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Stable, crystalline ethyl N-tert-butyl-4-nitrobenzenesulfonimidate has been prepared in high yield by direct O-ethylation of N-tert-butyl-4-nitrobenzenesulfonamide with iodoethane and silver(I) oxide in dichloromethane. This sulfonimidate directly ethylates various acids to esters; the stronger the acid, the faster it alkylates and in higher yield. It readily ethylates alcohols and phenols to ethers at room temperature in the presence of tetrafluoroboric acid catalyst without molecular rearrangements or racemization. We have defined these reactions as SNAAP alkylations: [substitution, nucleophilic of acids, alcohols and phenols]. The hard sulfonimidate alkylating agent is chemoselective, preferring oxygen > nitrogen > sulfur. The sulfonamide byproduct of alkylation is readily recycled to the sulfonimidate.

The adenovirus E1A protein has been shown to be involved in the potentiation of apoptosis induced by chemotherapeutic agents, yet the molecular events of E1A-mediated apoptosis are not very clear. A recent report has suggested that deamidation of the Bcl-X(L) protein inhibits its antiapoptotic ability and leads to apoptosis induced by alkylating agents in Rb-deficient tumor cells. Since Rb is known to interact with E1A, which interrupts Rb's normal function, we examined Bcl-X(L) deamidation and cell death induced by cisplatin in E1A transfectants. We found that the E1A transfectants became sensitive to cisplatin-induced apoptosis compared to the parental cells, SKOV3.ip1. Our data show that cisplatin treatment induced the modification of Bcl-X(L) in the E1A transfectants in dosage and time-dependent manner. Furthermore, phosphatase treatment had no effect on the level of Bcl-X(L) modification, whereas alkaline lysis buffer appeared to induce the same modification of Bcl-X(L). Ectopic expression of the deamidated forms of Bcl- X(L) in SKOV3.ip1 cells revealed that the modification to the Bcl- X(L) protein molecules was deamidation. Expression of the E1A mutant (dl1108) which contains deletion at CR2 domain suppressed Bcl-X(L) deamidation and apoptosis induced by cisplatin. We also found that expression of the nondeamidated Bcl-X(L) protected E1A transfectants from apoptosis. These findings suggest that Bcl-X(L) deamidation contributes to E1A-mediated cisplatin sensitization in SKOV3.ip1 cells.

Meeting the compliance limits of the U.S. Environmental Protection Agency's final rule revision to the National Emission Standards for Hazardous Air Pollutants from the Portland Cement Manufacturing Industry (Portland Cement MACT) regulation will be a challenge for cement kilns equipped with only dry particulate control devices (that is, electrostatic precipitator or fabric filter baghouse control devices). In addition to increases in operation and maintenance costs, installing additional control devices will require capital investment. Conventional control technologies, such as selective non-catalytic reduction (for nitrogen oxides control), wet flue gas desulfurization (for acid gases—sulfur dioxide /hydrogen chloride—control), and powder activated carbon with fiber filter baghouse collection (for mercury control), can be capital intensive. Limits on additional hazardous air pollutants (HAPs) such as dioxins/furans and surrogate HAPs, particulate matter, and total hydrocarbons further complicate air pollution control strategies for cement kilns.

The corrosion fatigue (CF) behaviour, under constant deflection bending conditions with a pulsating tension stress form, of 2024-T3 aluminium alloy, unanodized and anodized to form a thick porous film, in 3.5% NaCl solution has been investigated. It was found that E corr varies very little until specimen fracture under low frequency CF conditions, whereas E corr drops rapidly when approaching the later fracturing stage of the CF process under high-frequency conditions for unanodized specimens. However, a slow drop in E corr was detected from the commencement of the CF process, and lasted up to a much more rapid drop at a later fracturing stage for the anodized specimen. This behaviour presumably can be explained by the cracking of the anodic film and the theory of imperfect recovery of the surface film. It is suggested that the E corr monitoring technique may be useful for determining the remnant CF life for existing structural parts of this alloy or other aluminium alloys regardless of whether or not they are anodized. Furthermore, the T3 temper provides a microstructure which may retard main-crack formation and penetration in the CF process of the anodized alloy, thus mitigating partly the negative effect of the readily crackable anodic film.

