Phosvitin phosphate content. Implications for protein kinase assay.

The maximal rates of the protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) reaction studied with chicken egg yolk phosvitin as substrate are dependent on the level of dephosphorylation of phosvitin. 30 per cent dephospho-phosvitin gives the optimal initial rates. With varying levels of dephosphorylation, the apparent Km for the substrate also changes in a biphasic manner. If this factor is taken into account, and a suitable adjustment is made for the concentration of dephospho-phosvitin in the reaction it is possible to achieve maximal rates for the kinase reaction with phosvitin preparations of varying levels of dephosphorylation. Such a consideration is important for comparing the results of protein kinase studies using phosvitin as the substrate.     

Localization of protein phosphokinase activities in the nucleolus distinct from extra-nucleolar regions in rat ventral prostate nuclei

Rat ventral prostate nucleoli contain protein phosphokinase(s) which have distinctly different characteristics from protein phosphokinase(s) localized in the extra-nucleolar regions of the nucleus. The differences pertain to pH vs activity profiles and activation by divalent cations, utilizing dephospho-phosvitin as substrate. Lysine-rich and arginine-rich F3 histones are also phosphorylated by nucleolar protein phosphokinase(s). Phosphorylation of histones by either nucleolar or extra-nucleolar fractions was not stimulated by cAMP, whereas that of phosvitin was slightly inhibited.     

Neuroprotection in a Novel Mouse Model of Multiple Sclerosis

Multiple sclerosis is an immune-mediated, demyelinating and neurodegenerative disease that currently lacks any neuroprotective treatments. Innovative neuroprotective trial designs are required to hasten the translational process of drug development. An ideal target to monitor the efficacy of strategies aimed at treating multiple sclerosis is the visual system, which is the most accessible part of the human central nervous system. A novel C57BL/6 mouse line was generated that expressed transgenes for a myelin oligodendrocyte glycoprotein-specific T cell receptor and a retinal ganglion cell restricted-Thy1 promoter-controlled cyan fluorescent protein. This model develops spontaneous or induced optic neuritis, in the absence of paralytic disease normally associated with most rodent autoimmune models of multiple sclerosis. Demyelination and neurodegeneration could be monitored longitudinally in the living animal using electrophysiology, visual sensitivity, confocal scanning laser ophthalmoscopy and optical coherence tomography all of which are relevant to human trials. This model offers many advantages, from a 3Rs, economic and scientific perspective, over classical experimental autoimmune encephalomyelitis models that are associated with substantial suffering of animals. Optic neuritis in this model led to inflammatory damage of axons in the optic nerve and subsequent loss of retinal ganglion cells in the retina. This was inhibited by the systemic administration of a sodium channel blocker (oxcarbazepine) or intraocular treatment with siRNA targeting caspase-2. These novel approaches have relevance to the future treatment of neurodegeneration of MS, which has so far evaded treatment.     

Antimicrobial and antioxidant properties of novel synthesized nanocomposites based on polystyrene packaging material waste

This work aims at preparation and characterization of novel synthesized nanocomposites based on polystyrene (Psty) packaging material waste. Moreover, the antimicrobial and antioxidant properties were examined and evaluated. The Psty was grafted with two different monomers such as acrylic and maleic acids (AAc and MAc, respectively) in presence of montmorillonite (MMT) using potassium persulfate as initiator under nitrogen atmosphere. The prepared nanocomposites were characterized using Fourier transform infrared spectroscopy (FT-IR), transmission (TEM) and scanning electron microscopes (SEM). Moreover, the antimicrobial activity was evaluated using agar disc diffusion method against gram negative bacteria such as: Klebsiella pneumonia, and Escherichia coli and gram positive bacterium (Sarcina lutea); in addition to the yeast fungus (Candida albicans). Furthermore, the radical scavenging ability of the prepared nanocomposites has been examined using the DPPH assay.     

Design,synthesis and evaluation against Mycobacterium tuberculosis of azole piperazine derivatives as dicyclotyrosine (cYY) mimics

Three series of azole piperazine derivatives that mimic dicyclotyrosine (cYY), the natural substrate of the essential Mycobacterium tuberculosis cytochrome P450 CYP121A1, were prepared and evaluated for binding affinity and inhibitory activity (MIC) against M. tuberculosis. Series A replaces one phenol group of cYY with a C3-imidazole moiety, series B includes a keto group on the hydrocarbon chain preceding the series A imidazole, whilst series C explores replacing the keto group of the piperidone ring of cYY with a CH₂-imidazole or CH₂-triazole moiety to enhance binding interaction with the heme of CYP121A1. The series displayed moderate to weak type II binding affinity for CYP121A1, with the exception of series B 10a, which displayed mixed type I binding. Of the three series, series C imidazole derivatives showed the best, although modest, inhibitory activity against M. tuberculosis (17d MIC?=?12.5?μg/mL, 17a 50?μg/mL). Crystal structures were determined for CYP121A1 bound to series A compounds 6a and 6b that show the imidazole groups positioned directly above the haem iron with binding between the haem iron and imidazole nitrogen of both compounds at a distance of 2.2?Å. A model generated from a 1.5?Å crystal structure of CYP121A1 in complex with compound 10a showed different binding modes in agreement with the heterogeneous binding observed. Although the crystal structures of 6a and 6b would indicate binding with CYP121A1, the binding assays themselves did not allow confirmation of CYP121A1 as the target.     

