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...

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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...

Apolipoproteins have been often found to be highly enriched in the serum protein coronas produced on various engineered nanoparticles (NPs), which is also known to greatly influence the behaviors of protein corona NPs in the biological systems. As most of the apolipoproteins in blood are associated with lipoproteins, it suggests the active involvement of lipoproteins in the formation of biomolecular coronas on NPs. However, the interactions of lipoprotein complexes with NPs in the corona formation have been rarely understood. In this study, to obtain insights into the interactions, the formation of biomolecular coronas of high-density lipoproteins (HDLs) on the PEGylated gold NPs (PEG-AuNPs) of various sizes (20−150 nm dia.) was investigated as a model system. The results of this study revealed a noticeable size dependence, which is a drastic increase in the affinity of HDL for larger NPs and thus less-curved NP surfaces. For example, only a few HDLs per NP, which correspond to 5% surface coverage, were found to constitute the hard coronas of HDLs on 20 nm PEG-AuNPs, whereas 73% surface coverage was assessed for larger 150 nm PEG-AuNPs. However, the relative affinities of HDL and apolipoprotein A-1 (APOA1) examined in competition with human serum albumin exhibited the opposite size dependences, which suggests that the adsorption of HDLs is not driven by the constituent protein, APOA1. In fact, the total strength of non-covalent intermolecular interactions between a HDL particle and a NP relies on the physical contact between the two particles, which thus depends on the varying curvatures of spherical NPs in this case. Therefore, it was concluded that it is whole HDL complex that interacts with the spherical PEG-AuNPs in the initial stage of adsorption toward biomolecular coronas, which is unveiled by the distinct size dependence observed in this study.

The prolactin receptor (PRLR) is a member of the lactogen/ cytokine receptor family which mediates the diverse cellular actions of prolactin (PRL) in several target tissues. PRL is a major factor in the proliferation and differentiation of breast epithelium and is the primary hormone in the stimulation and maintenance of lactation. It is also a tumor promoter in rodents and has been implicated in the development of breast cancer (5). The human PRLR (hPRLR) has several forms, including a long form and several short forms, which are products of alternative splicing with variable lengths in their cytoplasmic domains containing some unique sequences (8, 9). The short forms S1a and S1b are dominant-negative repressors of the function of homodimers of the PRLR long form that mediates all known stimulatory functions of PRL through the Jak-2/Stat5 signaling pathway. The dominant-negative effect of the short forms results from their heterodimerization with the long form, leading to inactivation due to the lack of required cytoplasmic sequences for STAT signaling activation (23, 29). Intramolecular S-S bonds in the extracellular domain of the PRLR short forms were found to be required for their inhibitory action on PRL-induced PRLR long form-mediated STAT5-dependent action (29). The PRLR forms are expressed in normal and tumoral breast tissue, in most human breast cancer cells, and in several other tissues (8,17,19). In humans, hPRLR expression is controlled by a complex regulatory system at the transcriptional level which is governed by multiple promoters. These include the preferentially utilized generic promoter 1/exon 1 (PIII/hEI3), which also is present in rat and mouse, and five human-specific exon 1 promoters (hE1N1 to hE1N5) (11, 12). These promoters were found to be utilized in breast cancer tissue and cell lines, including MCF7 and T47D, and variably in other tissues (12). Among these promoters, the preferentially utilized human promoter III (hPIII), the human counterpart of rodent PIII, was functionally characterized in breast cancer cells. 17␤-Estradiol (E2) induced an increase in PRLR mRNA transcripts directed by the preferentially utilized hPIII promoter. Also, in transfection studies E2 activated the hPIII promoter, which lacks an estrogen response element (ERE) (16). This promoter contains functional SP1 and C/EBP␤ elements that bind SP1/SP3 and C/EBP␤, respectively (10, 12). The abolition of the E2 effect by the mutation of SP1 and C/EBP␤ elements within PIII indicated the cooperation in E2-induced transcription of hPRLR. E2 regulates PRLR expression through a nonclassical ERE-independent mechanism in target cells. The E2/estrogen receptor ␣ (ER␣) complex translocates to the nucleus and binds transfactors at the hPIII promoter, leading to the recruitment of coactivators (p300, SRC-1, and pCAF) to the complex with consequent region-specific changes in histone acetylation. These hormone/receptor-induced associations and chromatin changes favored TFIIB and RNA polymerase II recruitment, leading to the activa...

