-fixed or paraffin embedded tissues.The general electron microscopic appearances of the HBMCs in IC located in both mucosa and muscle layers were similar to those of HMCs observed in other sites where mastocytosis may occur due to various pathological processes (Fig. 1).

Structural and functional changes in root dentin following exposure to KTP/532 laser

The use of a new, modified Nd:YAG laser called the KTP/532 laser was evaluated within root canals to determine whether it would modify dentin permeability or alter the scanning electron microscopic appearance of canal dentin. Energies and exposure times were chosen which did not permit periodontal temperatures to increase above 5 degrees C (1 W x 1 s-5 W x 0.5 s). Half of the root canals were covered with smear layer and half were treated with EDTA/NaOCI to remove the smear layer. The results showed that this laser did not change the permeability of the smear layer-covered dentin, although scanning electron microscopic examination revealed modifications to the surface of smear layer. Lasing of etched dentin produced modest increases in root permeability which were associated with enlargement and cracking of tubule orifices.

Myocardial damage induced by tropical rattlesnake ( Crotalus durissus terrificus) venom in rats

This article describes the light and electron microscopic appearance of the rat myocardium at various time intervals after the administration of Crotalus durissus terrificus venom by intraperitoneal route. The crotalid envenomation produced small foci of myocardial necrosis scattered throughout the base of the ventricles. These lesions were predominantly perivascular and were associated with slight to moderate interstitial edema as well as infiltration of mononuclear cells and of a great number of mast cells. The first changes appeared 24 hours after envenomation and reached maximal severity after 4 days. By 8 days, the cardiac morphology was comparable to that of control animals except for small foci of interstitial fibrosis at the base of both ventricles—probably attributable to reabsorption of necrotic myofibers and healing—and a small number of degenerated myofibers. The main points concerning these findings are their preferential localization at the base of the heart and the association of the foci of myocytolytic necrosis with a large number of mast cells. On the basis of these data, although nonspecific, the mechanism of venom-induced myocardial damage is discussed. Moreover, these findings call attention to the potential cardiotoxic effect of crotalid poisoning in humans.

Disulfide bonds are essential for the stability of the sindbis virus envelope

Sindbis virus is a membrane-containing virus which has two glycoproteins organized in an icosahedral lattice. Protein-protein associations have been identified which participate in the formation of thtxte icosahedron and these associations are stabilized by intramolecular disulfide bridges (Anthony, R. P., and Brown, D. T., 1990, J. Virol. 65, 1187–1194). The present study further examines the role of disulfides in the structure and function of Sindbis virus by following the effect of dithiothreitol on the protease sensitivity of envelope proteins as well as the electron microscopic appearance and infectivity of Sindbis virus. Treatment of isolated virus with 5 m M dithiothreitol for 6 hr causes a marked increase in trypsin sensitivity of both E1 and E2, profound morphological alterations in the viral envelope, increased susceptibility of the nucleocapsid to RNase, and 95% loss of infectivity. These effects are greatly enhanced and accelerated when treatment with DTT is preceded by a brief exposure of the virus to pH 5.3, suggesting that acid-induced conformational changes render structurally critical disulfides more accessible to reductive cleavage by DTT. When compared to other manipulations known to change the conformation of the viral envelope, such as heating to 51 or 60° or exposure to acid pH, only the exposure to DTT with or without prior acid treatment caused marked structural changes correlated with a loss of infectivity. These data provide electron microscopic and functional evidence that intact disulfide bonds are critical for the stability of the virus envelope and suggest that the cleavage of critical disulfide(s) may play a role in the process of virus infection.

Structural differentiation of the epithelial cells of the testicular excurrent duct system of rats during postnatal development

The light and electron microscopic appearance of the various epithelial cells lining the efferent ducts and different regions of the epididymis were examined in rats on postnatal days 21, 39, 49, 56, and 90 to determine the role of androgens and/or spermatozoa, as well as other possible factors, on the structural differentiation of these cells. Five conclusions may be drawn from the observations made. First, on day 21 epithelial cells of all regions are structurally undifferentiated. Second, it was not until day 49 that nonciliated cells of the efferent ducts resembled those of adult animals, suggesting that more than one factor, such as androgens, testicular products, and/or spermatozoa, is needed for their full structural differentiation. Third, principal cells of the epididymis become structurally differentiated by day 39, i.e., these cells contained an elaborate Golgi apparatus, endoplasmic reticulum cisternae, and numerous 200-400 nm electron lucent secretory vesicles, as well as a full complement of endocytic organelles; this occurred in spite of the absence of spermatozoa in the epididymal lumen. The differentiation of these epididymal cells may be under the influence of androgens, which are known to be high at this time, but may also be due to specific secretions from Sertoli cells secreted directly into the efferent ducts. Fourth, clear cells of the cauda epididymidis are fully differentiated by day 39. The presence of degenerating germ cells in the lumen of the cauda epididymidis and various cellular debris, as well as high androgen levels, may be factors causing the differentiation of the cells of this region. Finally, clear cells of the corpus and cauda epididymidis only become fully differentiated by day 49, at a time when spermatozoa appear in the lumen, despite high levels of androgens at day 39; this observation indicates that the presence of spermatozoa in the lumen may be a necessary factor in causing their differentiation. Overall, these results suggest that a combination of different factors are necessary for the structural differentiation of the various epithelial cell types of the different regions of the epididymis.