The temperature rise in the substrate as well as in the bulk solution and the development of a local brown-coloured 'burnt' region on the anodic film are the characteristics of high rate anodizing of aluminum in oxalic acid. In this study, measurement of the barrier layer thickness in both the predicted burning region and the normal region indicates that they increase simultaneously as the voltage builds up to form the secondary voltage peak on the voltage/time transient; a depletion of oxalate anions allied with the temperature rise at the pore base is thought to govern this behaviour. Characteristic strong dotted anodic glows occurred simultaneously with a slight drop of voltage, about 1V, from the secondary peak value were observed just prior to the initiation of the local burning sector; also, the local substrate temperature always rises when burning is approaching. These studies suggest that a thermal modified electronic breakdown process is responsible for the burning phenomenon.

Introduction Toll-like receptors (TLRs) play important roles in innate immunity by eliciting inflammation. Upon receptor engagement, activation of IKKs and MAPKs control the production of cytokines. It has been shown that TAK1 is the essential mediator that transmits signal from receptor to downstream effectors. Meanwhile, in our previous study [1] , we implied that IKKs activation in TLR4 is independent to TAK1 phosphorylation. Therefore, the molecular mechanisms underlying how MAPKs and IKKs are differentially activated by TAK1 in TLR4 need for further investigation. Methods In order to examine the role of TAK1 in MAPKs and IKKs activation, TAK1-silencing cells were stable expressed with TAK1 mutants and stimulated with LPS. Additionally, LC-MS analysis was used to identify novel post-translation modification and binding partners of TAK1. Results As previous reports, we found that the production of cytokines in response to LPS was diminished in TAK1-silencing cells. Reconstituting cells with various mutated TAK1, mutants in either activation loop (T184 and T187) or kinase domain (D156) leaded to inhibition of MAPKs. The activation of IKKs was slightly decreased in D156A mutant, but not T (184; 187) A. Furthermore, mutation in phosphorylation sites of TAK1 enhanced the K63-linked ubiquitination. By in vivo ubiquitination assay, we showed that mutation in either K158 or K209 of TAK1 was unable to abolish polyubiquitination completely, but we confirmed that K158 of TAK1 as the critical site in TLR4 pathway for IKK activation and TAK1 phosphorylation that, thereby, activates of MAPKs. The proteomic analysis for binding partners of TAK1 in different modification status was in line with activity assay. Furthermore, we also identified K562 of TAK1 as a novel ubiquitination site. Conclusion We identified that ubiquitination in K209 of TAK1 regulates IKKs activation and the phosphorylation of TAK1 itself. The activation of TAK1 is able to transmit signal to activate MAPKs, and, in turn, block the K63-linked ubiquitination of TAK1 in feedback regulation. Our data suggested that TAK1-mediated activation of downstream effectors in TLR4 is carefully regulated by the crosstalk of phosphorylation and ubiquitination.

Postsynaptic densities (PSDs) isolated by biochemical means consist of a complex mixture of proteins that tightly bond to each other. The purpose of this report is to study whether the numerous interprotein disulfides found in the isolated PSDs contribute to the tight structure of the PSDs and whether these interprotein disulfides exist in vivo. PSDs were isolated from pig cerebral cortex by conventional methods except that iodoacetic acid (IAA) was added to all solutions to curtail the formation of disulfides during the isolation process. The PSDs thus isolated were fragmented easily by treatment with chaotropic reagents or ionic detergents, whereas the PSDs isolated in IAA-free solutions were resistant to these treatments. Electron microscopy revealed that the PSDs isolated in IAA-containing solutions were more fragmented than those isolated in IAA-free solutions. Furthermore, the PSD sample isolated in IAA-free solutions contained very large disulfide-linked aggregates that were virtually absent from the PSDs isolated in IAA-containing solutions. Our results suggest that the exceptionally tight structure of the PSDs isolated by conventional methods is due largely to the new disulfides formed during the isolation process and that the PSD proteins under in vivo conditions are held together primarily by noncovalent interactions.