Cytokines such as interleukin-1b (IL-1b) stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide overproduction leading to b-cell damage. Meanwhile, glucagon-like peptide-1 (GLP-1) and its potent analog exendin-4 (EX-4) were well known for b-cell proliferation. However, the protective mechanisms of GLP-1 in b-cells exposed to cytokines were not fully elucidated. Therefore, the effects of EX-4 on the IL-1b-induced iNOS gene expression were investigated employing RINm5F b-cells. EX-4 inhibited IL-1b-induced iNOS protein expression and nitrite production. However, northern blot and promoter analyses showed that EX-4 failed to inhibit IL-1b-induced iNOS mRNA expression and iNOS promoter activity. By electrophoretic mobility shift assay (EMSA), EX-4 did not alter the binding activity of NF-kB to the iNOS promoter. Consistent with the EMSA result, EX-4 did not inhibit nuclear translocation of p65. We also tested the effect of EX-4 on iNOS mRNA stability. Actinomycin D chase experiments showed that EX-4 did not affect the decay rate of iNOS mRNA and the promoter assay using the construct containing 3 0 -untranslated region of iNOS showed that EX-4 did not alter the stability of iNOS mRNA. Meanwhile, forskolin significantly inhibited IL-1b-induced iNOS protein, which was reversed by H-89, a protein kinase A (PKA) inhibitor. Moreover, EX-4 pretreatment restored IL-1b-induced decrease in cAMP toward control level. Additionally, the cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. We therefore concluded that EX-4 inhibited IL-1b-induced iNOS protein and nitrite production via cAMP/PKA system irrespective of both transcriptional and posttranscriptional mechanisms of iNOS gene, and this inhibitory effect of EX-4 appears to be regulated at posttranslational level.

cagon-like peptide-1 and its potent agonist exendin-4 induce several immediate early response genes (IEGs) that code for transcription factors implicated in cell proliferation, differentiation, and apoptosis. We recently observed that early growth response factor-1 (EGR-1), an IEG product, was required for transcriptional activation of Ccnd1 (cyclin D1) gene by exendin-4. Herein, the regulatory mechanism whereby exendin-4 activates the transcription of EGR-1 gene was investigated in the pancreatic ␤-cell line INS-1. Deletion analysis of rat EGR-1 promoter identified a critical region between Ϫ73 and Ϫ46 for the activation of EGR-1 in response to exendin-4. Mutation of the proximal putative cAMP response element (CRE, 5Ј-GTACGTCA-3Ј) located at Ϫ69 resulted in a significant decrease in the EGR-1 transcription, whereas the mutation of the distal putative CRE at Ϫ139 was without such an effect. In immune supershift assays using exendin-4-treated cells, binding of cAMP response element-binding protein (CREB) phosphorylated on Ser 133 to the proximal CRE was increased. Employment of a CREB mutant containing Ala substitution at Ser 133 or a dominant negative CREB mutant that inhibits the binding of endogenous CREB to DNA significantly decreased the exendin-4-induced EGR-1 transcription. In experiments using specific protein kinase inhibitors, the effect of H-89 was more prominent than PD-98059, indicating the predominance of the PKA signaling over the MEK/ERK in induction of EGR-1. Therefore, it appears that the proximal CRE site is critical and the binding with CREB phosphorylated on Ser 133 is necessary for induction of the EGR-1 transcription by exendin-4.