Point mutations of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) have been linked to familial amyotrophic lateral sclerosis (FALS). We reported that Cu,Zn-SOD can catalyze free radical generation and a FALS mutant, G93A, exhibits an enhanced free radical-generating activity, while its dismutation activity is identical to that of the wild-type enzyme (Yim, M. B., Kang, J.-H., Yim, H.-S., Kwak, H.-S., Chock, P. B., and Stadtman, E. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5709 -5714). The A4V mutation is both the most commonly detected of FALS-associated SOD1 mutations and among the most clinically severe (Rosen, D. R., Bowling, A. C., Patterson, D., Usdin, T. B., Sapp, P., Mezey, E., McKenna-Yasek, D., O'Regan, J. P., Rahmani, Z., Ferrante, R. J., Brownstein, M. J., Kowall, N. W., Beal, M. F., Horvitz, H. R., and Brown, R. H., Jr. (1994) Hum. Mol. Genet. 3, 981-987). We cloned the cDNA for the FALS A4V mutant, overexpressed the protein in Sf9 insect cells, purified the protein, and studied its enzymic activities. Our results show that the mutant and wild-type enzymes contain one copper ion per subunit and have identical dismutation activities. However, the free radical-generating activity of the mutant, as measured by the spin trapping method at low H 2 O 2 concentration, is enhanced relative to that of the wild-type and G93A enzyme (wild-type < G93A < A4V). This is due to the decrease in the K m value for H 2 O 2 , wild-type > G93A > A4V, while the k cat is identical for these enzymes. Thus, the FALS symptoms are not associated with the reduction in the dismutation activity of the mutant enzyme. The fact that the A4V mutant has the lowest K m for H 2 O 2 is correlated to the clinical severity observed with the A4V patients, if FALS is associated with a differential gain of the free radical-generating function of the Cu,Zn-SOD mutant. Familial amyotrophic lateral sclerosis (FALS)1 is an autosomal dominant disorder of motor neurons of cortex, brainstem, and spinal cord (1). Recent studies showed that FALS cases have missense mutations in the coding regions in SOD1, the gene for Cu,Zn-superoxide dismutase (Cu,Zn-SOD) (2, 3) that catalyzes the dismutation of superoxide radical anions (O 2 . ) to hydrogen peroxide and oxygen molecules (4). Cu,Zn-SOD also catalyzes free radical generation using H 2 O 2 and small anions as substrates (5-7). Most of the FALS mutants have pointmutation sites in conserved interaction regions critical to the subunit fold and dimer contact, rather than residues in the active-site or in the electrostatic active channel (3). Initial studies of Cu,Zn-SOD activity in erythrocytes and brain tissues of FALS patients carrying mutations at the SOD1 locus demonstrated reduced Cu,Zn-SOD dismutation activity compared with that of normal individuals (3, 8 -11). This reduction in SOD dismutation activity may facilitate the pathway of oxidative damage to cause FALS symptoms. However, several studies with transgenic mice (12, 13), transfected cells (14, 15), and lymphoblasts of patients (16) also demonstr...
