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International audience; Cyclic nucleotide phosphodiesterase type 4 (PDE4), which controls the intracellular level of cyclic adenosine monophosphate (cAMP), has aroused scientific attention as a suitable target for antiinflammatory therapy of respiratory diseases. This work describes the development and characterization of pyridazinone derivatives bearing an indole moiety as potential PDE4 inhibitors and their evaluation as antiinflammatory agents. Among these derivatives, 4-(5-methoxy-1H-indol-3-yl)-6-methylpyridazin-3(2H)-one possesses promising activity, and selectivity towards PDE4B isoenzymes and is able to regulate potent proinflammatory cytokine and chemokine production by human primary macrophages.

BACKGROUND: Cancer is a major risk factor for venous thromboembolism (VTE) and for all-cause mortality following a VTE event. Higher risk can be attributed to certain cancer types and to metastatic disease. Until recently the gold standard for VTE and active cancer was low molecular weight heparin (LMWH), shown to be superior to warfarin in preventing thrombosis (Lee et al. 2003). Previously only minimal data from the direct oral anticoagulant (DOAC) pivotal RCTs were available in this population. However recent RCTs have been conducted that provide rationale for the use of DOACs as an alternative to LMWH in this patient group (Agnelli et al. 2020; Raskob et al. 2018; Young et al. 2018). This evidence is reflected in international guidelines (ASH 2021; ESC 2019; ITAC 2019, NCCN 2021) where DOACs are now proposed in VTE and active cancer patients without gastrointestinal cancer. PURPOSE: To describe patient characteristics, anticoagulant (AC) treatment patterns and outcomes among patients with VTE and active cancer in France. The analysis included all relevant ACs that were available for the treatment of VTE to understand how treatment recommendations were being reflected in clinical practice. METHODS: A nationwide retrospective cohort study of all adult patients (identified via the French national health data system: SNDS) with VTE and active cancer prescribed LMWH, a vitamin K antagonist (VKA), or a DOAC (apixaban or rivaroxaban) from 2013 to 2018. AC treatment-naïve (defined as patients without an AC prescription 24 months prior to index VTE) and AC treatment-experienced patients were included. Active cancer was defined by the presence of medical claims for cancer diagnosis or cancer-specific treatment in the 6 months prior or 30 days after index VTE event. A modified Khorana VTE risk scale (based on ICD codes and not blood tests) was used to evaluate the distribution of baseline VTE risk associated with different cancers. Rates of bleeding (defined as principal diagnoses of hospital stays), recurrent VTE, and all-cause mortality were assessed at 6 months for the standard of care (LMWH) cohort. RESULTS: 39,023 patients with VTE and active cancer were included. Most patients were prescribed LMWH 31,771 (81.42%), followed by rivaroxaban 2,259 (5.79%), VKAs 1,591 (4.08%), and apixaban 678 (1.74%). 2,724 (6.98%) were prescribed other parenteral anticoagulants (PAC) or PAC combinations. A slightly lower proportion of patients prescribed LMWH or VKAs were AC treatment naïve (44.4% and 45% respectively) compared to those prescribed rivaroxaban (51.2%) or apixaban (51.6%). Median duration of treatment (months; IQR) was shortest for LMWH patients (3.84; 1.35-8.48) and similar amongst the other ACs: VKAs (5.26; 1.05-14.16), rivaroxaban (5.85; 1.91-10.74), and apixaban (5.73; 1.74-9.07). Patients initiating VKAs and apixaban were older than those initiating rivaroxaban or LMWH (mean age in years: VKAs, 74; apixaban, 73; rivaroxaban, 67; LMWH, 66) however, LMWH patients had a higher comorbidity burden (mean CCI score: LMWH 6.4, VKAs, 4.95; rivaroxaban, 4.20; apixaban, 4.31). The proportion of patients with pulmonary embolism (PE), with or without DVT, was lower for LMWH and VKA cohorts (58.74% and 61.85% respectively) compared to rivaroxaban (71.76%) and apixaban (69.32%). Amongst patients with a medical claim for cancer diagnosis, the majority receiving LMWH (19,300 (61.8%)) had cancers with very high risk (brain, pancreatic, stomach) or high risk (gynaecological, lung, lymphoma, testicular, renal cell) for VTE. This proportion was lower in patients receiving VKAs, 484 (40.27%); rivaroxaban, 719 (43.52%); or apixaban, 194 (40%). Metastatic disease was present in most LMWH patients 21,994 (72.14%) but only in about a third of those receiving other ACs: VKAs, 406 (34.18%); rivaroxaban, 591 (36.37%); and apixaban, 152 (31.73%). Event rates for LMWH are reported in the table. CONCLUSIONS: AC therapy for a significant majority of patients with VTE and active cancer was LMWH. Most LMWH patients had metastatic disease. Median LMWH treatment was less than 4 months and VTE-related clinical event rates remained high in this population, suggesting a key unmet medical need. Future studies reflecting potential changes in clinical practice, because of guideline updates and the emergence of new RCT evidence, are required to understand clinical outcomes with different AC treatments. Figure 1 Figure 1. Disclosures Bertoletti: Pfizer: Honoraria, Other: Personal fees; BMS: Honoraria, Other: Personal Fees; Aspen: Other: Personal Fees; Bayer: Other: Personal Fees; Leo Pharma: Other: Personal Fee. Gusto: Pfizer: Consultancy. Khachatryan: Pfizer: Consultancy. Quignot: Pfizer: Consultancy. Chaves: Pfizer: Current Employment. Moniot: Pfizer: Current Employment. Mokgokong: Pfizer: Current Employment.

DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD+ (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD+ to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate–ribose) polymerase], a critical DNA repair protein. As mice age and NAD+ concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD+. Thus, NAD+ directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging.

Sirt2 is a target for the treatment of neurological, metabolic, and age-related diseases including cancer. Here we report a series of Sirt2 inhibitors based on the 1,2,4-oxadiazole scaffold. These compounds are potent Sirt2 inhibitors active at single-digit μM level by using the Sirt2 substrate α-tubulin-acetylLys40 peptide and inactive up to 100 μM against Sirt1, -3, and -5 (deacetylase and desuccinylase activities). Their mechanism of inhibition is uncompetitive toward both the peptide substrate and NAD(+), and the crystal structure of a 1,2,4-oxadiazole analog in complex with Sirt2 and ADP-ribose reveals its orientation in a still unexplored subcavity useful for further inhibitor development. Tested in leukemia cell lines, 35 and 39 induced apoptosis and/or showed antiproliferative effects at 10 or 25 μM after 48 h. Western blot analyses confirmed the involvement of Sirt2 inhibition for their effects in NB4 and in U937 cells. Our results provide novel Sirt2 inhibitors with a compact scaffold and structural insights for further inhibitor improvement.

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.

cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3(-)); it thereby serves as a cellular sensor for HCO3(-), carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3(-) binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3(-) activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3(-) sensing and a basis for targeting this system with drugs.

Sirtuins are NAD+-dependent protein deacetylases that regulate metabolism and aging-related processes. Sirt2 is the only cytoplasmic isoform among the seven mamalian Sirtuins (Sirt1-7) and structural information concerning this isoform is limited. We crystallized Sirt2 in complex with a product analog, ADP-ribose, and solved this first crystal structure of a Sirt2 ligand complex at 2.3 A resolution. Additionally, we re-refined the structure of the Sirt2 apoform and analyzed the conformational changes associated with ligand binding to derive insights into the dynamics of the enzyme. Our analyses also provide information on Sirt2 peptide substrate binding and structural states of a Sirt2-specific protein region, and our insights and the novel Sirt2 crystal form provide helpful tools for the development of Sirt2 specific inhibitors.

Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression.

PfluDING is a bacterial protein isolated from Pseudomonas fluorescens that belongs to the DING protein family, which is ubiquitous in eukaryotes and extends to prokaryotes. DING proteins and PfluDING have very similar topologies to phosphate Solute Binding Proteins (SBPs). The three-dimensional structure of PfluDING was obtained at subangstrom resolution (0.88 and 0.98 A) at two different pH's (4.5 and 8.5), allowing us to discuss the hydrogen bond network that sequesters the phosphate ion in the binding site. From this high resolution data, we experimentally elucidated the molecular basis of phosphate binding in phosphate SBPs. The phosphate ion is tightly bound to the protein via 12 hydrogen bonds between phosphate oxygen atoms and OH and NH groups of the protein. The proton on one oxygen atom of the phosphate dianion forms a 2.5 A low barrier hydrogen bond with an aspartate, with the energy released by forming this strong bond ensuring the specificity for the dianion even at pH 4.5. In particular, contrary to previous theories on phosphate SBPs, accurate electrostatic potential calculations show that the binding cleft is positively charged. PfluDING structures reveal that only dibasic phosphate binds to the protein at both acidic and basic phosphate, suggesting that the protein binding site environment stabilizes the HPO(4)(2-) form of phosphate.

