Effects of cytoprotective antioxidants on lymphocytes from representative mitochondrial neurodegenerative diseases

Two new aza analogues of the neuroprotective agent idebenone have been synthesized and characterized. Their antioxidant activity, and ability to augment ATP levels have been evaluated in several different cell lines having suboptimal mitochondrial function. Both compounds were found to be good ROS scavengers, and to protect the cells from oxidative stress induced by glutathione depletion. The compounds were more effective than idebenone in neurodegenerative disease cells. These novel pyrimidinol derivatives were also shown to augment ATP levels in coenzyme Q₁₀-deficient human lymphocytes. The more lipophilic side chains attached to the pyrimidinol redox core in these compounds resulted in less inhibition of the electron transport chain and improved antioxidant activity.     

Synthesis and evaluation of verticipyrone analogues as mitochondrial complex I inhibitors

Verticipyrone has recently been isolated from the culture broth of Verticillium sp. and shown to inhibit NADH fumarate reductase, as well as NADH oxidoreductase (complex I) of the mitochondrial electron transport chain. In order to assess the structural elements in verticipyrone essential for complex I inhibitor, 15 structural analogues were prepared and analyzed for their effects on mitochondrial NADH oxidoreductase and NADH oxidase activities. Also measured were the abilities of several of the analogues to inhibit respiration as judged by a shift to glycolysis, and to inhibit the growth of several mammalian cell lines. The nature of the pyrone ring was shown to be important to potency of inhibition, as was the length and nature of substituents in the side chain of the analogues.     

Analysis of the structural and mechanistic factors in antioxidants that preserve mitochondrial function and confer cytoprotection

Selected pyridinol analogues of the experimental neuroprotective drug idebenone have been synthesized and evaluated as antioxidants capable of preserving mitochondrial function. The compounds, having a different redox core but the same side chain as idebenone, exhibited a range of potencies, reflecting differences in their structures. The results obtained provide guidance in the design of such analogues with improved properties. Analogues were identified that have significantly improved antioxidant activity compared with idebenone in cultured lymphocytes, and which exhibit lesser inhibition of the electron transport chain.     

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich’s ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF LEPTOSPIRA INTERROGANS SEROVAR POMONA ANTIBODIES IN BOVINE SERA

An indirect ELISA was developed and initially evaluated for the detection of bovine antibodies to Leptospira interrogans serovar pomona. The antigen used in this ELISA was extracted from a serovar pomona culture supernatant by a combination of centrifugation, digestion with proteinase K and ultra-centrifugation. The antigen showed little cross-reaction with immune rabbit sera to L. interrogans serovars copenhageni, grippotyphosa, hardjo and sejroe and, Leptospira biflexa serovar patoc. Some cross-reaction was observed with immune rabbit serum to L. interrogans serovar canicola. The relative sensitivity of the ELISA was 94.76% confidence interval =± 3.32%) when estimated with bovine sera (n=172) with serovar pomona microscopic agglutination test (MAT) titers of 100. The relative specificity of the ELISA was 99.28% (95% confidence interval = 1.40%) when estimated with bovine sera (n=139) with MAT titers of <100 to L. interrogans serovars canicola, copenhageni, grippotyphosa, hardjo, pomona and sejroe. Thirty six of 258 field sera (13.95%) with serovar pomona MAT titers of <100, gave positive reactions in the ELISA.     

DEVELOPMENT AND EVALUATION OF A RAPID ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme-linked immunofiltration assay (ELIFA) and a microtitre plate enzyme-linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.     

Simplified bicyclic pyridinol analogues protect mitochondrial function

Bicyclic pyridinol antioxidants have been reported to suppress the autoxidation of methyl linoleate more effectively than α-tocopherol in benzene solution. A few novel lipophilic analogues have recently been synthesized by conjugating a pyridinol core with the phytyl side chain of α-tocopherol; these have been shown to possess potent antioxidant activity. However, the complexity of the synthetic routes has hampered their further development. Herein, we describe a facile approach, involving only five synthetic steps, to simplified analogues (1a-1c) and their acetate ester precursors (2a-2c). Simple alkyl chains of different lengths were attached to the 6-methyl group of the antioxidant core via regioselective metalation. These analogues were found to retain biological activity and exhibit protective behaviour under conditions of induced oxidative stress, which could lead to the development of more readily accessible analogues as potential antioxidants capable of preserving mitochondrial function.     

Effects of alkyl side chain modification of coenzyme Q10 on mitochondrial respiratory chain function and cytoprotection

The effect of the alkyl side chain length of coenzyme Q₁₀ on mitochondrial respiratory chain function has been investigated by the use of synthetic ubiquinone derivatives. Three analogues (3, 4 and 6) were identified that exhibited significantly improved effects on mitochondrial oxygen consumption and mitochondrial membrane potential, and also conferred significant cytoprotection on cultured mammalian cells in which glutathione had been depleted by treatment with diethyl maleate. The analogues also exhibited lesser inhibition of the electron transport chain than idebenone. The results obtained provide guidance for the design of CoQ₁₀ analogues with improved activity compared to that of idebenone (1), the latter of which is undergoing evaluation in the clinic as a therapeutic agent.