Electron cytochemical studies of Cryptococcus neoformans grown on uric acid and related sources of nitrogen.

Cells of Cryptococcus neoformans grown on xanthine or urate as the sole sources of nitrogen produced numerous, single membrane-bound organelles, deemed to be microbodies. Electron images of these structures showed positive cytochemical staining for catalase and alpha-hydroxy acid oxidase, known marker enzyme activities for microbodies. Microbodies in xanthine and urate-grown cells were cytochemically reactive for the presence of the hydrogen peroxide-producing xanthine and urate oxidases. Molybdenum and phosphorus (elements associated with the cofactor common to nitrogen scavenging enzymes) were detected in the substrate-induced microbodies by X-ray dispersive microanalysis. The single limiting membrane of the substrate-induced microbody was stained by a modified Gomori reaction for the presence of alkaline phosphatase, thereby suggesting the participation of this enzymic activity in the events associated with microbody chemistry.

Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum.

Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.

Fibronectin and postpartum infection in rabbits: an animal model.

A rabbit model was explored in preliminary studies of uterine colonization and infection, following cesarean section (CS), by toxigenic Staphylococcus aureus (Harrisburg) and other bacterial species known to be isolated from traumatized human uteri; effects on host susceptibility of fibronectin prophylaxis were studied. Histological characteristic and plasma fibronectin levels were recorded in double-blinded studies of four groups of rabbits (55 total), all sacrificed 96 h after CS performed at term. Animals which were inoculated without fibronectin prophylaxis, showed stable or slightly elevated plasma fibronectin levels through 96 h. Rabbits inoculated with nontoxigenic S. aureus (Wiley) exhibited stable fibronectin levels and less microscopic evidence of infection. All inoculated animals were colonized, but those that received fibronectin cryoprecipitate intravenously at CS showed less microscopic evidence of infection. Fibronectin levels for rabbits scores as 'seriously infected' were nearly 1.5 times that of controls. This is in contrast to other clinical studies in which chronic infections and poor clinical outcome have been strongly correlated with decreased plasma fibronectin levels. Histochemical studies of antifibronectin binding suggested that less fibronectin is deposited throughout the endometrial stroma of animals that received fibronectin prior to bacterial challenge. An increased polymorphonuclear leukocytic reaction in the stroma near the incisions was observed in animals that received fibronectin prophylaxis, with or without bacterial challenge. This reaction may be characterized as an acute-phase reaction.

Mitochondrial morphology in Basidiobolus haptosporus.

Young hyphal cells of the potentially zoopathogenic fungus Basidiobolus haptosporus characteristically exhibit unusual proportions of annulate views of mitochondria in the two-dimensional perspective of thin sections. Such views exhibit a central space containing cytoplasmic ground substance and often profiles of other cytoplasmic organelles (lipid bodies, other mitochondrial forms, and especially crystalloid-containing microbodies). Three-dimensional projections are presented to suggest that these mitochondria have assumed the form of a goblet-shaped enclosure, and that the various annulate views are the consequence of plane of section viewed by electron microscopy. Their frequent occurrence and consistent morphology argues against their being random expressions of mitochondrial plasticity, but rather for close spatial associations amongst cytoplasmic organelles of young hyphae. When the fungus is grown on xanthine or its catabolites as sole sources of nitrogen, there is a proliferation of crystalloid-containing microbodies, double-membraned vesicles, and ovate to ellipsoidal mitochondria. Annulate views of mitochondria then are no longer observed, but microbodies again frequently appear in close association with mitochondria and at times in intimate contact with the mitochondrial outer membrane.

Virus-like particles in Basidiobolus species.

Transmission electron microscopy of ultrathin sections of hyphal cells of Basidiobolus haptosporus and B. ranarum revealed the presence of particulate inclusions consistent in morphology with that described in certain other fungi as virus-like particles. Two types of particles were observed which differed in substructure and distribution within the host cytoplasm. Relative numbers of these particles were never great, and their presence in the host was not associated with any demonstrable cytopathological changes detectable at the ultrastructural level. We believe this report to be the first description of an association of virus-like particles with fungi of the Entomophthoraceae.

Electron cytochemical demonstration of phosphatase activity with microbody membranes of Basidiobolus haptosporus.

