The activity of kinases is regulated by phosphorylation on Ser, Thr or Tyr residues within the activation loop. The ability to produce these enzymes recombinantly with a specific phosphorylation status is essential in order to understand structure and function. In this paper we describe a screening approach to co-express different phosphatases together with a kinase in the baculovirus expression system. This enabled the testing of different phosphatases as well as different levels of both phosphatase and kinase by varying the multiplicity of infection (MOI) of the different baculoviruses. This approach translated well to a larger scale. An unexpected observation was that co-expression of the phosphatase could have profound effects on expression levels even of heterologous target proteins that would not be a substrate for the phosphatase. This was most apparent with lambda phosphatase, an enzyme that removes phosphorylation from Ser and Thr residues, where expression was almost completely abolished for all proteins, even at modest MOIs. The effect of lambda phosphatase was observed irrespective of whether co-expression was from two separate baculoviruses or from two genes on the same vector. The effect was shown to be due, in part at least, to a decrease in transcription.

A new naphthalene derivative, named eucleanal (1), was isolated from Euclea divinorum Hiern., and its structure elucidated by detailed spectroscopic (1H, 13C NMR, COSY, HMQC, HMBC) and HREIMS analysis.

A series of polycyclic fluorescent ligands were synthesised and evaluated in murine striatal synaptoneurosomes for N-methyl-d-aspartate receptor (NMDAR) mediated calcium flux inhibition and inhibition of calcium influx through voltage gated calcium channels (VGCC). Amantadine (a) and N-(1-adamantyl)-1,3-propanediamine (c) substituted with 1-cyanoisoindole (3), indazole (5), dinitrobenzene (7, 8), dansyl (9, 10) and coumarin (11) moieties showed moderate to high inhibition of the NMDAR. A high degree of VGCC inhibition was observed for the cyanoisoindole compounds (3, 4) the dansyl compounds (9, 10) and the coumarin compound (12). Fluorophores conjugated to hydroxy-4-aza-8-oxoheptacyclotetradecane (13, 14) did not exhibit any significant VGCC inhibition, but the indazole conjugate (14) showed promising NMDAR activity. Dose response curves were calculated for selected NMDAR inhibitors (8-11) and N-[3-(1-adamantylamino)propyl]-5-dimethylaminonaphthalene-1-sulfonamide (10) exhibited the highest activity of the novel compounds. Compound 10 was further used as a fluorescent NMDAR ligand in a fluorescent competition assay utilizing MK-801, NGP1-01 and amantadine as known NMDAR inhibitors to demonstrate the possible applications of the novel fluorescent compounds. These small molecule fluorescent ligands can be considered as possible pharmacological tools in assay development and/or other investigations in the study of neurodegeneration.

Protein lysine methyltransferases are important regulators of epigenetic signaling. These enzymes catalyze the transfer of donor methyl groups from S-adenosylmethionine to specific acceptor lysines on histones, leading to changes in chromatin structure and transcriptional regulation. These enzymes also methylate nonhistone protein substrates, revealing an additional mechanism to regulate cellular physiology. The oncogenic protein SMYD2 represses the functional activities of the tumor suppressor proteins p53 and Rb, making it an attractive drug target. Here we report the discovery of AZ505, a potent and selective inhibitor of SMYD2 that was identified from a high throughput chemical screen. We also present the crystal structures of SMYD2 with p53 substrate and product peptides, and notably, in complex with AZ505. This substrate competitive inhibitor is bound in the peptide binding groove of SMYD2. These results have implications for the development of SMYD2 inhibitors, and indicate the potential for developing novel therapies targeting this target class.

Fumonisins are mycotoxins produced by various species of Fusarium and occur naturally in contaminated maize and maize-based foods. Ingestion of fumonisins has considerable health implications for humans and animals. Since fumonisins lack a useful chromophore or fluorophore, their determination in maize is routinely achieved via HPLC with fluorescence detection (FLD) after precolumn derivatization. This study optimized naphthalene-2,3-dicarboxaldehyde (NDA) derivatization of fumonisins in naturally contaminated maize following strong anion exchange (SAX) solid phase extraction (SPE) clean-up and utilizing diode array detection (DAD) as a practical alternative simultaneously to FLD. The limit of detection (LOD) for fumonisin B(1)(FB(1)), fumonisin B(2)(FB(2)) and fumonisin B(3)(FB(3)) with FLD was 0.11 ng, 0.50 ng and 0.27 ng, respectively, and with DAD it was 13.8 ng, 12.5 ng and 6.6 ng, respectively injected on column. The coefficient of variation (CV, n = 6) for FB(1), FB(2) and FB(3) in a naturally contaminated samples obtained with FLD was 2.6%, 1.8% and 5.3%, respectively, compared to 6.0%, 3.4% and 9.5%, respectively, obtained with DAD. Subsequently the optimized NDA derivatization was compared to the widely used o-phthaldialdehyde (OPA) derivatization agent as well as alternative sample clean-up with immunoaffinity column (IAC) by analyzing naturally contaminated maize samples (n = 15) ranging in total fumonisin (TFB = FB(1)+FB(2)+FB(3)) levels from 106 to 6000 μg/kg. After immunoaffinity column clean-up of extracted samples, the recoveries of spiked maize samples for NDA-FLD of FB(1), FB(2) and FB(3) were 62%, 94% and 64%, respectively. NDA proved to be an effective derivatization reagent of fumonisin in naturally contaminated maize samples following IAC clean-up, except for DAD at TFB levels below 1000 μg/kg. In contrast NDA derivatization following SAX clean-up produced results comparable to OPA only for levels below 1000 μg/kg. Aside from the difference in detection limits, FLD and DAD produced comparable results irrespective of the clean-up method or the derivatization agent.

