Stimulation of endocytosis in mouse blastocysts by insulin: a quantitative morphological analysis.

The effects of insulin on the endocytic activity of mouse blastocysts in vitro were investigated using confocal laser scanning microscopy, quantitative image analysis and electron microscopy. Confocal studies showed that fluorescein isothiocyanate-labelled markers, dextran (fluid phase) and albumin (combined membrane and fluid phase), were endocytosed by blastocysts and localized within vesicles (about 2.5 microns in diameter) in the outer trophectoderm cells. No labelling was detected in the inner cell mass cells or the blastocoel cavity. Treatment with 170 nmol insulin l-1 stimulated the endocytosis of fluorescently labelled dextran in freshly collected blastocysts, increasing mean vesicle diameter per embryo by 15% (P < 0.05) after incubation with insulin for 2.5 h and mean vesicle number per embryo by 56% (P < 0.01) after 6 h. Both effects were also evident in blastocysts that had been cultured from the late eight-cell stage. Blastocysts incubated for 6 h with insulin displayed increased convolutions in the trophectoderm apical membrane compared with controls, indicating increased membrane activity and suggesting macropinosome formation. Collectively, these results suggest that insulin enhances endocytosis in the trophectoderm by stimulating uptake at the apical membrane into larger and more numerous endocytic vesicles and with some evidence of vesicle fusion. This mechanism may provide a metabolic basis for the stimulation by insulin of biosynthesis, proliferation and morphological development in early embryos.

Characterization of a five-gene cluster required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa produces type 4 fimbriae which promote adhesion to epithelial cells and are associated with a form of surface translocation called twitching motility. Transposon mutagenesis was used to identify loci required for fimbrial assembly or function by screening for mutants that lack the spreading colony morphology characteristic of twitching motility. Six mutants were isolated that contain transposon insertions upstream of the previously characterized gene pilQ. This region contains four genes: pilM-P, which encode proteins with predicted sizes of 37.9, 22.2, 22.8 and 19.0 kDa, respectively. pilM-P appear to form an operon and to be expressed from a promoter in the intergenic region between pilM and the divergently transcribed upstream gene ponA. PilM-P were found to be required for fimbrial biogenesis by complementation studies using twitching motility and sensitivity to fimbrial-specific phage as indicators of the presence of functional fimbriae. This was confirmed by electron microscopy. PilO and PilP did not have homologues in the sequence databases, but the predicted PilN amino acid sequence displayed similarity to XpsL from Xanthamonas campestris, a protein required for protein secretion. PilP contained a hydrophobic leader sequence characteristic of lipoproteins, while PilN and PilO have long internal hydrophobic domains which may serve to localize them to the cytoplasmic membrane. PilM has shared sequence motifs with the cell division protein FtsA from Bacillus subtilis and Escherichia coli, as well as the rod-shape-determining protein MreB from E. coli. These motifs are also conserved in eukaryotic actin, in which they are involved in forming an ATPase domain. Deletion mutants of pilM and pilQ displayed a dominant negative phenotype when transformed into wild-type cells, suggesting that these genes encode proteins involved in multimeric structures.

In vivo endogenous spore formation by Coxiella burnetii in Q fever endocarditis.

AIMS: To determine whether Coxiella burnetii, the aetiological agent of Q fever, undergoes endogenous spore-like formation, the crucial stage of the developmental cycle, in the infected cardiac valves of patients with chronic Q fever endocarditis. METHODS: Surgically removed valves from three cases of Q fever endocarditis were processed for electron microscopy. Sections were stained with potassium permanganate and uranyl acetate before being extensively examined by transmission electron microscopy. RESULTS: In all three cases endogenous spore-like formation was seen in the infected cardiac valves. CONCLUSIONS: As the factors that govern sporogenesis in C burnetii are still largely unknown, it is uncertain how important are the implications of the discovery of endogenous spore-like formation in Q fever endocarditis. However, this finding may add new dimensions to current thinking about the treatment of chronic Q fever.

Comparison of type I and type II Chlamydia psittaci strains infecting koalas (Phascolarctos cinereus).