A recombinant DING protein from Pseudomonas fluorescens has been previously shown to have a phosphate-binding site, and to be mitogenic for human cells. Here we report the three-dimensional structure of the protein, confirming a close similarity to the “Venus flytrap” structure seen in other human and bacterial phosphate-binding proteins. Site-directed mutagenesis confirms the role of a key residue involved in phosphate binding, and that the mitogenic activity is not dependent on this property. Deletion of one of the two hinged domains that constitute the Venus flytrap also eliminates phosphate binding whilst enhancing mitogenic activity.

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies., Author summary Parkinson disease is an age-associated neurodegenerative disorder characterized by the loss of dopamine-producing neurons from a region in the brain known as the substantia nigra and by the accumulation of the protein alpha-synuclein in intracellular clumps called inclusions. Whether these inclusions are the cause or a consequence of the pathological processes is still unclear. Sirtuin proteins are considered master regulators of the ageing process and have previously been associated with neurodegeneration. In this study, we investigated the interplay between sirtuin 2 and alpha-synuclein in order to dissect the molecular mechanisms associated with protection against alpha-synuclein toxicity. We found that sirtuin 2 interacted with and removed acetyl groups from alpha-synuclein. By decreasing the levels of sirtuin 2, or by expressing mutant versions of alpha-synuclein that modulate its acetylation status, we found that acetylation reduces the aggregation of alpha-synuclein and its cytotoxicity in vitro. Next, we evaluated whether genetic inhibition of sirtuin 2 could prevent the deleterious effects of alpha-synuclein in vivo and found that, in two different models of Parkinson disease, deletion of sirtuin 2 was neuroprotective. Our data therefore suggest that strategies aimed at decreasing sirtuin 2 activity might prove valuable therapeutic avenues for intervention in Parkinson disease and other synucleinopathies.

A rapid and very simple method for extracting polycyclic aromatic hydrocarbons (PAHs) from soils, sediments, and air particulate matter has been developed by coupling static subcritical water extraction with styrene-divinylbenzene (SDB-XC) extraction discs. Soil, water, and the SDB-XC disc are placed in a sealed extraction cell, heated to 250 degrees C for 15 to 60 min, cooled, and the PAHs recovered from the disc with acetone/methylene chloride. If the cells are mixed during heating, all PAHs with molecular weights from 128 to 276 are quantitatively (90%) extracted and collected on the sorbent disc and are then recovered by shaking with acetone/methylene chloride. After water extraction, the sorbent discs can be stored in autosampler vials without loss of the PAHs, thus providing a convenient method of shipping PAH extracts from field sites to the analytical laboratory. The method gives good quantitative agreement with standard Soxhlet extraction, and with certified reference materials for PAH concentrations on soil, sediment (SRM 1944), and air particulate matter (SRM 1649a).

Anti-müllerian hormone type II receptor (AMHRII) is a serine/threonine receptor and a member of type II receptors of the transforming growth factor beta superfamily. AMHRII has been recently identified in humans, mice, rats, and rabbits. In the male embryo, the AMHRII gene has been shown to be expressed in Sertoli's cells, in Leydig's cells and in the mesenchymal cells surrounding the müllerian duct. To determine the functional region of the AMHRII promoter as well as the factors controlling AMHRII gene expression, we used a 1.1-kilobase DNA fragment from the 5'-flanking region of the human AMHRII gene to generate a series of deletion or mutation and analyzed the resulting transcriptional activities after transfection of the NT2/D1 teratocarcinoma cell line. Our results indicate that maximal expression of the AMHRII promoter in this particular cell line, a cell line positive for endogenous AMHRII expression, requires a conserved estrogen receptor half-site element (AGGTCA) identical to the binding element for steroidogenic factor-1 (SF-1). Studies of this SF-1 binding element using gel mobility shift, antibody supershift assays, and transient transfections of reporter constructs indicate that SF-1 can bind and transactivate the AMHRII promoter. Finally, SF-1 protein expression in human male embryos was shown to display a good coincidence with the previously reported AMHRII expression profile. We then propose that SF-1 may be a key transcriptional regulator of AMHRII gene expression during early human development.

