Orally active multi-functional antioxidants delay cataract formation in streptozotocin (type 1) diabetic and gamma-irradiated rats

Background

Age-related cataract is a worldwide health care problem whose progression has been linked to oxidative stress and the accumulation of redox-active metals. Since there is no specific animal model for human age-related cataract, multiple animal models must be used to evaluate potential therapies that may delay and/or prevent cataract formation.

Methods/Principal Findings

Proof of concept studies were conducted to evaluate 4-(5-hydroxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 4) and 4-(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 8), multi-functional antioxidants that can independently chelate redox metals and quench free radicals, on their ability to delay the progression of diabetic “sugar” cataracts and gamma radiation-induced cataracts. Prior to 15 Gy of whole head irradiation, select groups of Long Evans rats received either diet containing compound 4 or 8, or a single i.p. injection of panthethine, a radioprotective agent. Compared to untreated, irradiated rats, treatment with pantethine, 4 and 8 delayed initial lens changes by 4, 47, and 38 days, respectively, and the average formation of posterior subcapsular opacities by 23, 53 and 58 days, respectively. In the second study, select groups of diabetic Sprague Dawley rats were administered chow containing compounds 4, 8 or the aldose reductase inhibitor AL1576. As anticipated, treatment with AL1576 prevented cataract by inhibiting sorbitol formation in the lens. However, compared to untreated rats, compounds 4 and 8 delayed vacuole formation by 20 days and 12 days, respectively, and cortical cataract formation by 8 and 3 days, respectively, without reducing lenticular sorbitol. Using in vitro lens culture in 30 mM xylose to model diabetic “sugar” cataract formation, western blots confirmed that multi-functional antioxidants reduced endoplasmic reticulum stress.

Conclusions/Significance

Multi-functional antioxidants delayed cataract formation in two diverse rat models. These studies provide a proof of concept that a general cataract treatment focused on reducing oxidative stress instead of a specific mechanism of cataractogenesis can be developed.     

Redox status of the testes and sperm of rats following exposure to 2,5-hexanedione

Objectives: Exposure to 2,5-hexanedione (2,5-HD) is well known to be associated with reproductive dysfunctions in both humans and animals. However, the role of oxidative stress in 2,5-HD-induced toxicity in testes and sperm has not yet been studied.

Methodology: The present study investigated the influence of 2,5-HD on antioxidant systems in the testes and epididymal sperm of rats following exposure to 0, 0.25, 0.5, and 1% 2,5-HD in drinking water for 21 consecutive days.

Results: Administration of 0.5% 2,5-HD significantly (P?<?0.05) decreased epididymis weight, whereas 1% 2,5-HD-treated rats showed significantly decreased body weight, testis, and epididymis weights compared with the control group. Exposure to 2,5-HD caused a significant dose-dependent increase in the activities of superoxide dismutase, catalase, and glutathione peroxidase in both testes and sperm compared with the control group. Moreover, 2,5-HD-exposed rats showed significant decrease in glutathione-S-transferase activity and glutathione level with concomitant significant elevation in the levels of hydrogen peroxide and malondialdehyde in both testes and sperm. Testicular and epididymal atrophy with significant, dose-dependent, decrease in epididymal sperm number, sperm motility, and viability were observed in 2,5-HD-treated rats.

Conclusion: 2,5-HD exposure impaired testicular function and sperm characteristics by disruption of the antioxidant systems and consequently, increased oxidative stress in the treated rats.     

NSUN3 and ABH1 modify the wobble position of mt‐tRNAMet to expand codon recognition in mitochondrial translation

Mitochondrial gene expression uses a non‐universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt‐)tRNA^Met mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt‐tRNA^Met to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m⁵C34 of mt‐tRNA^Met to generate an f⁵C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m⁵C34 mt‐tRNA^Met in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt‐tRNA^Met function. Together, our data reveal how modifications in mt‐tRNA^Met are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNA^Met to recognise the different codons encoding methionine.     