The native Australian marsupial Phascolarctos cinereus, otherwise known as the koala, is prone to infection by the obligate intracellular parasite Chlamydia psittaci, which causes ocular 'pink eye' and urogenital 'dirty tail' diseases. Several chlamydial DNA probes to both chromosomal and plasmid sequences were used to type by Southern blot analysis 51 samples taken from wild and captive koalas from habitats on the eastern seaboard of Australia as far apart as Queensland and Victoria. Two types of C. psittaci were observed and called types I and II. Type II was found more frequently than type I and occurred in both ocular and urogenital samples, while type I showed a strong but not absolute preference for ocular sites. Cross-hybridization analyses indicated that type I and type II had about 10% DNA sequence identity to each other. DNA analyses showed that type II was very closely related to some ovine and bovine chlamydiae but type I could not be related to any other C. psittaci strain available. Light and electron microscopic analyses of infected BGM monolayers revealed that the two strains were similar in morphological characteristics. The type I strain was considerably more infectious than the type II strain in BGM cells and in the yolk sacs of embryonated eggs. A PCR based assay detected both type I and type II koala chlamydiae in samples that had been negative by Southern blot and tissue culture and provided the first evidence that both types can occur simultaneously at the one site of infection.

Electron microscopy of Coxiella burnetii in tissue culture. Induction of cell types as products of developmental cycle.

An in vitro ultrastructural study was carried out on tissue cultures (J774, murine macrophage-like tumour cell line, and BHK-21, baby hamster kidney cell line) persistently infected with C. burnetii to investigate whether the events of cellular differentiation could be visualized. At a given stage of the developmental cycle, a proportion of the cells within the affected phagolysosomes clearly underwent cellular differentiation. The cells initially showed asymmetrical septation, the primary stage of cellular differentiation, and ended with the formation of the differentiated product, a precursor to the small cell. The results verified our initial observation that the events occurring during growth in a phagolysosome represent stages of a complex developmental cycle consisting not only of i) vegetative growth by typical transverse binary fission, but also ii) cellular differentiation.

Antigenic differences between Coxiella burnetii cells revealed by postembedding immunoelectron microscopy and immunoblotting.

The aim of this study was to investigate the antigenic structures of the morphologically distinct cells of the Coxiella burnetii developmental cycle. Postembedding immunoelectron microscopy with polyclonal antibodies produced in rabbits to (i) phase I cells, (ii) a chloroform-methanol residue fraction of cells, (iii) the cell walls (CW) of large and small cells and small dense cells (SDC), and (iv) the peptidoglycan-protein complexes of small cells and SDC labelled the continuum of morphologically distinct cells. But these antibodies did not distinguish between the antigenic structures of the various cells. Monoclonal antibodies to the phase I lipopolysaccharide labelled the CW of a majority of the smaller cells, but there was diminished reactivity to the larger cells. Although monoclonal antibodies to a 29.5-kDa outer membrane protein labelled the CW of the large mother cells, the large cells, and the small cells, a minority of the SDC with compact CW were not labelled. The endogenous spore within the mother cell was not labelled by the polyclonal or monoclonal antibodies to cellular components. A selected population of SDC was prepared by osmotic lysis of large cells, differential centrifugation, Renografin step-gradient fractionation, and breakage of the small cells in a French press at 20,000 lb/in2. The pressure-resistant SDC collected as fraction CL did not contain the 29.5-kDa protein, as evidenced by the lack of (i) Coomassie brilliant blue staining of protein in the 29.5-kDa region of sodium dodecyl sulfate-polyacrylamide gels and (ii) reactivity of the 29.5-kDa protein antigenic epitopes in immunoblotting with monoclonal antibodies to the protein. In contrast, CW of the pressure-sensitive small cells contained the 29.5-kDa protein. Therefore, the observed ultrastructural differences between large and small cells and SDC reflect differences in sensitivity to breakage by pressure treatment and in cell-associated antigens. Although the process of differentiation in C. burnetii remains an enigma, we have taken steps toward identifying cellular antigens as markers of differentiation. The pressure-resistant SDC in fraction CL that are devoid of the 29.5-kDa protein may be useful for answering questions about the physiological events required for triggering outgrowth and sequential regulation of the Coxiella developmental cycle.

Localization of DNA in Coxiella burnetii by post-embedding immunoelectron microscopy.

