Enzymatic synthesis and curing of biodegradable epoxide-containing polyesters from renewable resources

Epoxide-containing polyesters were enzymatically synthesized via two routes using unsaturated fatty acids as starting substrate. Lipase catalysis was used for both polycondensation and epoxidation of the unsaturated fatty acid group. One route was synthesis of aliphatic polyesters containing an unsaturated group in the side chain from divinyl sebacate, glycerol, and the unsaturated fatty acids, followed by an epoxidation of the unsaturated fatty acid moiety in the side chain of the resulting polymer. In another route, epoxidized fatty acids were prepared from the unsaturated fatty acids and hydrogen peroxide in the presence of lipase catalyst, and subsequently the epoxidized fatty acids were polymerized with divinyl sebacate and glycerol. The polymer structure was confirmed by NMR and IR, and for both routes, the high epoxidized ratio was achieved. Curing of the resulting polymers proceeded thermally, yielding transparent polymeric films with high gloss surface. Pencil scratch hardness of the present films improved, compared with that of the cured film obtained from the polyester having an unsaturated fatty acid in the side chain. The obtained film showed good biodegradability, evaluated by BOD measurement in an activated sludge.     

Cloning, expression, and crystallization of Cpn60 proteins from Thermococcus litoralis

Two genes of the extreme thermophilic archaeon Thermococcus litoralis homologous to those that code for Cpn60 chaperonins were cloned and expressed in Escherichia coli. Each of the Cpn60 subunits as well as the entire Cpn60 complex crystallize in a variety of morphological forms. The best crystals diffract to 3.6 A resolution at room temperature and belong to the space group 1422 with unit cell parameters a = b = 193.5 A, c = 204.2 A.     

Analysis of JNK, Mdm2 and p14(ARF) contribution to the regulation of mutant p53 stability

Identification of Mdm2 and JNK as proteins that target degradation of wt p53 prompted us to examine their effect on mutant p53, which exhibits a prolonged half-life. Of five mutant p53 forms studied for association with the targeting molecules, two no longer bound to Mdm2 and JNK. Three mutant forms, which exhibit high expression levels, showed lower affinity for association with Mdm2 and JNK in concordance with greater affinity to p14(ARF), which is among the stabilizing p53 molecules. Monitoring mutant p53 stability in vitro confirmed that, while certain forms of mutant p53 are no longer affected by either JNK or Mdm2, others are targeted for degradation by JNK/Mdm2, albeit at lower efficiency when compared with wt p53. Expression of wt p53 in tumor cells revealed a short half-life, suggesting that the targeting molecules are functional. Forced expression of mutant p53 in p53 null cells confirmed pattern of association with JNK/Mdm2 and prolonged half-life, as found in the tumor cells. Over-expression of Mdm2 in either tumor (which do express endogenous functional Mdm2) or in p53 null cells decreased the stability of mutant p53 suggesting that, despite its expression, Mdm2/JNK are insufficient (amount/affinity) for targeting mutant p53 degradation. Based on both in vitro and in vivo analyses, we conclude that the prolonged half-life of mutant p53 depends on the nature of the mutation, which either alters association with targeting molecules, ratio between p53 and targeting/stabilizing molecules or targeting efficiency.     

Chemoenzymatical grafting of acrylamide onto lignin

Laccase (E.C.1.10.3.2.) from white-rot basidiomycete Trametes versicolor and dioxane peroxides were essential in the copolymerization of acrylamide and a derivative with lignin in a dioxane-H(2)O (7:3) mixture. Both a solubility test and an elemental analysis of the eluted and separated fractions provided evidence of grafting. Gel permeation chromatography on Sephadex G-100 showed that the side chain and the lignin backbone migrate as one unit through the column. Copolymers obtained were characterized by UV-VIS-spectroscopy as well as FT-IR, and 13C-CPMAS-NMR spectroscopy. Freeze-dried copolymers of lignin and acrylamide appeared as homogeneous fibril-like particulate. The mechanism of the enzymatical grafting is discussed.     

