4-Octyl itaconate protects chondrocytes against IL-1ß-induced oxidative stress and ferroptosis by inhibiting GPX4 methylation in osteoarthritis.

The role of oxidative stress and ferroptosis in osteoarthritis (OA) pathogenesis is increasingly recognized. Notably, 4-octyl Itaconate (OI) has been documented to counteract oxidative stress and inflammatory responses, highlighting its therapeutic potential in OA. This study explored the effects of OI on GPX4 methylation, oxidative stress, and ferroptosis in chondrocytes affected by OA. Our results demonstrated that OI mitigated IL-1ß-induced chondrocyte degeneration in a dose-dependent manner. It also suppressed reactive oxygen species (ROS) production and sustained GPX4 expression, thereby attenuating the degenerative impact of IL-1ß and Erastin on chondrocytes by curtailing ferroptosis. Moreover, we observed that blocking GPX4 methylation could alleviate IL-1ß-induced degeneration, oxidative stress, and ferroptosis in chondrocytes. The regulatory mechanism of OI on GPX4 expression in chondrocytes involved the inhibition of GPX4 methylation. In a mouse model of OA, OI's protective effects against OA were comparable to those of Ferrostatin-1. Thus, OI reduced chondrocyte degeneration, oxidative stress, and ferroptosis by inhibiting GPX4 methylation, offering a novel mechanistic insight into its therapeutic application in OA.

Design and evaluation of new analogs of the sweet protein brazzein.

We have previously modeled the interaction of the sweet protein brazzein with the extracellular domains of the sweet taste receptor. Here, we describe the application of that model to the design of 12 new highly potent analogs of brazzein. Eight of the 12 analogs have higher sweetness potency than wild-type brazzein. Results are consistent with our brazzein-receptor interaction model. The model predicts binding of brazzein to the open form of T1R2 in the T1R2-T1R3 heterodimer.

Unusually high frequency MHC class I alleles in Mauritian origin cynomolgus macaques.

Acute shortages of Indian origin Rhesus macaques significantly hinder HIV/AIDS research. Cellular immune responses are particularly difficult to study because only a subset of animals possess MHC class I (MHC I) alleles with defined peptide-binding specificities. To expand the pool of nonhuman primates suitable for studies of cellular immunity, we defined 66 MHC I alleles in Cynomolgus macaques (Macaca fascicularis) of Chinese, Vietnamese, and Mauritian origin. Most MHC I alleles were found only in animals from a single geographic origin, suggesting that Cynomolgus macaques from different origins are not interchangeable in studies of cellular immunity. Animals from Mauritius may be particularly valuable because >50% of these Cynomolgus macaques share the MHC class I allele combination Mafa-B*430101, Mafa-B*440101, and Mafa-B*460101. The increased MHC I allele sharing of Mauritian origin Cynomolgus macaques may dramatically reduce the overall number of animals needed to study cellular immune responses in nonhuman primates while simultaneously reducing the confounding effects of genetic heterogeneity in HIV/AIDS research.

Probing the sweet determinants of brazzein: wild-type brazzein and a tasteless variant, brazzein-ins(R18a-I18b), exhibit different pH-dependent NMR chemical shifts.

Brazzein is a small, intensely sweet protein. As a probe of the functional properties of its solvent-exposed loop, two residues (Arg-Ile) were inserted between Leu18 and Ala19 of brazzein. Psychophysical testing demonstrated that this mutant is totally tasteless. NMR chemical shift mapping of differences between this mutant and brazzein indicated that residues affected by the insertion are localized to the mutated loop, the region of the single alpha-helix, and around the Cys16-Cys37 disulfide bond. Residues unaffected by this mutation included those near the C-terminus and in the loop connecting the alpha-helix and the second beta-strand. In particular, several residues of brazzein previously shown to be essential for its sweetness (His31, Arg33, Glu41, Arg43, Asp50, and Tyr54) exhibited negligible chemical shift changes. Moreover, the pH dependence of the chemical shifts of His31, Glu41, Asp50, and Tyr54 were unaltered by the insertion. The insertion led to large chemical shift and pKa perturbation of Glu36, a residue shown previously to be important for brazzein's sweetness. These results serve to refine the known sweetness determinants of brazzein and lend further support to the idea that the protein interacts with a sweet-taste receptor through a multi-site interaction mechanism, as has been postulated for brazzein and other sweet proteins (monellin and thaumatin).

Monkey electrophysiological and human psychophysical responses to mutants of the sweet protein brazzein: delineating brazzein sweetness.

Responses to brazzein, 25 brazzein mutants and two forms of monellin were studied in two types of experiments: electrophysiological recordings from chorda tympani S fibers of the rhesus monkey, Macaca mulatta, and psychophysical experiments. We found that different mutations at position 29 (changing Asp29 to Ala, Lys or Asn) made the molecule significantly sweeter than brazzein, while mutations at positions 30 or 33 (Lys30Asp or Arg33Ala) removed all sweetness. The same pattern occurred again at the beta-turn region, where Glu41Lys gave the highest sweetness score among the mutants tested, whereas a mutation two residues distant (Arg43Ala) abolished the sweetness. The effects of charge and side chain size were examined at two locations, namely positions 29 and 36. The findings indicate that charge is important for eliciting sweetness, whereas the length of the side-chain plays a lesser role. We also found that the N- and C-termini are important for the sweetness of brazzein. The close correlation (r = 0.78) between the results of the above two methods corroborates our hypothesis that S fibers convey sweet taste in primates.

Critical regions for the sweetness of brazzein.

Brazzein is a small, heat-stable, intensely sweet protein consisting of 54 amino acid residues. Based on the wild-type brazzein, 25 brazzein mutants have been produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29-33 and 39-43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness of brazzein. Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor.

Expression of Anti-PCNA mRNA Ribozyme by T7 Vaccinia Virus System in HeLa Cells.

We have designed and synthesized a hammer-head Rz targeted to human proliferating cell nuclear antigen (PCNA) mRNA at site No.524. Results showed that Rz524 can cleave the transcribed substrate RNA site-specifically in vitro. A self trimming expression plasmid of this Rz was constructed and co-introduced into HeLa cells with T7 vaccinia virus. Dot-blotting hybridization showed that the Rz was expressed in HeLa cells.