Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-(3)H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.     

Modelling time‐varying low‐temperature‐induced mortality rates for pupae of Tuta absoluta (Gelechiidae,Lepidoptera)

In a series of laboratory experiments, acclimated pupae of Tuta absoluta were exposed to various constant low temperatures in order to estimate their maximum survival times (Kaplan–Meier, Lt_99.99). A Weibull function was fitted to the data points, describing maximum survival time as a function of temperature. In another experiment at ?6°C, the progress of mortality increasing with exposure time was identified. These values were fitted by a sigmoidal function converging asymptotically to 100% mortality for very long exposure times. Analysing mortality data from the maximum survival experiment by a generalized linear model showed a significant common slope parameter (p < .001) that reveals parallelism of the survival curves at each temperature if a log time axis is used. These curves appear stretched (time scaled) if plotted with a nonlogarithmic time axis. By combining these mathematical relations, it was possible to calculate a species‐specific ‘mortality surface’ which exhibits mortalities, depending on temperature and duration of exposure. In order to accumulate hourly mortalities for courses of varying temperatures, an algorithm was developed which yields mortality values from that surface taking into account the attained mortality level. In validation experiments, recorded mortalities were compared against modelled mortalities. Prediction of mortality was partially supported by the model, but pupae experiencing intensely fluctuating temperatures showed decreased mortality, probably caused by rapid cold hardening during exposure. Despite this observation, mortality data converged to distinct levels very close to 100% depending on the intensity of temperature fluctuations that were characteristic for different types of experiments. The highest mortality limit occurred at intensely fluctuating temperatures in laboratory experiments. This constituted a benchmark that was not reached under various field conditions. Thus, it was possible to identify temperature limits for the extinction of field populations of Tuta absoluta pupae.     

Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling

The dimerisation of Raf kinases involves a central cluster within the kinase domain, the dimer interface (DIF). Yet, the importance of the DIF for the signalling potential of wild-type B-Raf (B-Raf(wt)) and its oncogenic counterparts remains unknown. Here, we show that the DIF plays a pivotal role for the activity of B-Raf(wt) and several of its gain-of-function (g-o-f) mutants. In contrast, the B-Raf(V600E), B-Raf(insT) and B-Raf(G469A) oncoproteins are remarkably resistant to mutations in the DIF. However, compared with B-Raf(wt), B-Raf(V600E) displays extended protomer contacts, increased homodimerisation and incorporation into larger protein complexes. In contrast, B-Raf(wt) and Raf-1(wt) mediated signalling triggered by oncogenic Ras as well as the paradoxical activation of Raf-1 by kinase-inactivated B-Raf require an intact DIF. Surprisingly, the B-Raf DIF is not required for dimerisation between Raf-1 and B-Raf, which was inactivated by the D594A mutation, sorafenib or PLX4720. This suggests that paradoxical MEK/ERK activation represents a two-step mechanism consisting of dimerisation and DIF-dependent transactivation. Our data further implicate the Raf DIF as a potential target against Ras-driven Raf-mediated (paradoxical) ERK activation.     

Anti-proliferative effect of cytohesin inhibition in gefitinib-resistant lung cancer cells

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib, have been proven to efficiently inhibit the proliferation of a subset of non small-cell lung cancers (NSCLC). Unfortunately, the majority of NSCLC expressing wild type EGFR is primarily resistant to EGFR-TKI treatment. Here, we show that the proliferation of the gefitinib-resistant NSCLC cell lines H460 and A549 is reduced by the small molecule SecinH3 which indirectly attenuates EGFR activation by inhibition of cytohesins, a class of recently discovered cytoplasmic EGFR activators. SecinH3 and gefitinib showed a synergistic antiproliferative effect, which correlated with a profound inhibition of Akt activation and survivin expression. Treating mice bearing H460 xenografts with SecinH3 showed the antiproliferative and pro-apoptotic effect of SecinH3 in vivo. Our data suggest that targeting the EGFR indirectly by inhibiting its cytoplasmic activators, the cytohesins, has the potential to improve the treatment of primarily EGFR-TKI resistant lung cancers.     

Spermatozoa production in male <Emphasis Type="Italic">Varroa destructor</Emphasis> and its impact on reproduction in worker brood of <Emphasis Type="Italic">Apis mellifera</Emphasis>

Reproduction in Varroa destructor exclusively takes place within the sealed honey bee brood cell and is, therefore, limited by the duration of the postcapping period. Oogenesis, ontogenetic development and mating must be optimized to ensure the production of as many mated daughter mites as possible. One adult male mite has to mate with up to five sister mites and transfer 30–40 spermatozoa to each female. We analyzed the production and transfer of male spermatozoa during a reproductive cycle by counting all spermatozoa in the genital tracts of the male and daughter mites in 80 worker brood cells at defined times after cell capping. We could show that spermatozoa production in male mites is an ongoing process throughout their adult lifetime starting after the adult molt. The spermatozoa are transferred to the females in an early non-capacitated stage and require further maturation within the female’s genital tract. Our study points out that a Varroa male has at any time in the brood cell enough spermatozoa to inseminate all daughter mites but does not waste energy in producing a big surplus. In total one male produced, on average, 125 spermatozoa during a reproductive cycle in worker brood which is sufficient for successful matings with at least three daughter mites. Spermiogenesis in Varroa males represents therefore a further adaptation to the limited time available for reproduction.     

Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthesis and Growth of Arabidopsis

Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate.     

Terpene Conjugates of the Nigella sativa Seed‐Oil Constituent Thymoquinone with Enhanced Efficacy in Cancer Cells

Thymoquinone (TQ; 1 ) is a weak anticancer constituent of black seed oil. Derivatives bearing terpene‐terminated 6‐alkyl residues were tested in cells of human HL‐60 leukemia, 518A2 melanoma, multidrug‐resistant KB‐V1/Vbl cervix, and MCF‐7/Topo breast carcinomas, as well as in non‐malignant human foreskin fibroblasts. Derivatives with a short four‐atom spacer between quinone and cyclic monoterpene moieties were more antiproliferative than analogues with longer spacers. 6‐(Menthoxybutyryl)thymoquinone ( 3a ) exhibited single‐digit micromolar IC₅₀ (72 h) values in all four cell lines. It was seven times more active than TQ ( 1 ) in 518A2 melanoma cells and four times in KB‐V1/Vbl cervix carcinoma cells, while only half as toxic in the fibroblasts. Compound 3a was also not a substrate for the P‐gp and BCRP drug transporters of the resistant cancer cells. The caryophyllyl and germacryl conjugates 3e and 3f specifically inhibited the growth of the resistant MCF‐7 breast carcinoma cells. Conjugation of TQ with the triterpene betulinic acid via the OH group as in 3g led to a loss in activity, while conjugation via the carboxylic acid afforded compound 4 with nanomolar IC₅₀ (72 h) activity against HL‐60 cells. All anticancer‐active derivatives of TQ ( 1 ) induced apoptosis associated with DNA laddering, a decrease in mitochondrial membrane potential and a slight increase in reactive oxygen species.