We have previously shown (Smith et al., 1994) that antibodies raised against the growth arrest and DNA damage inducible protein Gadd45 co-precipitate proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and repair. Here we demonstrate that Gadd45 can directly bind to PCNA using a Far-western blotting approach. In this assay, a Gadd45 bacterial expression vector was modified to allow synthesis of purified 32P-labeled Gadd45 fusion protein. This protein was used to detect filter bound PCNA protein, while filter bound Gadd45 protein could also be detected by free PCNA molecules. Using recombinant proteins in conjunction with immunoprecipitation and immunoblotting, we show that Gadd45 competes with p21 for binding to PCNA and conversely, p21 blocks the ability of Gadd45 to bind PCNA. In addition, p21 appears to disrupt PCNA trimers whereas Gadd45 has a lesser effect. PCNA trimer disruption was also observed in UV-irradiated cells but not in repair-defective xeroderma pigmentosum group A (XP-A) cells.

Exon sizes in vertebrate genes are, with a few exceptions, limited to less than 300 bases. It has been proposed that this limitation may derive from the exon definition model of splice site recognition. In this model, a downstream donor site enhances splicing at the upstream acceptor site of the same exon. This enhancement may require contact between factors bound to each end of the exon; an exon size limitation would promote such contact. To test the idea that proximity was required for exon definition, we inserted random DNA fragments from Escherichia coli into a central exon in a three-exon dihydrofolate reductase minigene and tested whether the expanded exons were efficiently spliced. DNA from a plasmid library of expanded minigenes was used to transfect a CHO cell deletion mutant lacking the dhfr locus. PCR analysis of DNA isolated from the pooled stable cotransfectant populations displayed a range of DNA insert sizes from 50 to 1,500 nucleotides. A parallel analysis of the RNA from this population by reverse transcription followed by PCR showed a similar size distribution. Central exons as large as 1,400 bases could be spliced into mRNA. We also tested individual plasmid clones containing exon inserts of defined sizes. The largest exon included in mRNA was 1,200 bases in length, well above the 300-base limit implied by the survey of naturally occurring exons. We conclude that a limitation in exon size is not part of the exon definition mechanism.

We describe a Drosophila DNA clone of tandemly duplicated genes encoding an amino acid sequence nearly identical to human ribosomal protein S14 and yeast rp59. Despite their remarkably similar exons, the locations and sizes of introns differ radically among the Drosophila, human, and yeast (Saccharomyces cerevisiae) ribosomal protein genes. Transcripts of both Drosophila RPS14 genes were detected in embryonic and adult tissues and are the same length as mammalian S14 message. Drosophila RPS14 was mapped to region 7C5-9 on the X chromosome. This interval also encodes a previously characterized Minute locus, M(1)7C.

We describe sequences of two human ribosomal proteins, S14 and S17, and messenger RNAs that encode them. cDNAs were used as molecular hybridization probes to recognize complementary genes in rodent, Drosophila, and yeast chromosomal DNAs. Human ribosomal protein sequences are compared to analogous Chinese hamster, yeast, and bacterial genes. Our observations suggest that some ribosomal protein genes have been conserved stringently in the several phylogenetic lines examined. These genes apparently were established early in evolution and encode products that are fundamental to the translational apparatus. Other ribosomal protein genes examined, although similar enough to heterologous DNA sequences to indicate their structural relationships, appear to have diverged substantially during evolution, probably reflecting adaptations to different genetic environments.