Growth of cells of the potentially zoopathogenic fungus Basidiobolus haptosporus on a nutritionally defined medium with xanthine or urate as the nitrogen source results in greatly increased populations of microbodies. Modified Gomori procedures at the electron microscopic level suggested the single limiting membrane (and in some cases the granular matrix) of immature microbodies to be the exclusive subcellular locale(s) of alkaline phosphatase, 5'-nucleotidase and nucleoside diphosphatase activities. When grown in the presence of low inorganic phosphate, additional alkaline phosphatase activity was further identified cytochemically at and along profiles of endoplasmic reticulum and on inclusions previously described as "double-membraned vesicles". Cytochemical localization of acid phosphatase at microbody membranes was minimal if not ambiguous; Mg++-dependent adenosine triphosphatase and glucose-6-phosphatase were not identified at these locales. Quantitative biochemical estimates of alkaline phosphatase activity levels in particulate fractions initially increased with age of cells, perhaps as a function of the cultural induction and marked increase in immature microbody populations. We suggest that this enzyme may participate in some manner with protein translocation mechanisms associated with microbody biogenesis, ontogeny, and/or physiological function.

Possible role of cholesterol in the susceptibility of a human acute lymphoblastic leukemia cell line to dexamethasone.

Because the absence of high affinity glucocorticoid receptors in lymphoid leukemia cells does not always correlate with the resistance to glucocorticoids in vitro, an effort was made to identify an alternate biochemical marker which, independently from the receptor system, would improve the reliability of the existing in vitro sensitivity assays as predictors of the patients' response to glucocorticoid therapy. Two receptor-containing lines of acute lymphoblastic leukemia, one glucocorticoid sensitive (CEM-C7) and one glucocorticoid resistant (CEM-C1), were used to study endogenous synthesis of cholesterol with [14C]-acetate as a cholesterol precursor. We found that dexamethasone inhibited cholesterol synthesis in the glucocorticoid-sensitive clone but was without effect on the resistant clone. In sensitive cells, this reduction in synthesis was paralleled by a decreased activity of 3-hydroxy-3-methylglutaryl coenzyme A synthase (EC 1.1.3.5) and was followed by a moderate decrease in cellular free cholesterol. Moreover, sonic dispersions of cholesterol in delipidized serum when added to the cultures reversed the growth-inhibitory effect of dexamethasone on CEM-C7 cells.

Localization of alkaline phosphatase activity at microbody membranes of Neurospora crassa and Aspergillus nidulans.

Hyphal cells of Neurospora crassa and Aspergillus nidulans, grown in Sabouraud glucose broth or in a defined medium with xanthine or its catabolites as the nitrogen source, contained single membrane-bound organelles cytochemically identified as microbodies. Modified Gomori procedures at the ultrastructural level revealed putative alkaline phosphatase activity sites in thin sections of cells of both species of fungi. Microbody membranes displayed electron opaque deposits (lead phosphate) which were absent in control preparations either lacking the substrate (beta-glycerophosphate) or the lead capture ion. Inhibition of this enzymic activity was achieved in parallel incubations fortified with the inhibitor levamisole. Cells grown in media containing limiting levels of inorganic phosphate revealed additional alkaline phosphatase activity at and along the nuclear membrane and endoplasmic cisternae. Hexagonal inclusions found in the cytoplasm of N. crassa (but not A. nidulans) and believed to arise from microbody precursors were without demonstrable cytochemical staining for microbody marker oxidases.

Double-membraned vesicles and their possible role in the ontogeny of microbodies of Basidiobolus haptosporus.

A concomitant but transient occurrence of large numbers of double-membraned vesicles with immature, culturally induced microbodies was demonstrated in thin sections of Basidiobolus haptosporus soon after transfer of the fungus to a defined medium containing xanthine or its catabolites as the sole source of nitrogen. Double-membraned vesicles were rapidly formed as derivatives of endoplasmic cisternae later to occur free in the cytoplasm, often as the most predominant cytoplasmic inclusion. Densitometric measurements revealed that heavy metal-binding cytoplasmic constituents were concentrated within the vesicular lumen. Most of the double-membraned vesicles appeared to undergo degeneration; they were rare to absent in thin sections of older cells. At times, double-membraned vesicles were seen connected to newly formed microbodies in a manner suggesting ontogenic relationships. The outer vesicular membrane was continuous with the single limiting membrane of the microbody via a short tubule. We interpret these observations as empirical ultrastructural evidence to suggest that some double-membraned vesicles become specialized and function as precursor inclusions in the biogenesis of new microbody populations committed to purine salvage.

Ultrastructural cytology of Basidiobolus haptosporus: morphology and electron cytochemistry of microbodies.

Vegetative and reproductive cells of Basidiobolus haptosporus possess naturally occurring organelles identified as microbodies. Cells of four other species of the genus contained morphologically indistinguishable organelles. Microbodies were invariably present in cells of the fungi grown on routine mycological media. The constitutive microbody was characterized by a single, intensely electron-opaque crystalloid body which rapidly enlarged to fill the organellar compartment. The microbody then underwent degeneration by an autolytic-like process. Growth of the fungi on xanthine and its catabolites as sole nitrogen sources (but not urea) greatly enhanced the production of new microbodies in which protein was initially accumulated as paracrystalline arrays. These inclusions then underwent reorganization and compaction to form crystalloid bodies. Key enzymes of the purine degradation pathway are believed to be core proteins of the crystalloid. D-amino acid oxidase, alpha-hydroxy acid oxidase, xanthine oxidase and urate oxidase (but not catalase) were detected cytochemically in mature microbodies. Significant levels of phosphorus and molybdenum were present in the microbody crystalloid by X-ray dispersive microanalysis; iron and copper were not detected. The ability of Basidiobolus species to assimilate xanthine and its catabolites might explain their ecological association with the gut and cloacal contents of various amphibia, reptiles and fish.