A new paediatric formulation of roxithromycin was tested for acceptability, efficacy and safety in a multicentre, prospective, non-comparative trial in 210 children, aged between 2 and 8 years, with a variety of respiratory tract infections. Most children were diagnosed as having rhinobronchitis, acute pharyngitis or acute tonsillitis. A dose of 5–8 mg/kg/day (mean ± SD, 5.92 ± 1.12) roxithromycin was given orally for 5–10 days (mean ± SDL 6.86 ± 1.80). The formulation consists of a tablet for suspension in a small volume of water, administered using a spoon, twice daily. Acceptability was good, with only eight children refusing their medication because of the taste or because of vomiting. The method of administration was found to be convenient by 76% of parents. The clinical success rate was 89.1% in the intent-to-treat analysis. There were only 18 adverse events reported by 14 patients; of these, only 10 events (all gastrointestinal) in eight patients were classified as drug related. A total of eight patients discontinued treatment because of an adverse event, but in only four were the events drug related.

The human Y-linked testis determining gene SRY encodes a protein with a DNA binding domain from the high mobility group box family. To date, no function has been assigned to amino acid sequences located outside this DNA binding motif. Here, we identify in a yeast two-hybrid screen a PDZ protein termed SIP-1, as an interacting protein with human SRY. In vitro, biochemical analysis, immunoprecipitation experiments, as well as expression of SIP-1 in human embryonic testis confirm that the two proteins can interact together. Interacting domains were mapped to the C-terminal seven amino acids of SRY and to the PDZ domains of SIP-1, respectively. We hypothesize that SIP-1 could connect SRY to other transcription factors providing SRY for its missing trans-regulation domain.

The opportunistic bacterial pathogen Pseudomonas aeruginosa employs three transcriptional regulators, LasR, RhlR and PqsR, to control the transcription of a large subset of its genes in a cell-density-dependent process known as quorum sensing. Here, the recombinant production, crystallization and structure solution of the ligand-binding domain of PqsR (MvfR), the LysR-type transcription factor that responds to the Pseudomonas quinolone signal (PQS), a quinolone-based quorum-sensing signal that is unique to P. aeruginosa and possibly a small number of other bacteria, is reported. PqsR regulates the expression of many virulence genes and may therefore be an interesting drug target. The ligand-binding domain (residues 91-319) was produced as a fusion with SUMO, and hexagonal-shaped crystals of purified PqsR_91-319 were obtained using the vapour-diffusion method. Crystallization in the presence of a PQS precursor allowed data collection to 3.25 Å resolution on a synchrotron beamline, and initial phases have been obtained using single-wavelength anomalous diffraction data from seleno-L-methionine-labelled crystals, revealing the space group to be P6(5)22, with unit-cell parameters a = b = 116-120, c = 115-117 Å.

Sialic acids are essential components of membrane glycoconjugates. They are responsible for the interaction, structure, and functionality of all deuterostome cells and have major functions in cellular processes in health and diseases. The key enzyme of the biosynthesis of sialic acid is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase that transforms UDP-N-acetylglucosamine to N-acetylmannosamine (ManNAc) followed by its phosphorylation to ManNAc 6-phosphate and has a direct impact on the sialylation of cell surface components. Here, we present the crystal structures of the human N-acetylmannosamine kinase (MNK) domain of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase in complexes with ManNAc at 1.64 Å resolution, MNK·ManNAc·ADP (1.82 Å) and MNK·ManNAc 6-phosphate · ADP (2.10 Å). Our findings offer detailed insights in the active center of MNK and serve as a structural basis to design inhibitors. We synthesized a novel inhibitor, 6-O-acetyl-ManNAc, which is more potent than those previously tested. Specific inhibitors of sialic acid biosynthesis may serve to further study biological functions of sialic acid.