The deoxyribonucleic acid of Coxiella burnetii was detected with monoclonal antibodies against single-stranded and double-stranded DNA by post-embedding immunoelectron microscopy. The antibody labeled the nucleoid of the spore, the small cell variant (SCV), and the large cell variant (LCV). The DNA was segregated completely in both the spore and the mother cell. At the terminal stage of development of the spore, the loss of nuclear morphology in the mother cell was related to the reduction in the labeling of the nucleoid. The mother cell was therefore killed for the development and release of the spore.

Inactivation of Coxiella burnetii by gamma irradiation.

The gamma radiation inactivation kinetics for Coxiella burnetii at -79 degrees C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0.64 to 1.2 kGy depending on the phase of the micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing 10(11) C. burnetii ml-1 was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes.

Roles of the Fc receptor and respiratory burst in killing of Rickettsia prowazekii by macrophagelike cell lines.

It is known that the virulent strain of Rickettsia prowazekii grows in macrophagelike cell lines, but if the rickettsiae are treated with antirickettsial serum before infection, the intracellular rickettsiae fail to grow and are destroyed. The uptake of rickettsiae by macrophagelike cell lines was increased by treatment of the rickettsiae with immune serum and with purified immunoglobulin G (IgG) from this serum but not by treatment with the F(ab')2 fragment derived from this IgG. This suggested that the normal rickettsial pathway of entry could be augmented by the Fc receptor-mediated pathway. However, rickettsiae treated with these F(ab')2 fragments which contained no Fc region were destroyed as effectively as those treated with immune serum or IgG. Internalization of R. prowazekii (whether virulent, avirulent, treated, or untreated) did not lead to an increased release of CO2 from [1-14C]glucose, an increase that would have been indicative of a respiratory burst. Furthermore, a mutant macrophagelike cell line, incapable of a respiratory burst, was able to destroy rickettsiae treated with immune serum as effectively as did the parental cell line. Electron micrographs of macrophagelike cells which had been incubated with either antirickettsial IgG or with F(ab')2 fragments derived from this IgG both demonstrated marked deterioration of the rickettsiae, which were primarily within vacuoles but occasionally free in the cytoplasm. In contrast, untreated rickettsiae displayed morphologically normal rickettsiae which were mostly in the cytoplasm but occasionally in the intact and damaged vacuoles. These results indicated that (i) a respiratory burst was not a significant part of the mechanism used by macrophagelike cells to destroy R. prowazekii treated with immune serum, (ii) the destruction of the rickettsiae by the macrophage was not dependent on a diversion to the Fc receptor-mediated pathway of entry, and (iii) the locus of damage to the rickettsiae was most likely the phagolysosome of the macrophagelike cell line.

Fulminant hepatitis. An ultrastructural study.

Ultrastructural changes were observed in 23 consecutive patients who died from fulminant hepatic failure due to hepatitis B virus (4 cases), sporadic non-A, non-B (7), or paracetamol (acetaminophen) overdose (12) and in 3 patients with subacute hepatic necrosis of unknown cause. The findings are described in detail in 12 of these patients. Fatal fulminant hepatitis was characterised by massive confluent necrosis accompanied by collapse of reticulin framework and sudden drop-out of liver cells. No aetiological distinction could be made between different viral causes of fulminant hepatitis on the basis of ultrastructural pathology. Parenchymal changes in viral cases varied from reversible non-specific necrosis to irreversible changes where fragmentation of endoplasmic reticulum, mitochondria and nuclei had occurred. Differences in ultrastructural pathology between non-viral (paracetamol overdose-induced) and viral fulminant hepatitis were apparent. Modification of endoplasmic reticulum with enlarged attached polyribosomes, breakdown of plasma membrane, accumulation of cytoplasmic amorphous material and karyorrhexis and karyolysis of nuclei were the most prominent features in non-viral cases.

Acute glandular fever-like illness in a patient with HTLV-III antibody.