Compactin (ML-236B) reduces the content of filipin-cholesterol complexes in the plasma membrane of chronic lymphocytic leukemia cells.

The polyene antibiotic filipin was used to visualize the presence and distribution of cholesterol in the plasma membrane of glutaraldehyde-fixed human chronic lymphocytic leukemia (CLL) cells. Both compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and 25-hydroxycholesterol reduced the content of filipin-cholesterol complexes in the plasma membrane of CLL cells grown in media supplemented with either 15% delipidized horse serum or 15% normal (whole) horse serum. The reduction due to compactin was reversed by the concomitant addition of mevalonolactone. The ability of compactin to reduce the relative cholesterol content (as judged by filipin labeling) in CLL cells grown in lipoprotein-containing (normal) serum suggest that either CLL cells are different from other cells in that they predominantly utilize endogenously synthesized cholesterol for incorporation into the plasma membrane, or that a separate pool of endogenously synthesized cholesterol provides cholesterol for the plasma membrane.

Ultrastructural aspects of cytoplasmic ribosomes from Histoplasma capsulatum and Blastomyces dermatitidis as revealed by heavy metal staining.

Cytoplasmic ribosomes were isolated and purified from sonicates of the mycelial and yeastlike growth forms of the pathogenic dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Similar ribosomal fractions were prepared from Neurospora crassa and Saccharomyces cerevisiae. These latter organisms were selected as typical filamentous and yeastlike monophasic fungi, and their ribosomes were used as reference standards. High resolution electron microscopy permitted a comparison of both positively and negatively-stained ribosomes to those dehydrated without heavy metal salt. Such studies revealed statistically significant differences in physical dimensions. Cautious interpretations of substructural detail of the various ribosomal preparations suggested both interphasic and interspecies differences.

A model for the structure of bovine parathormone derived by dark field electron microsocpy.

We have analyzed images of parathormone obtained by dark field electron microscopy in order to determine the three-dimensional structure of the molecule. The technique of autocorrelation was used to differentiate hormone particles from the background of noise in the electron micrographs. Our data suggest that parathormone is about 36 A in maximum dimension and is comprised of two interconnected domains of different mass that occur in a consistent orientation to each other. By means of the formulation of Chou and Fasman (Chou, P.Y., and Fasman, G.D. (1974) Biochemistry 13, 211-222), we predicted the secondary structure of the hormone and fitted this into the three-dimensional structure developed by microscopy. The resultant speculative model can explain certain physical and chemical properties of parathormone.

Real-space filtering of electron images from optical autocorrelation patterns.

The capability for filtering electron images, in real space, is demonstrated to be inherent to a two plate, incoherent optical procedure (originally described by Meyer-Eppler and Darius) for recording autocorrelation functions from transparencies. If an image contains features that occur with more than random frequency, these features may give rise to a resolvable peak in the optically recorded autocorrelation function. The optical requirements for obtaining an image of the transparency that is filtered to observe only features giving rise to some set of such peaks, or to exclude them, are described. The principle is to form an image of the transparency, with a properly placed plano-convex lens, from the incoherent light transmitted through apertures positioned over the peaks in the autocorrelation plane. The application of the method in defining the position and orientation of specific projections of protein molecules, as observed in negative stain by bright-field, or unstained by dark-field electron microscopy, is also described.

Cd-metallothionein-a test object for dark-field studies of protein structure.

Cadmium-metallothionein contains about six metal atoms per 6,000 molecular weight, reflecting the high proportion of cystein residues in the structure. Because of the strong scattering from probable Cd-S (Cyst)3 complexes, the protein is unambiguously visualized by conventional tilted beam dark-field electronmicroscopy. The projections of the structure correspond to free, aggregated and partially denatured forms of the presumed native structure, a hexahedral mass, 36 multiplied by 25 multiplied by 16 A. Relaxed states of this structure show that the molecule is comprised of two similar, covalently linked "half-metallothineins", each comprised of three domains. Each of the six domains of the native structure is evidently formed by stacking of two characteristic scattering centers together, at a separation of 9 A. A speculative scheme for the folding of the native structure is presented. The results are interpreted as substantiating the fidelity of dark-field images of small proteins, and the cadmium-metallothionein molecule is suggested as a standard test object for the method.