Electron Microscopic Appearances of Human Corneal Endothelium Following Nd:YAG Laser Iridotomy

We looked at the electron microscopic appearances of the corneal endothelium following neodymium:YAG (Nd:YAG) laser iridotomy in a patient with a shallow anterior chamber. An area of denuded endothelial cells approximately 500 microns in diameter was produced when no contact lens was used. The area of cell loss was reduced by approximately 50% when an Abraham YAG goniolens was used. We conclude that the use of a positive plus contact lens can help minimize cell loss following Nd:YAG laser iridotomy.

Ultrastructural aspects and amino acid composition of the purified inner and outer membranes of human liver mitochondria as compared to rat liver mitochondria

1. 1. The mitochondria isolated from human or rat liver were fractionated into submitochondrial particles and purified inner and outer membrane. According to different marker enzymes the inner membranes were enriched about 5–6-fold and the outer membranes about 12–14-fold. The electron microscopical appearance of the membranes was that expected on the basis ofenzymic characterization. 2. 2. A comparison of the average amino acid composition of the membrane proteins from the two types of mitochondria has been made. In the case of submitochondrial particles there were statistically significant differences between the human and rat hydrolysates for only five amino acids. Analysing the purified mitochondrial membranes there were significant differences between the two species for nine amino acids in the case of outer membranes and for 12 amino acids in the case of inner membranes. 3. 3. With one exception all amino acids that were increased or decreased in the outer membrane exhibited a similar trend in the inner membrane of human compared with rat liver mitochondria. It appears that liver mitochondrial membranes have a species-dependent pattern of amino acid composition of their proteins.

Light Microscopy, Immunohistochemistry and Electron Microscopy of the Valves of the Lower Limb Veins and Jugular Veins

Objectives: To demonstrate the light, electron microscopy and immunohistochemistry appearances of the valves of lower limb veins and jugular veins of subject with evidence of venous disease. Design: A prospective study of saphenous veins obtained at post mortem examination and jugular vein obtained at time of surgery. Setting: Health Service district general hospital. Patients: Patients with no evidence of venous disease being treated in the hospital for unrelated conditions, or examined at autopsy in the Department of Pathology. Interventions: Sections of saphenous vein from the lower limb and jugular vein were examined using conventional histology, immunohistochemical staining and electron microscopy. Main outcome measures: Microscopic and electron microscopic appearances of histological sections of venous valves. Results: The valve agger contains an increased amount of fibrous tissue compared with the vein wall and saphenous and jugular veins. However, in saphenous veins the muscle layer of the wall could be traced across the base of the agger, but this was deficient in jugular valves. Electron microscopic examination showed that most of the valve cusp was composed of collagen fibrils, aggregated in irregularly arranged bundles. Elastic fibre material was present in the subendothelial zones. Nerve and muscle fibres were not present in the cusp. Conclusions: Valve cusps in both high and low pressure systems are similar, and the authors suggest that cusp/agger/vein wall complex is important for valve function.

Fractionation and antigenic characterization of organelles ofEimeria tenellasporozoites

Sporozoites of Eimeria tenella were disrupted by sonication and subcellular fractions were separated by sucrose gradient ultracentrifugation. Fractions from gradients were characterized by electron microscopical appearance and their polypeptide and antigenic profiles determined by PAGE and Western blotting with antisera to sporozoites and 1st- and 2nd-generation merozoites. Fractions containing micronemes, rhoptries or membranes showed markedly different polypeptide content and antigenic reactivity. Microneme epitopes were strongly conserved between sporozoites and 2nd-generation merozoites whereas the majority of rhoptry epitopes and many membrane epitopes were sporozoite specific. The only polypeptide of sporozoites which was strongly recognized by antisera raised to 1st generation merozoites was a microneme antigen of molecular weight approximately 100 kDa.

White sponge nevus presenting in the esophagus — case report and literature review

White sponge nevus (WSN) is a rare inherited disorder which usually presents as non-painful white plaques primarily involving the buccal mucosa, gingiva and palate. Extra-oral lesions most often occur in the esophagus or anogenital area but almost invariably follow the development of typical buccal lesions. We present a non-familial case in which the first manifestation of the disease was in the esophagus in the absence of oral lesions. This sequence of events has not previously been reported in the literature. The cytohistological and electron microscopical appearances which allow the recognition and differentiation of WSN from other conditions presenting as esophageal white lesions are discussed.

