Electron microscopic observations of surface projections on Chlamydia psittaci reticulate bodies.

Electron microscopic observations were carried out to confirm the presence of surface projections on Chlamydia psittaci reticulate bodies (RBs). The morphology of the projections on RBs was identical with that on elementary bodies (EBs); one end of each projection was connected with the cytoplasmic membrane, but the other end of the projection protruded beyond the cell wall through a fine hole or rosette in the cell wall. The results demonstrated that the rosettes seen in RB cell walls were morphological markers indicating the presence of the surface projections. A statistical anaylsis of the number of projections on EBs and the number of rosettes in RB cell walls prepared at 10, 15, and 20 h after infection demonstrated that all RBs had the projections and that the number of projections was maximal by 10 h after infection and then decreased gradually to approximately the same number of projections on EBs.     

Arrays of hemispheric surface projections on Chlamydia psittaci and Chlamydia trachomatis observed by scanning electron microscopy.

Scanning microscopy of two strains of Chlamydia psittaci and four strains of Chlamydia trachomatis representative of the wide diversity in origin and behavior of members of the genus revealed patches of regular arrays of hemispheric projections on the surfaces of elementary bodies of all six strains. These distinctive and perhaps unique surface structure are probably present in all populations of chlamydiae.     

Scanning electron microscopic observations of polyacrylamide gels.

Structures of polyacrylamide gels have been viewed in a scanning electron microscope. Structures observed after freeze-drying of the gels have been substantiated by different experiments as the preexisting structure of the gels in their state of hydration. Parameters of content and polymerization of polyacrylamide gels have been varied to reveal their effects on the observed morphology. The structural patterns revealed by electron microscopy suggest a model of both the polymerization and the molecular-sieving properties of these gels.     

Pyrimidine metabolism by intracellular Chlamydia psittaci.

Pyrimidine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined mutations affecting pyrimidine metabolism. C. psittaci AA Mp cannot synthesize pyrimidines de novo, as assessed by its inability to incorporate aspartic acid into nucleic acid pyrimidines. In addition, the parasite cannot take UTP, CTP, or dCTP from the host cell, nor can it salvage exogenously supplied uridine, cytidine, or deoxycytidine. The primary source of pyrimidine nucleotides is via the salvage of uracil by a uracil phosphoribosyltransferase. Uracil phosphoribosyltransferase activity was detected in crude extracts prepared from highly purified C. psittaci AA Mp reticulate bodies. The presence of CTP synthetase and ribonucleotide reductase is implicated from the incorporation of uracil into nucleic acid cytosine and deoxycytidine. Deoxyuridine was used by the parasite only after cleavage to uracil. C. psittaci AA Mp grew poorly in mutant host cell lines auxotrophic for thymidine. Furthermore, the parasite could not synthesize thymidine nucleotides de novo. C. psittaci AA Mp could take TTP directly from the host cell. In addition, the parasite could incorporate exogenous thymidine and thymine into DNA. Thymidine kinase activity and thymidine-cleaving activity were detected in C. psittaci AA Mp reticulate body extract. Thus, thymidine salvage was totally independent of other pyrimidine salvage.     

Purine metabolism by intracellular Chlamydia psittaci.

Purine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined deficiencies in purine metabolism. C. psittaci AA Mp cannot synthesize purines de novo, as assessed by its inability to incorporate exogenous glycine into nucleic acid purines. C. psittaci AA Mp can take ATP and GTP, but not dATP or dGTP, directly from the host cell. Exogenous hypoxanthine and inosine were not utilized by the parasite. In contrast, exogenous adenine, adenosine, and guanine were directly salvaged by C. psittaci AA Mp. Crude extract prepared from highly purified C. psittaci AA Mp reticulate bodies contained adenine and guanine but no hypoxanthine phosphoribosyltransferase activity. Adenosine kinase activity was detected, but guanosine kinase activity was not. There was no competition for incorporation into nucleic acid between adenine and guanine, and high-performance liquid chromatography profiles of radiolabelled nucleic acid nucleobases indicated that adenine, adenosine, and deoxyadenosine were incorporated only into adenine and that guanine, guanosine, and deoxyguanosine were incorporated only into guanine. Thus, there is no interconversion of nucleotides. Deoxyadenosine and deoxyguanosine were cleaved to adenine and guanine before being utilized, and purine (deoxy)nucleoside phosphorylase activity was present in reticulate body extract.     

