Roy D. LeGrand

The superantigen Pseudomonas exotoxin a requires additional functions from accessory cells for T lymphocyte proliferation

We have examined the functions required of accessory cells (AC) for murine thymocyte proliferation induced by Pseudomonas exotoxin A (PE) and have compared these functions to those required of a known superantigen, staphylococcal enterotoxin B (SEB). We demonstrate that PE, like SEB, preferentially stimulates PNA+ thymocytes expressing a specific V beta element within the T cell receptor. However, PE requires functions from AC that are distinct from those required by SEB. AC treated with paraformaldehyde (PCHO) prior to stimulation supported thymocyte proliferation induced by SEB but not PE. However, when AC were treated with PCHO subsequent to stimulation with PE, thymocyte proliferation was observed, which suggests that PE requires antigen processing in addition to presentation. Furthermore, treatment of AC with lysosomotropic agents abrogated thymocyte proliferation induced by PE but not SEB. Antibodies to MHC class II molecules inhibited thymocyte proliferation induced by both PE and SEB. In addition, we observed that interleukin 1 alpha (IL-1 alpha) participated in the proliferation of thymocytes induced by PE but not SEB. Thus, our data indicate that PE is a unique microbial superantigen that requires additional AC functions for T lymphocyte proliferation.

Glutathionylation of lens proteins through the formation of thioether bond

Formation of lanthionine, a dehydroalanine crosslink, is associated with aging of the human lens and cataractogenesis. In this study we investigated whether modification of lens proteins by glutathione could proceed through an alternative pathway: that is, by the formation of a nonreducible thioether bond between protein and glutathione. Direct ELISA of the reduced water-soluble and water-insoluble lens proteins from human cataractous, aged and bovine lenses showed a concentration-dependent immunoreactivity toward human nonreducible glutathionyl-lens proteins only. The reduced water-insoluble cataractous lens proteins showed the highest immunoreactivity, while bovine lens protein exhibited no reaction. These data were confirmed by dot-blot analysis. The level of this modification ranged from 0.7 to 1.6 nmol/mg protein in water-insoluble proteins from aged and cataractous lenses. N-terminal amino acid determination in the reduced and alkylated lens proteins, performed by derivatization of these preparations with dansyl chloride followed by an exhaustive dialysis, acid hydrolysis and fluorescence detection of dansylated amino acids by RP-HPLC, showed that N-terminal glutamic acid was present in concentration of approximately 0.2 nmol/mg of lens protein. This evidence points out that at least some of the N-terminal amino groups of nonreducible glutathione in the reduced human lens proteins are not involved in a covalent bond formation. Since disulfides were not detected in the reduced and alkylated human lens proteins, GSH is most likely attached to lens proteins through thioether bonds. These results provide, for the first time, evidence that glutathiolation of human lens proteins can occur through the formation of nonreducible thioether bonds.

Inhibition of adenylate cyclase activity in the corneal epithelium by anti-inflammatory steroids

Abstract Adenylate cyclase activity in bovine corneal epithelial or rat kidney particulate fractions was completely inhibited by clinically employed levels of anti-inflammatory steroids. Dose-response studies using several steroids showed that dexamethasone phosphate was the most potent enzyme inhibitor, followed respectively by prednisolone phosphate and hydrocortisone phosphate. Enzyme inhibition occurred regardless of the level or mechanism of adenylate cyclase activation, although agonist-stimulated activity was more sensitive to inhibition than fluoride-stimulated activity. Steroids appear to cause a direct, reversible and non-competitive inhibition of adenylate cyclase, affecting both the apparent substrate affinity and maximum velocity of the enzyme. Application of high levels of anti-inflammatory steroids may block the reported cyclic AMP-mediated effects of increased epithelial wound closure rates and stimulated epithelial basement membrane protein synthesis in a healing cornea.

Distribution of cyclic AMP-dependent protein kinase in the bovine cornea

Soluble and particulate fractions of bovine corneal epithelium, stroma and endothelium were prepared and assayed for protein kinase activity. All preparations exhibited measurable activity in the absence of cyclic AMP (cyclic AMP-independent activity) as well as significant enzyme stimulation in its presence (cyclic AMP-dependent activity). The epithelial layer contained over 90% of the total corneal cyclic AMP-dependent protein kinase activity, almost all of it in the soluble fraction. In contrast, over one-third of the endothelial activity was expressed by the particulate fraction. All preparations showed high affinity and high specificity for, cyclic AMP in terms of binding cyclic [3H]AMP and stimulating protein kinase activity. Elution characteristics of soluble preparations on DEAE cellulose columns demonstrated that epithelial and endothelial layers contained predominantly ‘Type II cyclic AMP-dependent protein kinase, whereas stroma had the ‘Type I enzyme. Each preparation studies also exhibited endogenous substrates for cyclic AMP-dependent protein kinase.

