Geographical variation in circadian eclosion rhythm and photoperiodic adult diapause inDrosophila littoralis

Summary The time measuring system ofDrosophila littoralis strains originating between 40–70° N was found to be highly variable and latitude dependent. The critical daylength for photoperiodic adult diapause varied from 12 h or no diapause response in the south to 20 h in north. The median timing of pupal eclosion rhythm varied correspondingly from 21 h to 12 h from lights off in LD 321, and the period of free-running rhythm of eclosion from 24 h to 19 h. The phase of the free-running rhythm was also variable, and correlated with the phase of the entrained rhythm. Latitudinal variation in the entrained rhythm of eclosion and in diapause is adaptive, leading to eclosion early in the morning and to overwintering at the adult stage. In some strains with a late phase of eclosion, strong transient cycles were seen following the transition from LL to DD. A total damping of the free-running eclosion rhythm within 2–7 days was common to all strains. This damping was more pronounced in the northern strains. The phase and period of eclosion rhythms were statistically independent. Diapause was not correlated with any parameters of the eclosion rhythm in the analysis. Diapause may still be influenced by the period of the eclosion rhythm, even though its minor contribution may be masked by a more variable, eclosion rhythm independent system in the determination of diapause.Abbreviations, symbols and terms LD Light/dark; as in LD 321 meaning a cycle of 3 h light21 h darkness - LL Continuous light - DD Continuous darkness - T Period of a Zeitgeber cycle - Natural period of eclosion rhythm in constant conditions - _EL Phase of the free-running rhythm of eclosion - A Amplitude of the free-running rhythm of eclosion; possible range is from 4.17% (no rhythmicity) to 20% (the daily eclosion peaks 2–6 within 5 h each) - P Persistence of the free-running rhythm of eclosion; the number of daily eclosion peaks where the mean for five highest hourly percentages still exceed 6% - A phase shift, expressed in h; a re-setting of a rhythm; either as an advance shift (i.e. earlier= +), or as a delay shift (i.e. later = –) - PRC Phase-response curve - _LD Phase of entrained rhythm of eclosion; e.g. _{LD 321} is the median hour of eclosion peak from lights off at LD 321 - SD _ecl Amplitude of the entrained rhythm of eclosion; the smaller SD_ecl the higher the amplitude - PPRC Photoperiodic response curve; proportion of females in diapause displayed as a function of daylength - CDL Critical photoperiod; the photoperiod in the 24 h LD cycle at which 50% of the population studied diapauses - SD _diap Accuracy of diapause response of a strain; the smaller the SD_diap the more accurate the response - Cdl The main locus controlling CDL inD. littoralis     

Penicillium subrubescens, a new species efficiently producing inulinase

Inulin is a reserve carbohydrate in about 15 % of the flowering plants and is accumulated in underground tubers of e.g. chicory, dahlia and Jerusalem artichoke. This carbohydrate consists of linear chains of β-(2,1)-linked fructose attached to a sucrose molecule. Inulinases hydrolyse inulin into fructose and glucose. To find efficient inulin degrading fungi, 126 fungal strains from the Fungal Biotechnology Culture Collection (FBCC) at University of Helsinki and 74 freshly isolated strains from soil around Jerusalem artichoke tubers were screened in liquid cultures with inulin as a sole source of carbon or ground Jerusalem artichoke tubers, which contains up to 19 % (fresh weight) inulin. Inulinase and invertase activities were assayed by the dinitrosalicylic acid (DNS) method and a freshly isolated Penicillium strain originating from agricultural soil (FBCC 1632) was the most efficient inulinase producer. When it was cultivated at pH 6 and 28 °C in 2 litre bioreactors using inulin and Jerusalem artichoke as a carbon source, inulinase and invertase activities were on day 4 7.7 and 3.1 U mL^?1, respectively. The released sugars analysed by TLC and HPLC showed that considerable amounts of fructose were released while the levels of oligofructans were low, indicating an exoinulinase type of activity. Taxonomic study of the inulinase producing strain showed that this isolate represents a new species belonging in Penicillium section Lanata-divaricata. This new species produces a unique combination of extrolites and is phenotypically and phylogenetically closely related to Penicillium pulvillorum. We propose the name Penicillium subrubescens sp. nov. (CBS 132785^T = FBCC 1632^T) for this new species.     