Next Section Abstract The crystal structure of the thioacylenzyme intermediate of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been solved at 1.8A resolution. Formation of the intermediate was obtained by diffusion of the natural substrate within the crystal of the holoenzyme in the absence of inorganic phosphate. To define the soaking conditions suitable for the isolation and accumulation of the intermediate, a microspectrophotometric characterization of the reaction of GAPDH in single crystals was carried out, following NADH formation at 340 nm. When compared with the structure of the Michaelis complex ( Didierjean, C., Corbier, C., Fatih, M., Favier, F., Boschi-Muller, S., Branlant, G., and Aubry, A. (2003) J. Biol. Chem. 278, 12968-12976Abstract/FREE Full Text ) the 206-210 loop is shifted and now forms part of the so-called “new Pi” site. The locations of both the O1 atom and the C3-phosphate group of the substrate are also changed. Altogether, the results provide evidence for the flipping of the C3-phosphate group occurring concomitantly or after the redox step.

Organophosphates are the largest class of known insecticides, several of which are potent nerve agents. Consequently, organophosphate-degrading enzymes are of great scientific interest as bioscavengers and biodecontaminants. Recently, a hyperthermophilic phosphotriesterase (known as SsoPox), from the Archaeon Sulfolobus solfataricus, has been isolated and found to possess a very high lactonase activity. Here, we report the three-dimensional structures of SsoPox in the apo form (2.6 A resolution) and in complex with a quorum-sensing lactone mimic at 2.0 A resolution. The structure also reveals an unexpected active site topology, and a unique hydrophobic channel that perfectly accommodates the lactone substrate. Structural and mutagenesis evidence allows us to propose a mechanism for lactone hydrolysis and to refine the catalytic mechanism established for phosphotriesterases. In addition, SsoPox structures permit the correlation of experimental lactonase and phosphotriesterase activities and this strongly suggests lactonase activity as the cognate function of SsoPox. This example demonstrates that promiscuous activities probably constitute a large and efficient reservoir for the creation of novel catalytic activities.

PfluDING is a phosphate-binding protein expressed in Pseudomonas fluorescens. This protein is clearly distinct from the bacterial ABC transporter soluble phosphate-binding protein PstS and is more homologous to eukaryotic DING proteins. Interestingly, bacterial DING proteins have only been detected in certain Pseudomonas species. Although DING proteins seem to be ubiquitous in eukaryotes, they are systematically absent from eukaryotic genomic databases and thus are still quite mysterious and poorly characterized. PfluDING displays mitogenic activity towards human cells and binds various ligands such as inorganic phosphate, pyrophosphate, nucleotide triphosphates and cotinine. Here, the crystallization of PfluDING is reported in a monoclinic space group (P2(1)), with typical unit-cell parameters a = 36.7, b = 123.7, c = 40.8 A, alpha = 90, beta = 116.7, gamma = 90 degrees. Preliminary crystallographic analysis reveals good diffraction quality for these crystals and a 1.43 A resolution data set has been collected.

SOX proteins belong to a multigenic family characterized by a unique DNA binding domain, known as the high mobility group box, that is related to that of the testis determining gene SRY. cDNA sequences for more than 30SOX genes have been identified, and some are known to have diverse roles in vertebrate differentiation and development. Here, we report the isolation and characterization of mouse Sox15that was uncovered during a screen for high mobility group box containing transcription factors that are expressed at different levels during skeletal muscle differentiation. Sox15 cDNAs were found at a much higher frequency in myoblasts prior to their differentiation into myotubes. Electrophoretic mobility shift assays indicated that recombinant SOX15 protein was capable of binding to a consensus DNA binding site for SOX proteins. When overexpressed in C2C12 myoblasts, wild type SOX15, but not a C-terminal truncated form or the related protein SOX11, specifically inhibited activation of muscle-specific genes and expression of the basic helix-loop-helix myogenic factors myogenin and MyoD, resulting in a failure of the cells to differentiate into myotubes. These results suggest a specific and repressive role for SOX15, requiring the C-terminal domain, during myogenesis.

Of the several strategies that eukaryotes have evolved to modulate transcription factor activity, phosphorylation is regarded as one of the major mechanisms in signal-dependent transcriptional control. To conclusively demonstrate that the human sex-determining gene SRY is affected by such a post-translational control mechanism, we have analyzed its phosphorylation status in living cells. In the present study, we show that the cyclic AMP-dependent protein kinase (PKA) phosphorylates the human SRY protein in vitro as well as in vivo on serine residues located in the N-terminal part of the protein. This phosphorylation event was shown to positively regulate SRY DNA-binding activity and to enhance the ability of SRY to inhibit a basal promoter activity located downstream of an SRY DNA-binding site concatamer. Together these results strongly support the hypothesis that human SRY is a natural substrate for PKA in vivo and that this phosphorylation significantly modulates its major activity, DNA-binding, thereby possibly altering its biological function.