Gonadotropin-regulated testicular RNA helicase (GRTH/ DDX25), a testis-specific member of the DEAD-box family, is an essential post-transcriptional regulator of spermatogenesis. Failure of expression of Transition protein 2 (TP2) and Protamine 2 (Prm2) proteins (chromatin remodelers, essential for spermatid elongation and completion of spermatogenesis) with preservation of their mRNA expression was observed in GRTHnull mice (azoospermic due to failure of spermatids to elongate). These were identified as target genes for the testis-specific miR-469, which is increased in the GRTH-null mice. Further analysis demonstrated that miR-469 repressed TP2 and Prm2 protein expression at the translation level with minor effect on mRNA degradation, through binding to the coding regions of TP2 and Prm2 mRNAs. The corresponding primary-microRNAs and the expression levels of Drosha and DGCR8 (both mRNA and protein) were increased significantly in the GRTH-null mice. miR-469 silencing of TP2 and Prm2 mRNA in pachytene spermatocytes and round spermatids is essential for their timely translation at later times of spermiogenesis, which is critical to attain mature sperm. Collectively, these studies indicate that GRTH, a multifunctional RNA helicase, acts as a negative regulator of miRNA-469 biogenesis and consequently their function during spermatogenesis.Mammalian spermatogenesis is a complex process in which primary germ cells undergo mitotic and meiotic divisions to generate haploid round spermatids, and proceed to the differentiation process of spermiogenesis that produces elongating spermatids and mature sperm. This process is regulated at the transcriptional and post-transcriptional levels by the integrated expression of an array of testicular genes in a precise temporal sequence (1, 2). Chromatin compactation that occurs in elongated spermatids during spermiogenesis is essential for nuclear condensation to generate mature spermatozoa. This repackaging event is achieved by replacing histones with transition proteins (TP1 and TP2), which in turn are replaced by protamines (Prm1 and Prm2). The initial active transcription phase with translational repression is followed by cessation of transcription associated with chromatin modifications. mRNA of genes that are essential for later stages of spermiogenesis are generated well prior their translation. Several mRNAs that associate with messenger ribonuclear proteins are repressed translationally at cytoplasmic sites, presumably in the chromatoid body of round spermatids.Gonadotropin-regulated testicular RNA helicase (GRTH 2 / DDX25), a testis-specific member of the DEAD (Asp-Glu-GlyAsp)-box family present in Leydig and germ cells (meiotic spermatocytes and round spermatids) is regulated developmentally by androgen at the transcriptional level (3-6). GRTH is a multifunctional protein, and as component of messenger ribonuclear protein, it transports target mRNAs from the nucleus to cytoplasmic sites (chromatoid bodies, a perinuclear organelle of nuage structure in spermatids for stor...
Two complementary sets of conditions for radical additions of thiols to terminal ynamides are described. The use of 1 equiv of thiol affords the cis-β-thioenamide adducts in rapid fashion (10 min) and good dr, whereas employing excess thiol and longer reaction times favors the trans products.Ynamides are a class of compounds that have gained prominence in recent years. 1 They are electron-rich alkynes, 2 although their nucleophilicity can be tuned by varying the nature of the N-acyl group. Malacria has demonstrated the utility of ynamides as radical acceptors. 3,4 We reasoned that they should react readily with thiyl radicals, which are electrophilic in nature. 5 A recent report by Yorimitsu and Oshima detailing radical additions of arenethiols to internal tosylynamides 6 lent support to this hypothesis.The addition of a thiyl radical to an ynamide produces a β-thioenamide. This moiety is present in unusual cyclic peptides such as thioviridamide 7 (Figure 1) and the lantibiotics. 8 Inspired by the striking architecture of these natural products, we investigated additions of thiyl radicals to terminal ynamides. Herein, we report the initial results of our study, which demonstrate that both cis and trans β-thioenamides can be obtained selectively by simply varying the reaction conditions.The proposed reaction is shown in Figure 2. Regioselective addition of a thiyl radical to the terminal carbon of ynamide A would provide vinyl radicals B and/or C. These intermediates would rapidly equilibrate, and hydrogen atom abstraction from the thiol by the less hindered radical C, according to the precedent of Montevecchi and co-workers, 9 should afford cis-β-thioenamide D as the kinetic product. In contrast, known methods of β-thioenamide construction based on imine acylation 10 or Pummerer rearrangement 11 chemistry deliver predominantly the trans isomers. We also recognized that the presence of excess thiol in the reaction mixture would permit isomerization of D to the thermodynamically more stable trans isomer via a radical addition-β-thiyl radical elimination pathway. 12 Accordingly, we pursued a stereoselective synthesis of both cis-and trans-β-thioenamides by seeking two complementary sets of reaction conditions. We began by studying the additions of commercially available thiols to simple ynamides. Our results are collected in Table 1. Addition of excess n-butyl thiol (4 equiv) to acyclic amide- derived ynamide 1 13 in refluxing t-BuOH with AIBN as initiator 14 afforded β-thioenamide E-3a as the major product of a separable mixture (72%, 15:1 E:Z) after 3 h. In contrast, employing 1 equiv of thiol and 0.5 equiv of AIBN led to Z-3a in good yield (76%, 1:11 E:Z) after only 10 min. Similar trends were observed with thiophenol, although greater quantities of the E isomer were obtained under both sets of conditions. When the radical addition of tert-butyl thiol to 1 was performed under the Z-selective conditions, no reaction was observed. Subjection of this bulky thiol to the typically E-selective conditions a...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.