Proteomic identification of lynchpin urokinase plasminogen activator receptor protein interactions associated with epithelial cancer malignancy

Urokinase plasminogen activator (uPA) and its high affinity receptor (uPAR) play crucial proteolytic and non-proteolytic roles in cancer metastasis. In addition to promoting plasmin-mediated degradation of extracellular matrix barriers, cell surface engagement of uPA through uPAR binding results in the activation of a suite of diverse cellular signal transduction pathways. Because uPAR is bound to the plasma membrane through a glycosyl-phosphatidylinositol anchor, these signalling sequelae are thought to occur through the formation of multi-protein cell surface complexes involving uPAR. To further characterize uPAR-driven protein complexes, we co-immunoprecipitated uPAR from the human ovarian cancer cell line, OVCA 429, and employed sensitive proteomic methods to identify the uPAR-associated proteins. Using this strategy, we identified several known, as well as numerous novel, uPAR associating proteins, including the epithelial restricted integrin, alphavbeta6. Reverse immunoprecipitation using anti-beta6 integrin subunit monoclonal antibodies confirmed the co-purification of this protein with uPAR. Inhibition of uPAR and/or beta6 integrin subunit using neutralizing antibodies resulted in the inhibition of uPA-mediated ERK 1/2 phosphorylation and subsequent cell proliferation. These data suggest that the association of beta6 integrin (and possibly other lynchpin cancer regulatory proteins) with uPAR may be crucial in co-transmitting uPA signals that induce cell proliferation. Our findings support the notion that uPAR behaves as a lynchpin in promoting tumorigenesis by forming functionally active multiprotein complexes.     

Functional expression and direct visualization of the human alpha 2B -adrenergic receptor and alpha 2B -AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors

The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology.     

CMPF Does Not Associate with Impaired Glucose Metabolism in Individuals with Features of Metabolic Syndrome

Objective

3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) is a metabolite produced endogenously from dietary sources of furan fatty acids. The richest source of furan fatty acids in human diet is fish. CMPF was recently shown to be elevated in fasting plasma in individuals with gestational diabetes and type 2 diabetes, and mechanistically high level of CMPF was linked to β cell dysfunction. Here we aimed to study the association between plasma CMPF level and glucose metabolism in persons with impaired glucose metabolism.

Methods

Plasma CMPF concentration was measured from plasma samples of the study participants in an earlier controlled dietary intervention. All of them had impaired glucose metabolism and two other characteristics of the metabolic syndrome. Altogether 106 men and women were randomized into three groups for 12 weeks with different fish consumption (either three fatty fish meals per week, habitual fish consumption or maximum of one fish meal per week). Associations between concentration of CMPF and various glucose metabolism parameters at an oral glucose tolerance test at baseline and at the end of the study were studied.

Results

Fasting plasma CMPF concentration was significantly increased after a 12-week consumption of fatty fish three times per week, but the concentration remained much lower compared to concentrations reported in diabetic patients. Increases of plasma CMPF concentrations mostly due to increased fish consumption were not associated with impaired glucose metabolism in this study. Instead, elevated plasma CMPF concentration was associated with decreased 2-hour insulin concentration in OGTT.

Conclusions

Moderately elevated concentration of CMPF in plasma resulting from increased intake of fish is not harmful to glucose metabolism. Further studies are needed to fully explore the role of CMPF in the pathogenesis of impaired glucose metabolism.