A very mild and facile method for the preparation of phosphinic acids via Michael addition of phosphonous acids as well as esters, through an intermediate silyl alkyl phosphonite, to activated conjugated systems is described.

A very mild and simple method for the preparation of phosphinic acids from phosphonous acids through the intermediacy of silylalkyl phosphonites is described.

The reaction of lithium diphenylcuprate with trans-4- and cis-4-tosyloxy-L-prolines gives excellent yields of 4-phenyl substituted L-prolines and the reaction proceeds with net retention of configuration at the carbon center bearing the tosyloxy group.

Berberine chloride ( 1 ) upon treatment with NaOAc-Ac 2 O yields naphthalene derivatives 4 and 5 In like fashion, 8 gives naphthalene 10 ; isoquinoline methiodide leads to β-naphthyl acetate; and 12 provides 14 . The reaction has been extended to the 3-carbonyl pyridinium series where 3-acetylpyridine methiodide and 3-pyridinecarboxaldehyde methiodide furnish lactones 16 and 17 , respectively. All these transformations proceed by initial nucleophilic attack of the acetic anhydride anion on the immonium carbon atom.

The isolation and structural elucidation of six new cytotoxic bufadienolides from Bersama abyssinica are reported. Bersaldegenin 1,3,5-orthoacetate (1), 16β-hydroxy-bersaldegenin 1,3,5-orthoacetate (10), bersamagenin 1,3,5-orthoacetate (18), and 16β-hydroxybersamagenin 1,3,5-orthoacetate (19) appear to be the first recognized naturally occurring orthoacetates. Interrelations of bersaldegenin 3-acetate (5) with 1 and of 16β-hydroxybersaldegenin 3-acetate (15) with 10 are described. The bersaldegenin derivatives 1 and 10 were interrelated with the corresponding bersamagenin derivatives 18 and 19 via the common reduction products 24 and 25. A novel intramolecular facilitation of equilibration of bersaldegenin 1,3,5-orthoacetate (1) and bersaldegenin 1-acetate (2) is described, and a plausible rationalization which involves participation of the aldehyde group is presented. The parallelism between cytotoxic activity and inhibition of transport ATPase among the foregoing and other bufadienolides is noted, and a speculative rationalization is discussed.

One new and 5 known flavone C -glycosides were isolated from leaves and stems of Coronilla varia . The new compound was shown to be isoorientin 2″- O -rhamnoside. The known compounds were isovitexin, isoorientin, isovitexin 4′- O -glucoside, isoorientin 4′- O -glucoside, and isoorientin 7- O -glucoside.

Three new 3-nitropropanoyl- d -glucopyranoses, 2,3,6-tri(3-nitropropanoyl)-α- d -glucopyranose (corollin), 1,2,6-tri(3-nitropropanoyl)-α- d -glucopyranose (coronillin) and 2,6-di(3-nitropropanoyl)-α- d -glucopyranose (coronarian) were isolated from the aerial parts of Coronilla varia . Structural assignments were made on the basis of 220 MHz NMR spectra.

There are currently no disease-modifying therapies for the neurodegenerative disorder Huntington's disease (HD). This study identified novel thiazole-containing inhibitors of the deacetylase sirtuin-2 (SIRT2) with neuroprotective activity in exvivo brain slice and Drosophila models of HD. A systems biology approach revealed an additional SIRT2-independent property of the lead-compound, MIND4, as an inducer of cytoprotective NRF2 (nuclear factor-erythroid 2 p45-derived factor 2) activity. Structure-activity relationship studies further identified a potent NRF2 activator (MIND4-17) lacking SIRT2 inhibitory activity. MIND compounds induced NRF2 activation responses in neuronal and non-neuronal cells and reduced production of reactiveoxygen species and nitrogen intermediates. These drug-like thiazole-containing compounds represent an exciting opportunity for development of multi-targeted agents with potentially synergistic therapeutic benefits in HD and related disorders.