Infectious endophthalmitis: an ocular emergency.
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Anti-Bacterial Agents↗
Biomedical subjects
R E Morris
Publications and source records attributed to R E Morris.
Trafficking of Pseudomonas exotoxin A in mammalian cells.
Experiments designed to elucidate cellular internalization of Pseudomonas aeruginosa exotoxin A are described. Inhibition of protein synthesis was used as an index of the biological activity of exotoxin A, and a biotinyl-toxin: avidin-gold system to follow its movement on the ultrastructural level. Addition of amantadine, methylamine and dansylcadaverine to cells enhanced the toxicity of exotoxin A at lower concentrations, while protecting cells at higher concentrations. In general, both sensitive and resistant cell lines responded similarly. Exposure of LM or Vero cells to an acidic extracellular pH did not overcome the protection afforded by ammonium chloride against exotoxin A cytotoxicity. This and other data suggest that sensitive and resistant cells may internalize exotoxin A in a similar manner, the toxin entering the cytosol from a prelysosomal acidic vacuole.
ADP Ribose Transferases↗
Receptor-mediated entry of diphtheria toxin into monkey kidney (Vero) cells: electron microscopic evaluation.
To express toxicity in living cells, diphtheria toxin (DT) must cross a membrane barrier and reach its target in the cytosol. Here we examine the entry of DT into the toxin-sensitive monkey kidney (Vero) cells. Using electron microscopy we directly demonstrated for the first time that DT is internalized by receptor-mediated endocytosis, i.e., via clathrin-coated pits, and enters the endosomal system. Methylamine, which is known to protect cells from DT, stopped the movement of toxin to coated areas of the cell membrane. In the presence of amine, prebound biotinyl-DT was internalized, but toxicity was inhibited. Biochemical evidence revealed that methylamine maintained toxin molecules at a site accessible to neutralization by antitoxin. The data suggest that DT entering Vero cells in the presence of methylamine is sequestered within the cell and does not express toxicity.
Animals↗
Route of pseudomonas and diphtheria toxin entry into mammalian cells: basis for susceptibility to toxin.
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Acrylates↗
Cellular enzyme-linked immunospecific assay (CELISA). IV. Inhibition of endogenous cellular alkaline phosphatase activity.
The cellular enzyme-linked immunospecific assay (CELISA) uses ligands conjugated to calf intestinal alkaline phosphatase to quantitate antibody bound to antigens on the surfaces of whole cells. Since the endogenous alkaline phosphatase present in many cell types contributes unwanted background noise to the assay, ways of inhibiting endogenous cellular alkaline phosphatase were investigated. We found that the endogenous alkaline phosphatase in human peripheral blood mononuclear cells was incompletely inhibited by EDTA or L-cysteine. Levamisole, however, inhibited endogenous cellular alkaline phosphatase completely without impairing the sensitivity of the CELISA. The use of levamisole is recommended for assays that use alkaline phosphatase conjugates to detect molecules on the surfaces of cells that also contain endogenous alkaline phosphatase.
Alkaline Phosphatase↗
Abortive ectromelia virus infection in peritoneal macrophages activated by Corynebacterium parvum.
We have previously demonstrated that peritoneal macrophages (M phi S) from C3H mice were resistant to in vitro infection by ectromelia virus, following activation by intraperitoneal injection of the immunomodulator Corynebacterium parvum. In contrast, resident and mineral oil-elicited M phi S were fully susceptible to virus infection. This report analyzes the infectious cycle of ectromelia virus in C parvum-activated and mineral oil-elicited M phi S and demonstrates that an abortive infection occurred in the activated M phi S that blocked the infectious cycle prior to the release of DNA from the infecting virions. The kinetics of adsorption of radiolabeled virus were similar in both susceptible and resistant M phi cultures; however, viral-induced incorporation of uridine and thymidine occurred only in the mineral oil-elicited and not the C parvum-activated M phi S. In addition, the late protein hemagglutinin was only detected in infected cultures of susceptible mineral oil-elicited M phi S. An electron micrographic analysis of the infectious cycle indicated that the adsorption of virus to the plasma membrane, uptake into lysosomes, and the primary undercoating and release of viral cores into the M phi cytoplasm were identical in both M phi types. In contrast, secondary uncoating (release of genomic DNA from the viral cores into the cytoplasm) was never detected in infected C parvum M phi S. These data are consistent with our previous findings and with the hypothesis that activation of M phi S by C parvum induces an interferon-mediated resistance to ectromelia virus infection.