A lymph node biopsy obtained from a patient with human T-cell lymphocytotropic virus III/lymphadenopathy-associated virus (HTLV-III/LAV) antibody, presenting with an acute glandular fever-like illness, was examined by electron microscopy. Numerous pathological changes were present in the biopsy, including hypertrophy of smooth endoplasmic reticulum, intracytoplasmic rod-like inclusions within the cisternae of endoplasmic reticulum, multivesicular bodies, test-tube and ring-shaped forms, and tubulo-reticular structures. Intranuclear and intracytoplasmic viral-like particles measuring 105-120 nm in diameter and small cytoplasmic particles measuring 50-70 nm in diameter were found in some degenerating lymph node cells. These pathological findings may reflect a host cell response to various pathological and viral stimuli resulting from immune deficiency owing to infection with HTLV-III/LAV.

Maternal transmission of duck hepatitis B virus in pedigree Pekin ducks.

A 14-month old female Pekin duck experimentally infected as an embryo with duck hepatitis B virus via the amniotic route has been a chronic carrier of duck hepatitis B virus with very high (P/N) values of DNA polymerase activity since hatching. All the progeny were, on evaluation for congenital infection, found to be duck hepatitis B virus positive by endogenous DNA polymerase reaction and electron microscopy. These offspring remained persistently viremic throughout the study. Maternal transmission therefore bred true to a total of 49 offspring--24 ducklings (less than 24 hr old) and 25 ducks--studied. Six of these 25 ducks matched for age and sex and bled weekly for 6 weeks exhibited fluctuating plasma levels of DNA polymerase activity. Higher DNA polymerase activity was detected in newly hatched ducklings than in older viremic ducks. This observation was corroborated with the results of electron microscopic examination of thin sections of liver. Duck hepatitis B virus particles, located within vesicles of rough endoplasmic reticulum in the cytoplasm of hepatocytes, were more abundant, and therefore more readily observed, in ducklings than in older ducks.

Experimental in ovo transmission of duck hepatitis B virus.

Inoculation of fertile Pekin duck eggs with diluted serum containing DHBV into eggs incubated for 24 h and into the extra-embryonic cavities of 14-day-old embryos resulted in a high proportion of viraemic ducklings irrespective of the route of inoculation. Long-term observation of som of the ducks established that the viraemia induced experimentally is long-lasting and has persisted for periods up to 16 mth post-hatch. Separation of DHBV from the plasma of carrier ducks by rate zonal centrifugation was examined by DNA polymerase (DNAP) activity. Particles in the fraction with peak DNAP activity had a buoyant density of 1.16 g X cm-3 in sucrose and an estimated sedimentation coefficient, S20.w of 77. DHBV particles, the morphology of which could be resolved under the electron microscope, consisted of a coat (about 10 nm in thickness) surrounding a core with a diameter measuring 40 nm but not 27 nm as previously reported. Spike-like projections were found on the surface of the core as described previously by W.S. Mason, G. Seal and J. Summers, 1980, J. Virol. 36, 829-836.

Studies by electron microscopy on the assembly of duck hepatitis B virus in the liver.

Liver specimens from 1-day-old ducklings infected in ovo with maternally transmitted duck hepatitis B virus (DHBV) were examined by electron microscopy. Complete and incomplete DHBV particles were located within hypertrophied cisternae of the endoplasmic reticulum of the hepatocytes. The complete viral particles found intracellularly have inner cores with a diameter ranging from 35 to 37.5 nm and an outer coat or envelope. The whole particle measures approximately 45-65 nm in diameter. Naked core particles were located in the nuclei, free in the cytoplasm, and also near or on the cisternal membrane of the endoplasmic reticulum on the cytoplasmic face. Duck hepatitis B virions appear to share morphological characteristics with the viral coat and core of human hepatitis B virus (HBV). Electron microscopy suggested that the core particles of DHBV migrate from the nucleus into the cytoplasm through the nuclear pores. The complete viral particles are probably formed by protrusion of the core particles through the endoplasmic reticulum and by simultaneous encapsulation with a coat derived from the endoplasmic reticulum.

Biochemical and immunological properties of Coxiella burnetii cell wall and peptidoglycan-protein complex fractions.