Synthesis of DNA interactive C3-trans-cinnamide linked β-carboline conjugates as potential cytotoxic and DNA topoisomerase I inhibitors

A series of new C3-trans-cinnamide linked β-carboline conjugates has been synthesized by coupling between various β-carboline amines and substituted cinnamic acids. Evaluation of their anti-proliferative activity against a panel of selected human cancer cell lines such as A549 (lung cancer), MCF-7 (breast cancer), B16 (melanoma), HeLa (cervical cancer) and a normal cell line NIH3T3 (mouse embryonic fibroblast cell line), suggested that the newly designed conjugates are considerably active against all the tested cancer cell lines with IC₅₀ values 13–45?nM. Moreover, the conjugates 8v and 8x were the most active against MCF-7 cells (14.05?nM and 13.84?nM respectively) and also even potent on other cell lines tested. Further, detailed investigations such as cell cycle analysis, apoptosis induction study, topoisomerase I inhibition assay, DNA binding affinity and docking studies revealed that these new conjugates are DNA interactive topoisomerase I inhibitors.     

Synthesis of oxidative metabolites of CRA13 and their analogs: Identification of CRA13 active metabolites and analogs thereof with selective CB2R affinity

CRA13; a peripheral dual CB₁R/CB₂R agonist with clinically proven analgesic properties, infiltrates into CNS producing adverse effects due to central CB₁R agonism. Such adverse effects might be circumvented by less lipophilic compounds with attenuated CB₁R affinity. Metabolism produces less lipophilic metabolites that might be active metabolites. Some CRA13 oxidative metabolites and their analogues were synthesized as less lipophilic CRA13 analogues. Probing their CB₁R and CB₂R activity revealed the alcohol metabolite 8c as a more potent and more effective CB₂R ligand with attenuated CB₁R affinity relative to CRA13. Also, the alcohol analogue 8b and methyl ester 12a possessed enhanced CB₂R affinity and reduced CB₁R affinity. The CB₂R binding affinity of alcohol analogue 8b was similar to CRA13 while that of methyl ester 12a was more potent. In silico study provided insights into the possible molecular interactions that might explain the difference in the elicited biological activity of these compounds.     

Structure activity relationship studies on rhodanines and derived enethiol inhibitors of metallo-β-lactamases

Metallo-β-lactamases (MBLs) enable bacterial resistance to almost all classes of β-lactam antibiotics. We report studies on enethiol containing MBL inhibitors, which were prepared by rhodanine hydrolysis. The enethiols inhibit MBLs from different subclasses. Crystallographic analyses reveal that the enethiol sulphur displaces the di-Zn(II) ion bridging ‘hydrolytic’ water. In some, but not all, cases biophysical analyses provide evidence that rhodanine/enethiol inhibition involves formation of a ternary MBL enethiol rhodanine complex. The results demonstrate how low molecular weight active site Zn(II) chelating compounds can inhibit a range of clinically relevant MBLs and provide additional evidence for the potential of rhodanines to be hydrolysed to potent inhibitors of MBL protein fold and, maybe, other metallo-enzymes, perhaps contributing to the complex biological effects of rhodanines. The results imply that any medicinal chemistry studies employing rhodanines (and related scaffolds) as inhibitors should as a matter of course include testing of their hydrolysis products.     

<Emphasis Type="Italic">In Planta</Emphasis> transformation for conferring salt tolerance to a tissue-culture unresponsive indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivar

Many farmer-popular indica rice (Oryza sativa L.) cultivars are recalcitrant to Agrobacterium-mediated transformation through tissue culture and regeneration. In planta transformation using Agrobacterium could therefore be a useful alternative for indica rice. A simple and reproducible in planta protocol with higher transformation efficiencies than earlier reports was established for a recalcitrant indica rice genotype. Agrobacterium tumefaciens containing the salt tolerance-enhancing Pea DNA Helicase45 (PDH45) gene, with the reporter and selectable marker genes, gus-INT (β-glucuronidase with intron) and hygromycin phosphotransferase (hpt), respectively, were used. Overnight-soaked mature embryos were infected and allowed to germinate, flower, and set T₁ seeds. T₀ plants were considered positive for the transgene if the spikelets of one or more of their panicles were positive for gus. Thereafter, selection at T₁ was done by germination in hygromycin and transgenic status re-confirmation by subjecting plantlet DNA/RNA to gene-specific PCR, Southern and semi-quantitative RT-PCR. Additionally, physiological screening under saline stress was done at the T₂ generation. Transformation efficiency was found to be 30–32% at the T₀ generation. Two lines of the in planta transformed seedlings of the recalcitrant rice genotype were shown to be saline tolerant having lower electrolyte leakage, lower Na⁺/K⁺, minimal leaf damage, and higher chlorophyll content under stress, compared to the WT at the T₂ generation.