Selective activation of murine Vβ8.2 bearing T cells by Pseudomonas exotoxin A

We have determined that Pseudomonas aeruginosa exotoxin A (PE) can selectively stimulate the proliferation of V beta bearing T lymphocytes. Murine thymocytes were fractionated by selective agglutination with peanut agglutinin (PNA) and the PNA- thymocytes, which represent mature thymocytes, were shown to be responsive to PE stimulation. In addition, mature peripheral T lymphocytes (nylon wool nonadherent splenocytes) were also observed to respond to PE stimulation. Both CD4+ and CD8+ splenic T lymphocyte populations proliferated in response to PE. Flow microfluorimetry analysis of PNA- thymocytes stimulated with PE indicated that V beta 8.2 bearing T cells were preferentially expanded. Thus, our data indicate that PE represents a microbial super antigen which stimulates murine thymocytes which bear the V beta 8.2 element of the T cell receptor.

Regulation of cyclic AMP-dependent protein kinase and glycogen synthase by cyclic AMP in the bovine cornea

Bovine corneas were treated in vitro with various drug regimens and cyclic AMP, protein kinase and glycogen synthase activities were determined in each tissue extract. Isoproterenol (alone) did not increase corneal cyclic AMP under any condition tested. The phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (IBMX), produced a maximal 2·7-fold increase over control cyclic AMP values. Combinations of isoproterenol (or other β -adrenergic agonists) and IBMX stimulated cyclic AMP up to 25-fold over control. These increases were blocked completely by propranolol. Prostaglandin E 2 (with IBMX) was the only other drug tested which caused a significant increase (6·7-fold over control) in corneal cyclic AMP. Protein kinase activity was stimulated proportionally by cyclic AMP increases up to 8-fold over control. At this point the protein kinase activity ratio was essentially 1·0, indicating complete activation of the enzyme. Glycogen synthase activity decreased sharply in response to increases in corneal cyclic AMP and protein kinase activity. Maximal enzyme inhibition required only fourfold increases in cyclic AMP and approximately half-maximal stimulation of cyclic AMP-dependent protein kinase. These data indicate the conditions necessary to stimulate cyclic AMP in the cornea and the physiological relevance of cyclic AMP increases in terms of its effects on other corneal enzymes.

Sugar-mediated crosslinking of α-biotinylated-lys to cysteamine-agarose support

Advanced glycation end products (AGEs) and, specifically, protein-protein AGE crosslinks have long been studied for their potential role in aging, diabetic complications and Alzheimer disease. With few exceptions, the chemical nature of these structures remains unknown. We report here a simple approach that allows the preparation and isolation of milligram quantities of sugar-mediated AGE Lys-Lys-like crosslinks from glycation mixtures. The method is based on a sugar-dependent incorporation of Nα-biotinyl-l-Lys into cysteaminyldisulfide Sepharose 6B (AE-S-S-Sepharose 6B). Glycation mixtures with six different sugars showed a time- and sugar-dependent decrease in the concentration of the support-bound primary amino groups and accounted for almost 90% loss of cysteaminyl amino groups at the end of the various incubation periods. 4-Hydroxyazobenzene-2-carboxylic acid-avidin assays indicated the incorporation of Nα-biotinyl-l-Lys equal to 8% of the total support amino groups with methylglyoxal after 7d and 1% with fructose and glucose after 1 mo of incubation. Treatment of the washed, sugar-modified supports with 2-mercaptoethanol released the bulk of the bound AGE modifications and the crosslinks. Subsequent fractionation of these preparations over a monomeric avidin column afforded a complete separation of sugar-mediated AGE modifications and the crosslinks. Depending on the sugar employed, micromolar amounts of biotinylated Lys-Lys-like crosslinks were generated by this two-step procedure from 8 mL of the original AE-S-S-Sepharose 6B.Advanced glycation end products (AGEs) and, specifically, protein-protein AGE crosslinks have long been studied for their potential role in aging, diabetic complications and Alzheimer disease. With few exceptions, the chemical nature of these structures remains unknown. We report here a simple approach that allows the preparation and isolation of milligram quantities of sugar-mediated AGE Lys-Lys-like crosslinks from glycation mixtures. The method is based on a sugar-dependent incorporation of N(alpha)-biotinyl-L-Lys into cysteaminyldisulfide Sepharose 6B (AE-S-S-Sepharose 6B). Glycation mixtures with six different sugars showed a time- and sugar-dependent decrease in the concentration of the support-bound primary amino groups and accounted for almost 90% loss of cysteaminyl amino groups at the end of the various incubation periods. 4-Hydroxyazobenzene-2-carboxylic acid-avidin assays indicated the incorporation of N(alpha)-biotinyl-L-Lys equal to 8% of the total support amino groups with methylglyoxal after 7 d and 1% with fructose and glucose after 1 mo of incubation. Treatment of the washed, sugar-modified supports with 2-mercaptoethanol released the bulk of the bound AGE modifications and the crosslinks. Subsequent fractionation of these preparations over a monomeric avidin column afforded a complete separation of sugar-mediated AGE modifications and the crosslinks. Depending on the sugar employed, micromolar amounts of biotinylated Lys-Lys-like crosslinks were generated by this two-step procedure from 8 mL of the original AE-S-S-Sepharose 6B.