C57BL/6 congenic mouse NRASQ61K melanoma cell lines are highly sensitive to the combination of Mek and Akt inhibitors in vitro and in vivo

RAS is frequently mutated in various tumors and known to be difficult to target. NRAS^Q61K/R are the second most frequent mutations found in human skin melanoma after BRAF^V600E. Aside from surgery, various approaches, including targeted therapies, immunotherapies, and combination therapies, are used to treat patients carrying NRAS mutations, but they are inefficient. Here, we established mouse NRAS^Q61K melanoma cell lines and genetically derived isografts (GDIs) from Tyr::NRAS^Q61K mouse melanoma that can be used in vitro and in vivo in an immune‐competent environment (C57BL/6) to test and discover novel therapies. We characterized these cell lines at the cellular, molecular, and oncogenic levels and show that NRAS^Q61K melanoma is highly sensitive to the combination of Mek and Akt inhibitors. This preclinical model shows much potential for the screening of novel therapeutic strategies for patients harboring NRAS mutations that have limited therapeutic options and resulted in poor prognoses.     

A newly constructed Agrobacterium-mediated transformation system revealed the influence of nitrogen sources on the function of the LaeA regulator in Penicillium chrysogenum

Penicillium chrysogenum is not only an industrially important filamentous fungus for penicillin production, but it also represents as a promising cell factory for production of natural products. Development of efficient transformation systems with suitable selection markers is essential for genetic manipulations in P. chrysogenum. In this study, we have constructed a new and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system with two different selection markers conferring the resistance to nourseothricin and phleomycin for P. chrysogenum. Under the optimized conditions for co-cultivation at 22 °C for 60 h with acetosyringone concentration of 200 μM, the transformation efficiency of the ATMT system could reach 5009 ± 96 transformants per 10⁶ spores. The obtained transformants could be exploited as the T-DNA insertion mutants for screening genes involved in morphogenesis and secondary metabolism. Especially, the constructed ATMT system was applied successfully to generate a knockout mutant of the laeA regulatory gene and relevant complementation strains in a wild strain of P. chrysogenum. Our results indicated that the LaeA regulator controls growth, sporulation, osmotic stress response and antibiotic production in P. chrysogenum, but its function is reliant on nitrogen sources. Furthermore, we showed that the laeA orthologous genes from the citrus postharvest pathogen P. digitatum and from the industrial fungus Aspergillus niger could recover the phenotypic defects in the P. chrysogenum laeA deletion mutant. Conclusively, this work provides a new ATMT system, which can be employed for T-DNA insertional mutagenesis, heterologous gene expression or for molecular inspections of potential genes related to secondary metabolism in P. chrysogenum.     

Specific interaction with the nuclear transporter importin α2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles

Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMPα proteins exhibit dynamic synthesis and subcellular localization during cellular differentiation, including during spermatogenesis, little is known of how this affects cell fate. To investigate how IMPαs control cellular development, we conducted a yeast two-hybrid screen for IMPα2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPα2 expression coincident with germ-line masculization. We identified paraspeckle protein 1 (PSPC1), the original defining component of nuclear paraspeckles, as an IMPα2-binding partner. PSPC1-IMPα2 binding in testis was confirmed in immunoprecipitations and pull downs, and an enzyme-linked immunosorbent assay–based assay demonstrated direct, high-affinity PSPC1 binding to either IMPα2/IMPβ1 or IMPα6/IMPβ1. Coexpression of full-length PSPC1 and IMPα2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High-throughput image analysis of >3500 cells indicated IMPα2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMPα2 isoform or small interfering RNA knockdown of IMPα2 each reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPα2 nuclear import cargo in fetal testis provides novel evidence that PSPC1 delivery to paraspeckles, and consequently paraspeckle function, may be controlled by modulated synthesis of specific IMPs.