A series of fluorescent heterocyclic adamantane amines were synthesised with the goal to develop novel fluorescent ligands for neurological assay development. These derivatives demonstrated multifunctional neuroprotective activity through inhibition of the N-methyl-d-aspartate receptor/ion channel, calcium channels and the enzyme nitric oxide synthase. It also exhibited a high degree of free radical scavenging potential. N-(1-adamantyl)-2-oxo-chromene-3-carboxamide (8), N-adamantan-1-yl-5-dimethyl-amino-1-naphthalenesulfonic acid (11) and N-(1-cyano-2H-isoindol-2-yl) adamantan-1-amine (12) were found to possess a high degree of multifunctionality with favourable physical-chemical properties for bioavailability and blood-brain barrier permeability. The ability of these heterocyclic adamantane amine derivatives as nitric oxide synthase inhibitors, calcium channel modulators, NMDAR inhibitors and effective antioxidants, indicate that they may find application as multifunctional drugs in neuroprotection.

The effects of anacardic acids and cardols isolated from the cashew nut and apple Anacardium occidentale (Anacardiaceae) on murine B16-F10 melanoma cells were tested. Although anacardic acids and cardols were found to inhibit tyrosinase, a key enzyme in melanin synthesis, melanogenesis in melanocytes was not suppressed in cultured cells but rather enhanced. Both anacardic acids and cardols exhibited moderate cytotoxicity.

The recent decrease in the total number of living kidney transplants coupled with the increase in the number of candidates on the waiting list underscores the importance of eliminating barriers to living kidney donation. We report what we believe to be the first pure right-sided laparoscopic live donor nephrectomy with extraction of the kidney through the vagina. The warm ischemia time was 3 min and the renal vessels and ureter of the procured kidney were of adequate length for routine transplantation. The donor did not receive any postoperative parenteral narcotic analgesia, was discharged home within 24 h and was back to normal activity in 14 days. The kidney functioned well with no complications or infections. Laparoscopic live donor nephrectomy with vaginal extraction may be a viable alternative to open and standard laparoscopic approaches. Potential advantages include reduced postoperative pain, shorter hospital stay and convalescence and a more desirable cosmetic result. These possible, but yet unproven, advantages may encourage more individuals to consider live donation.

Chronic pelvic pain (CPP) has many potential causes and is often a complex disorder with multiple contributing etiologies. The evaluation and treatment of women with CPP often requires a multidimensional approach. The treatment of CPP consists of two approaches: 1) treatment of pain itself or global treatment, and 2) treatment of disease-specific etiologies. Most often, treatment requires a combination of both approaches. This article reviews recent literature in the global treatment of CPP, including pharmacologic, psychotherapy, and neuroablative, as well as specific interventions for endometriosis, interstitial cystitis, pelvic adhesive disease, adenomyosis, and pelvic venous congestion.

Hyperlipidemia in the general population has been linked to the development of chronic kidney disease with both oxidative and endoplasmic reticulum stress implicated. Physiological levels (50-300 micromol/L) of saturated fatty acids such as palmitic acid (PA) cause cytotoxicity in vitro. We investigated cell type- and stimulus-specific signaling pathways induced by PA in renal proximal tubular cells and whether oxidative stress leads to ER stress or vice versa and which pathways predominate in signaling for PA-induced apoptosis and necrosis. NRK-52E cells were incubated with PA or hydrogen peroxide (H(2)O(2)) combined with SP600125 which blocks c-Jun N-terminal kinase (JNK) activation; salubrinal, which maintains eukaryotic initiation factor 2alpha in its phosphorylated state and the antioxidant EUK-134 - a superoxide dismutase mimetic with catalase activity. We found that (i) PA causes both oxidative and ER stress leading to apoptosis which is mediated by phosphorylated JNK;(ii) oxidant-induced apoptosis generated by H(2)O(2) involves ER stress signaling and CHOP expression;(iii) the ER stress mediated by PA is largely independent of oxidative stress;(iv) in contrast, the apoptosis produced by PA is mediated partly via oxidative stress. PA-mediated cell signaling in renal NRK-52E cells therefore differs from that identified in neuronal, hepatic and pancreatic beta cells.