Animals↗
Evidence for pseudomonas exotoxin A receptors on plasma membrane of toxin-sensitive lm fibroblasts.
Pseudomonas exotoxin A enters mouse LM fibroblasts by receptor-mediated endocytosis and ultimately causes cell death. Here we present evidence for the existence of a specific receptor for the toxin. Toxin association with LM cells at 18 and 37 degrees C, but not at 4 degrees C, was highly specific. At 37 degrees C, the association increased with time, reaching a steady state by 5 h. Binding to paraformaldehyde-fixed cells at 37 degrees C was saturable (Kd = 5.4 nM), was reversible, and indicated ca. 100,000 binding sites per cell. It is believed that receptor-bound toxin is responsible for cell death. Once the kinetics of toxin entry were described, we examined the effect of reduced temperatures on the intracellular processing of toxin and thus its expression. Toxin-induced inhibition of protein synthesis was minimal at temperatures below 20 degrees C. This was seen even though at 20 degrees C sufficient toxin was internalized to kill cells, and toxin enzyme activity was maximal. Internalization of 125I-labeled toxin, but not of 125I-labeled horseradish peroxidase (marker of fluid-phase endocytosis), became rate limiting at 20 degrees C or below. These data suggest that reduced temperatures block a step in the receptor-mediated endocytic pathway essential for the expression of Pseudomonas toxin activity.
Animals↗
Traumatic dural tears: CT diagnosis using metrizamide.
Computed tomography of the spine using intrathecal metrizamide was performed on six patients with spinal trauma. Dural tears with contrast material escaping outside the subarachnoid space were documented in five cases. In one case, the thecal laceration had healed and the contrast material was confined in a post-traumatic meningocele. The characteristics of dural tears are demonstrable by metrizamide computed tomography, either in conjunction with myelography or as a separate procedure. Neurological deficits following spinal trauma may be aggravated by dural tears with entrapment of the spinal nerve roots.
Adult↗
Visualization of intracellular trafficking: use of biotinylated ligands in conjunction with avidin-gold colloids.
A simple, sensitive method to visualize the binding and internalization of protein ligands by cells in culture is described. A biotinylated toxin was used as ligand, and succinoylated avidin adsorbed onto 5.2 nm gold sols was the electron-dense marker. This method affords direct localization of proteins that are on the cell surface or intracellular without need for techniques that alter membrane integrity.
Animals↗
Erysipeloid.
Erysipelothrix infections have been a well-known clinical entity for over a century. Only a few cases have been studied histologically, and the agent has rarely been cultured from infected patients. We present here a case of erysipeloid of Rosenbach, in which histologic study and electron microscopy document the presence of microorganisms. We also present a complete review of the clinical and pathologic features of Erysipelothrix infections and their treatment. Our observations suggest that Erysipelothrix rhusiopathiae is capable of producing L forms that may revert to a bacterial form and produce sepsis at a later time.
Erysipeloid↗
Receptor-mediated entry of Pseudomonas toxin: methylamine blocks clustering step.
Clustering of ligands into coated regions of the plasma membrane is an early step in receptor-mediated endocytosis. The association of Pseudomonas exotoxin A (PE) with mouse LM fibroblasts was visualized by using biotinyl-PE and avidingold. Movement of PE into coated regions occurred within 30 s of warming monolayers to 37 degrees C. This clustering was stopped by the primary amines methylamine and ammonium chloride but was not altered by the tertiary amine chloroquine. Toxin internalization was rapid, with a half-time of approximately 5 min. Although primary amines stopped clustering, they did not alter the rate of toxin internalization; they did alter the route followed after entry. We have shown previously that methylamine protects cells from the lethal action of PE. Here we suggest that methylamine protects, at least in part, by blocking clustering, and that receptor-mediated endocytosis is required for efficient expression of PE toxicity.
ADP Ribose Transferases↗
Age-related loss of Lyt-1,2 cells in C58 mice results in susceptibility to lactic dehydrogenase virus-induced polioencephalomyelitis.
C58 mice (aged greater than or equal to 5 months) are susceptible to age-dependent polioencephalomyelitis, a paralytic central nervous system disease induced by lactic dehydrogenase virus. Susceptibility results from the loss of protective T cells. Data are presented showing a positive correlation between the age-related loss of Lyt-1,2 cells and the development of susceptibility to neuroparalytic lactic dehydrogenase virus infection.