Surface projections and internal structure of Chlamydia psittaci.

The outermost surface of the small infectious forms of Chlamydia psittaci contain geometrically arranged spikes distributed over approximately 50% of the bacterial surface. The spikes are located opposite the concave side of an electron-dense crescent-shaped chlamydial core.     

Transmission-scanning electron microscopic observations of selected Eikenella corrodens strains.

The morphology of Eikenella corrodens 333/54-55 (ATCC 23834) and two human periodontal lesion isolates, strains 470 and 373, was examined by transmission and scanning electron microscopy. All strains exhibited a cell envelope characteristic of gram-negative bacteria. Staining with ruthenium red and alcian blue revealed a loosely organized fibrous slime layer associated with the outer surface of the outer membrane. Slime "stabilization" was achieved by incubation of cells with antisera prepared against whole cells of the Eikenella strains. The stabilized slime appeared as a thick, electron-opaque layer juxtaposed to the outer membrane. Negative staining and heavy metal shadow-casting revealed an interwoven network of fibrils approximately 4 nm in diameter. These fibrils appeared to represent subunits of a larger fibril. Scanning electron microscopy after antibody slime stabilization confirmed the presence and location of the slime layer.     

Adenine nucleotide and lysine transport in Chlamydia psittaci.

Isolated reticulate bodies of Chlamydia psittaci were found to transport ATP and ADP by an ATP-ADP exchange mechanism. ATP uptake activity was not detected in elementary bodies. The apparent Km of transport for both ATP and ADP was approximately 5 microM, and the calculated Vmax for both was about 1 nmol of nucleotide transported per min per mg of protein. ADP competitively inhibited ATP transport with a Ki of 4.5 microM. Other nucleotides tested had no effect on the uptake of ATP. A magnesium-dependent, oligomycin-sensitive ATPase (ATP phosphohydrolase, EC 3.6.1.3) was associated with reticulate bodies, and most of the transported ATP was hydrolyzed to ADP, which was exchanged for additional, extracellular nucleotide. Some ADP was hydrolyzed to AMP, which exited the cells slowly. Lysine was transported against the electrochemical gradient by reticulate bodies in the presence of ATP. Oligomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited ATP-dependent lysine transport. Lysine exited reticulate bodies when the reticulate bodies were incubated in the presence of ADP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or a reduced concentration of ATP. The results support the concept that chlamydiae are energy parasites which are capable of drawing upon the adenine nucleotides of their hosts, hydrolyzing ATP, and establishing an energized membrane.     

Analytical electron microscopic study of mitochondrial inclusions in canine myocardial infarcts.

An analytical electron microscopic study, utilizing scanning transmission electron microscopy and energy-dispersive x-ray spectroscopy, was made of two types of mitochondrial inclusions identified in canine myocardial infarcts. The data were obtained from thin sections of tissues that were fixed in aldehyde, osmicated and embedded in epoxy resin. Calcium peaks of variable intensity were detected in inclusions which contained very electron-dense spicular material and which were localized to muscle cells at the peripheries of the infarcts. These findings indicate that the spicular inclusions represent early stages in the process of mitochondrial calcification in myocardial infarcts. In contrast, calcium or other trace elements were not detected in moderately electron-dense amorphous inclusions which were present in mitochondria of muscle cells throughout the infarcts. With the tissue preparative techniques employed, the possibility cannot be excluded that the amorphous inclusions contained calcium, either in small amounts or in a readily diffusable state, in vivo. The data, however, are in accord with the previously advanced hypothesis that the amorphous inclusions represent precipitates of denatured mitochondrial protein formed during the evolution of irreversible cellular injury. This study provides further evidence that analytical electron microscopy can yield important information regarding the nature of various inclusions occurring in normal and diseased tissues.     