The acute phase reactant response to respiratory infection with Chlamydia pneumoniae: implications for the pathogenesis of atherosclerosis

The acute phase response to Chlamydia pneumoniae infection was analyzed over a 72 h period post-infection in C57BL/6J mice. A single intra-nasal inoculation stimulated statistically significant increases in the plasma levels of IL-2, IL-5, IL-6, IL-10, IL-12, GM-CSF, IFN-γ, and serum amyloid A but not TNF-α, IL-1β, IL-4 and serum amyloid P. There was also a decrease in the activity of the HDL protective enzyme paraoxonase as well as a reduced ability of HDL to prevent oxidation of palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine by hydroperoxyoctadecadienoic acid at 48 and 72 h post-infection. To determine whether the C. pneumoniae induced acute phase response had any effect on atherosclerotic plaque stability, we measured the frequency of intra-plaque hemorrhage as a marker of plaque disruption in the innominate arteries of apolipoprotein E deficient mice at 29–30 weeks and 1.5–2.0 years of age. There was an increased frequency of intra-plaque hemorrhage only in the older mice infected with the live organism (8/14) as compared to mice treated with killed C. pneumoniae (2/11) or sham inoculated with PBS (2/12). These results suggest that acute phase reactant proteins produced in response to pulmonary infection with C. pneumoniae may contribute to the progression and destabilization of atherosclerotic lesions.     

DNA barcoding of blastocystis

We have developed a simple method for subtyping the intestinal protistan parasite Blastocystis using an approach equivalent to DNA barcoding in animals. Amplification of a 600 bp region of the small subunit ribosomal RNA gene followed by single primer sequencing of the PCR product provides enough data to assign isolates to specific subtypes unambiguously. We believe that this approach will prove useful in future epidemiological studies.     

Patterns of evolution in the unique tRNA gene arrays of the genus Entamoeba

Genome sequencing of the protistan parasite Entamoeba histolytica HM-1:IMSS revealed that almost all the tRNA genes are organized into tandem arrays that make up over 10% of the genome. The 25 distinct array units contain up to 5 tRNA genes each and some also encode the 5S RNA. Between adjacent genes in array units are complex short tandem repeats (STRs) resembling microsatellites. To investigate the origins and evolution of this unique gene organization, we have undertaken a genome survey to determine the array unit organization in 4 other species of Entamoeba-Entamoeba dispar, Entamoeba moshkovskii, Entamoeba terrapinae, and Entamoeba invadens-and have explored the STR structure in other isolates of E. histolytica. The genome surveys revealed that E. dispar has the same array unit organization as E. histolytica, including the presence and numerical variation of STRs between adjacent genes. However, the individual repeat sequences are completely different to those in E. histolytica. All other species of Entamoeba studied also have tandem arrays of clustered tRNA genes, but the gene composition of the array units often differs from that in E. histolytica/E. dispar. None of the other species' arrays exhibit the complex STRs between adjacent genes although simple tandem duplications are occasionally seen. The degree of similarity in organization reflects the phylogenetic relationships among the species studied. Within individual isolates of E. histolytica most copies of the array unit are uniform in sequence with only minor variation in the number and organization of the STRs. Between isolates, however, substantial differences in STR number and organization can exist although the individual repeat sequences tend to be conserved. The origin of this unique gene organization in the genus Entamoeba clearly predates the common ancestor of the species investigated to date and their function remains unclear.     

Fever response to intravenous prostaglandin E2 is mediated by the brain but does not require afferent vagal signaling

PGE2 produced in the periphery triggers the early phase of the febrile response to infection and may contribute to later phases. It can be hypothesized that peripherally synthesized PGE2 transmits febrigenic signals to the brain via vagal afferent nerves. Before testing this hypothesis, we investigated whether the febrigenic effect of intravenously administered PGE2 is mediated by the brain and is not the result of a direct action of PGE2 on thermoeffectors. In anesthetized rats, intravenously injected PGE2 (100 microg/kg) caused an increase in sympathetic discharge to interscapular brown adipose tissue (iBAT), as well as increases in iBAT thermogenesis, end-expired CO2, and colonic temperature (Tc). All these effects were prevented by inhibition of neuronal function in the raphe region of the medulla oblongata using an intra-raphe microinjection of muscimol. We then asked whether the brain-mediated PGE2 fever requires vagal signaling and answered this question by conducting two independent studies in rats. In a study in anesthetized rats, acute bilateral cervical vagotomy did not affect the effects of intravenously injected PGE2 (100 microg/kg) on iBAT sympathetic discharge and Tc. In a study in conscious rats, administration of PGE2 (280 microg/kg) via an indwelling jugular catheter caused tail skin vasoconstriction, tended to increase oxygen consumption, and increased Tc; none of these responses was affected by total truncal subdiaphragmatic vagotomy performed 2 wk before the experiment. We conclude that the febrile response to circulating PGE2 is mediated by the brain, but that it does not require vagal afferent signaling.