Ultrastructure of human osteoblasts and associated matrix in culture.

The ultrastructure, as visualized by transmission electron microscopy, of cells obtained from human bone explants and subsequently cultured is described along with the electron microscopic appearance of the associated intercellular matrix. The cells were characterized as osteoblasts on the basis of immunohistochemical, enzymatic, and functional criteria. Although the osteoblasts could be cultured in standard culture media and always appeared singly, not forming syncytia, the cultures were eventually confluent and formed multilayers. The cells were fusiform or cuboidal with diameters ranging between 10-15 microns. The cytoplasm was characterized by numerous large mitochondria, and especially by a very prominent RER. The intercellular matrix was woven with collagen fibres surrounding large numbers of matrix vesicles. In areas with matrix vesicles, evidence for osteoblast activity, i.e. mineralization related to matrix vesicles, could be observed after incubation with beta-glycerophosphate. In conclusion, we provide evidence that human osteoblasts cultured in vitro synthesize collagen and produce a matrix with vesicles capable of initiating mineralization processes.

Embryonic ability to prolong the interoestrous interval in virgin and repeat breeder heifers

Fourteen virgin heifers (VH) and eight repeat breeder heifers (RBH) were used for repeated non-surgical embryo collections. The heifers were flushed on a total of 61 occasions, 14 or 17 days after ovulation. The two categories of heifers were compared for degree of embryonic development, the ability of these embryos to prolong the interoestrous interval, the plasma progesterone levels after insemination, and the light and electron microscopical morphological appearance of the embryos. The RBH gave fewer and smaller embryos than the VH. Moreover, RBH embryos presented a higher incidence of morphological deviations than VH embryos. Day 17 RBH embryos were less successful in prolonging the interoestrous interval than the VH embryos, in particular, those 17-day-olcl RBH embryos which did not prolong the interoestrous interval showed a clearly deviated morphology of the embryonic disc. Plasma progesterone levels were positively correlated with pregnancy before maternal recognition of pregnancy. In the VH group, plasma progesterone levels at Days 6–11 were positively correlated with the length of 17-day-old embryos. Despite the strict control of oestrus and ovulation and the use of additional artificial insemination (Al) when necessary in this study, differences in pregnancy results between the RBH and VH still remained. It is concluded that the uterine environment in RBH does not provide proper support for embryo development up to the time when maternal recognition of pregnancy occurs.

Ultrastructure of Chlamydia pneumoniae in cell culture

The electron microscopic appearance of Chlamydia pneumoniae elementary bodies with pear-shaped, loose outer membrane has been suggested as one criterion of its classification as a new chlamydial species. The study of the original strain TW 183 in LCL 929 and HL cells and a low-passage isolate of Kajaani-6 isolate in HL cells revealed spherical compact elementary bodies common to other chlamydia.

Ultrastructure ofChlamydia pneumoniaein cell culture

The electron microscopic appearance of Chlamydia pneumoniae elementary bodies with pear-shaped, loose outer membrane has been suggested as one criterion of its classification as a new chlamydial species. The study of the original strain TW 183 in LCL 929 and HL cells and a low-passage isolate of Kajaani-6 isolate in HL cells revealed spherical compact elementary bodies common to other chlamydia.

Transmission electron microscopic appearance of dentin matrix in type II dentin dysplasia

Dentin matrix of demineralized primary and permanent teeth with type II dentin dysplasia was studied by transmission electron microscopy. The coronal dentin of a maxillary third molar exhibited a normal structure. In the radicular dentin, tubules were few in number; the major part of the dentin was composed of thick, curvy bundles of cross-striated collagen fibers. In the most aberrant areas of the radicular dentin, coarse collagen fibers measuring up to 140 nm in thickness were observed. The dentin of the primary tooth showed a similar, but somewhat less irregular, structure.

Immunohistological and electron microscopical study of nodular fasciitis of the orbit.

We report the case of a 7-year-old Japanese girl with nodular fasciitis which was investigated by immunohistological and electron microscopical methods. An excised nodular lesion in her right orbit showed characteristic histological features of the disease. The fibroblastic cells showed myofibroblastic characteristics, such as immunohistochemically positive reactions against muscle specific actin and vimentin and characteristic electron microscopical appearances. The multinuclear giant cells did not react against any histiocytic markers, including HLA-DR, antimacrophage antigen, lysozyme, and S-100 protein, but the myofibroblastic markers and the electron microscopical study did reveal myofibroblastic characters.