Melting of myosin and tropomyosin: electron microscopic observations

A method was devised to maintain a very low angle (2-3 degrees) during the metal casting of specimens for electron microscopy. With this modified rotary shadowing procedure the melting of myosin and tropomyosin (TM) was investigated. When protein solutions were sprayed on mica sheets and then heated to melt alpha-helices, myosin molecules did not show any sign of chain separation but appeared to have collapsed into loose clumps. A few molecules showed separation of the two chains at the light meromyosin-heavy meromyosin hinge region. Heating myosin in bulk solution at 65 degrees C before spraying caused extensive fusing of the myosin heads. In contrast, in the case of TM, separation of the chains appeared to occur at temperatures at which the unfolding of alpha-helices had been shown by circular dichroism. Dissolution of TM and myosin in 0.5% SDS followed by 150-fold dilution led to single chain species. This method capable of detecting single chain peptides of melting TM whose thickness is of the order of 1 nm may be applicable to the study of the structure of proteins previously not considered possible.     

Immunoelectron microscopy of Chlamydia psittaci with monoclonal antibodies.

An immunoelectron microscopic study was performed to determine the distribution of antigenic components on particles of Chlamydia psittaci and infected cells using a number of monoclonal antibodies (MAbs). Of three anti-lipopolysaccharide (LPS) antibodies (4D5, A2 and 4G5), two antibodies (4D5 and A2) reacted with the surface of reticulate bodies (RBs) but not with that of elementary bodies (EBs). The other antibody (4G5) reacted with both EBs and RBs. Examination of infected cells in thin sections revealed that 4D5 and A2 combined with the membranes of both EBs and RBs. These results indicate that each LPS epitope localized at a different position in the chlamydial membrane. Most MAbs directed to protein antigens reacted on the surface of both EBs and RBs though 3E9 specific for the 90 kDa and 50 kDa protein components combined with RBs only.     

Detection of Chlamydia psittaci by Immunofluorescence

A direct fluorescent-antibody (FA) test was developed to detect Chlamydia psittaci in dural impressions from specimen-inoculated mice. Technical procedures for the test were compared. C. psittaci was found in mice after infection as early by the FA technique as it was by cytochemical staining methods usually used. The lymphogranuloma venereum organism was also stained by conjugated antibody to C. psittaci. A distinctive advantage of the described FA test is that organisms are identified immunologically as members of the genus Chlamydia simultaneously with their detection.     

Light and electron microscopic observations on hydrocortisone-induced cleft palate in hamsters.

Palatal histogenesis in hydrocortisone-treated hamster fetuses was studied by both light and electron microscopy. At an early stage in the hydrocortisone-affected fetuses, when the palatal shelves hung vertically on either side of the tongue, necortic changes could be seen in some of the basal epithelial cells which lay adjacent to the fragmented basal lamina. The normal looking cells lay on an intact basal lamina and were attached to the contiguous necrotic cells by desmosomes. With horizontal reorientation of the palatal shelves and their approach to the midline, cellular necrosis and fragmentation of the basal lamina increased. When compared with normal cells, the hydrocortisone-affected ones were seen to be lighter, to contain fewer ribosomes and no lysosomes. At a later stage, when midline palatal fusion was lacking, the epithelium underwent stratification and keratinization while the necrotic debris was removed by mesenchymal macrophages. It appears that the normal process of protein synthesis is inhibited following hydrocortisone administration and that this, in turn, during palatogenesis, disrupts normal cellular differentiation and the integrity of the basal lamina, which are associated with the production of a cleft palate.