Coxiella burnetii morphological cell types were fractionated into large-cell variant cell walls, two fractions of small-cell variant cell walls, and one fraction of small-cell variant whole cells. Based on the contents of peptidoglycan (PG)-constituents and the yields of the sodium dodecyl sulfate-insoluble PG-protein complex (PG-PC) from cell walls, the fraction of large-cell variant cell walls contained significantly less PG than did the fraction of small-cell variant cell walls. The yields of PG-PC from the fractions of large-cell variant cell walls and small-cell variant cell walls were 2 and 32%, respectively. These results indicated that the PG of the large-cell variant cell walls may be partially digested by PG-lytic enzymes or incompletely synthesized, whereas the small-cell variant cell walls appeared to have intact PG. Proteins associated with PG-PC were resistant to proteolysis by trypsin, protease VI, and proteinase K. Saturated and unsaturated fatty acids were detected in whole cells and cell walls but not in PG-PC, which contained a 3-deoxy-D-mannooctulosonic acid-like component that is also present in phase I lipopolysaccharide. Immunogenicity of the fractions was tested by measuring the temporal sequence of phase II and phase I antibody responses in vaccinated rabbits. Both phase II and phase I antibody responses were demonstrated with all fractions except the sodium dodecyl sulfate supernatant of the small-cell variant cell walls, whereas PG-PC elicited a pure phase II antibody response up to 29 days postvaccination. The immunogenicity of these fractions may reflect a quantitative difference in antigen concentration or may be due to a qualitative difference in phase II and I determinants.

Persistent lymphadenopathy in homosexual men: a clinical and ultrastructural study.

Ultrastructural changes (tubuloreticular structures and tube and ring shaped forms) previously described in patients with acquired immunodeficiency syndrome (AIDS) are described for the first time in the lymph nodes and circulating lymphocytes of patients with persistent lymphadenopathy. These observations support the view that the persistent lymphadenopathy syndrome and AIDS are caused by the same transmissible agent(s).

Intracytoplasmic inclusions in human hepatocytes in non-A, non-B hepatitis: an ultrastructural study.

An ultrastructural study was carried out on 114 liver biopsies obtained for diagnostic purposes from patients with various pathological disorders of the liver including hepatitis B-related liver disease, non-A, non-B hepatitis, alcoholic liver disease, fatty change, and cryptogenic cirrhosis. The opportunity was taken to evaluate the significance of intracytoplasmic crystalline structures found in the hepatocytes of nine patients with a variety of liver disorders. The cytoplasmic inclusions varied in size up to 2 microns in length and shape and were not limited by membranes. The presence of these inclusions cannot, however, be correlated either specifically with non-A, non-B hepatitis or with other known nonviral liver disease. The functional, physiological, and pathological significance of the crystalline structures remain to be elucidated.

A study of ultrastructural alterations in experimental non-A, non-B hepatitis by electron-beam analysis.

An electron-beam X-ray microanalysis was carried out on sections of liver biopsy specimens obtained from chimpanzees infected with non-A, non-B hepatitis. The microanalysis was concentrated over areas where typical derangement of the endoplasmic reticulum, with the formation of tubular forms possessing walls with electron-dense central membrane, was visualized. These tubular structures are regarded as the most notable pathological alteration in affected hepatocytes. However, the electron-probe microanalysis showed no deviation of the energy spectrum when compared with the background or control analysis.

Application of electron microscopy to the study of structural changes in the liver in non-A, non-B hepatitis.

Ultrastructural studies employing techniques such as alternative electron metal stain, high-angle tilting and high-voltage electron microscopy were carried out on liver biopsies obtained from chimpanzees infected with non-A, non-B hepatitis. Typical derangement of the endoplasmic reticulum leading to the formation of tubular structures in hepatocytes was observed. The use of potassium permanganate as an alternative stain revealed two features which have not been previously described. The first of these shows the wall of the tubular structures to be composed of a well-defined fibrillar-like meshwork with a periodicity of approximately 15 nm. The second feature is the demonstration of clusters of fibrin-like inclusions consisting of striated fibrils in the neighborhood of the tubular structures. The presence of intracytoplasmic fibrin may indicate non-specific morphological evidence of cell injury. Crystalline structures containing arrays of particles with an average size of 24 nm were also observed in the endoplasmic reticulum of endothelial cells of the hepatic sinusoids. Morphological differences between the crystalline lattice and the reticular arrangement, demonstrated with the use of high-angle tilting of the specimen in the electron microscopy suggest that the arrays may not be viral particules but a reflection of pathological response of the host cell.