Aging↗
Diphtheria toxin does not enter resistant cells by receptor-mediated endocytosis.
Diphtheria toxin (DT) and pseudomonas toxin are two distinct microbial toxins which inhibit protein synthesis in an identical manner, i.e., by the inactivation of cytoplasmic elongation factor 2. Although murine cells bind both toxins, they are 10,000-fold less sensitive to DT than to pseudomonas toxin. This suggests that the level of resistance resides at some event after binding. We have previously shown that pseudomonas toxin enters mouse LM fibroblasts by receptor-mediated endocytosis, a process in which ligand is internalized via specialized clathrin-coated pits and moves to the Golgi region and then to the lysosomes. Here, we visually follow the entry and trafficking of DT by resistant mouse fibroblasts. A biotinyl-toxin-avidin-gold system was used to visualize DT on the ultrastructural level. DT entered resistant cells through non-clathrin-coated regions of the plasma membrane and within 2.5 to 5 min was seen in lysosomes. Only rarely was DT seen in coated pits or in the Golgi region. Furthermore, the temperature dependence of internalization of 125I-labeled horseradish peroxidase and 125I-labeled DT by LM cells was similar. On the basis of these observations, we postulate that DT does not enter DT-resistant LM cells by receptor-mediated endocytosis and that receptor-mediated endocytosis is required for efficient expression of toxicity.
Animals↗
Cellular enzyme-linked immunospecific assay (CELISA). III. Use of the CELISA to quantitate monoclonal antibodies bound to HLA antigens and to subset-specific antigens on cell surfaces.
The cellular enzyme-linked immunospecific assay (CELISA) quantitates the binding of antibody to cell-surface antigens. When conditions that produced the maximum specific enzymatic activity of the conjugate were used, the CELISA was able to screen hybridoma supernatant fluids for polymorphic anti-HLA monoclonal antibodies using as few as 20,000 normal human peripheral blood mononuclear target cells per well. The CELISA detected as little as 24 pg (0.55 fM or 330 X 10(6) molecules) of cell-surface HLA alloantigen and as little as 40 pg of monoclonal antibody bound to cell-surface antigen. In conjunction with various monoclonal antibodies to human peripheral blood mononuclear cell differentiation antigens, the CELISA was used to quantitate levels of different peripheral blood mononuclear cell subsets in normal and diseased people objectively, thus providing a simple, sensitive, and inexpensive alternative to immunofluorescence microscopic and flow cytometric techniques normally used to phenotype peripheral blood mononuclear cells.
Animals↗
The role of endoscopic vitreous microsurgery in proliferative diabetic retinopathy.
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Diabetic Retinopathy↗
A simple reliable method for producing electron dense markers of uniform size for use in immunoelectron microscopy.
A method is presented for the preparation of electron dense markers of defined size for use in immunoelectron microscopy, The electron dense markers were formed by the covalent binding of rabbit IgG and horse spleen ferritin mediated by glutaraldehyde. We demonstrated that the conjugate size, i.e. the number of ferritin grains per unit conjugate, was directly related to the time of incubation at 4 degrees C in a 1000 molar excess of glutaraldehyde. Conjugates were separated on the basis of molecular size by Sepharose 4B column chromatography. Three different sized conjugates were used to delineate pseudomonas toxin receptors on the surface of mouse LM fibroblasts; similar numbers of receptors were observed with all conjugates.
Animals↗
Inhibition of cellular protein synthesis by vaccinia virus surface tubules.
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Cell Line↗
Tumor and histocompatibility antigen interaction: direct visualization by a double-label bridging technique for immuno-electron microscopy.
The nature of the interaction between the Ib tumor-associated surface antigen (Ib-TASA) and the Dk and Kk (Dk/Kk) regions of the major histocompatibility complex on the surface of line Ib lymphocytes was evaluated by immuno-electron microscopy using a double-label bridging technique. Quantitation of antigenic sites by direct counts showed 44% of the Ib-TASA sites coincident with Dk/Kk antigens. This is greater than the predicted random interaction of two independent cell surface determinants suggesting an associative interaction, i.e. 'altered-self' antigen. The remaining 56% of the Ib-TASA sites were in a non-associative mode. On the basis of data presented here, we postulate that the interaction between 'self'- and 'non-self' antigens is not an 'all or none' phenomenon.
Animals↗