Trial Registration

ClinicalTrials.gov NCT00573781     

Predictive modelling of Fe(III) precipitation in iron removal process for bioleaching circuits

In this study, the applicability of three modelling approaches was determined in an effort to describe complex relationships between process parameters and to predict the performance of an integrated process, which consisted of a fluidized bed bioreactor for Fe³⁺ regeneration and a gravity settler for precipitative iron removal. Self-organizing maps were used to visually evaluate the associations between variables prior to the comparison of two different modelling methods, the multiple regression modelling and artificial neural network (ANN) modelling, for predicting Fe(III) precipitation. With the ANN model, an excellent match between the predicted and measured data was obtained (R ² = 0.97). The best-fitting regression model also gave a good fit (R ² = 0.87). This study demonstrates that ANNs and regression models are robust tools for predicting iron precipitation in the integrated process and can thus be used in the management of such systems.     

Delayed stigma receptivity in Collinsia heterophylla (Plantaginaceae): genetic variation and adaptive significance in relation to pollen competition, delayed self-pollination, and mating-system evolution

To increase our knowledge about mating-system evolution, we need to understand the relationship between specific floral traits and mating system. Species of Collinsia (Plantaginaceae) vary extensively in mating system; this variation is associated with variation in floral morphology and development and with the timing of self-pollination. Counterintuitively, large-flowered, more outcrossing species tend to have delayed stigma receptivity, reducing the amount of time that the stigma is receptive to cross-pollination before autonomous self-pollination. To understand how the timing of stigma receptivity is related to mating-system evolution, we studied in detail the timing of both stigma receptivity and self-pollination (anther-stigma contact) in two greenhouse-grown populations of large-flowered Collinsia heterophylla. Crosses on emasculated flowers at different stages of floral development always produced seeds, suggesting that cross-fertilization can be effected by pollen arriving prior to physiological receptivity. Phenotypic and genetic variation within populations in the timing of stigma receptivity and anther-stigma contact was substantial, although slightly less for the contact. Despite strong interspecific and interpopulation correlations, we did not find an among-genet phenotypic correlation between the traits. This indicates that each trait may respond independently to selection, and the trait association may be the result of correlational selection.     

Specific recognition of malondialdehyde and malondialdehyde acetaldehyde adducts on oxidized LDL and apoptotic cells by complement anaphylatoxin C3a

Oxidatively modified low-density lipoproteins (Ox-LDL) and complement anaphylatoxins C3a and C5a are colocalized in atherosclerotic lesions. Anaphylatoxin C3a also binds and breaks bacterial lipid membranes and phosphatidylcholine liposomes. The role of oxidized lipid adducts in C3a binding to Ox-LDL and apoptotic cells was investigated. Recombinant human C3a bound specifically to low-density lipoprotein and bovine serum albumin modified with malondialdehyde (MDA) and malondialdehyde acetaldehyde (MAA) in chemiluminescence immunoassays. No binding was observed to native proteins, LDL oxidized with copper ions (CuOx-LDL), or phosphocholine. C3a binding to MAA-LDL was inhibited by two monoclonal antibodies specific for MAA-LDL. On agarose gel electrophoresis, C3a comigrated with MDA-LDL and MAA-LDL, but not with native LDL or CuOx-LDL. C3a bound to apoptotic cells in flow cytometry. C3a opsonized MAA-LDL and was taken up by J774A.1 macrophages in immunofluorescence analysis. Complement-activated human serum samples (n=30) showed increased C3a binding to MAA-LDL (P<0.001) and MDA-LDL (P<0.001) compared to nonactivated samples. The amount of C3a bound to MAA-LDL was associated with total complement activity, C3a desArg concentration, and IgG antibody levels to MAA-LDL. Proteins containing MDA adducts or MAA adducts may bind C3a in vivo and contribute to inflammatory processes involving activation of the complement system in atherosclerosis.     

ADAM12 produced by tumor cells rather than stromal cells accelerates breast tumor progression

Expression of ADAM12 is low in most normal tissues but is markedly increased in numerous human cancers, including breast carcinomas. We have previously shown that overexpression of ADAM12 accelerates tumor progression in a mouse model of breast cancer (PyMT). In this study, we found that ADAM12 deficiency reduces breast tumor progression in the PyMT model. However, the catalytic activity of ADAM12 seems to be dispensable for its tumor-promoting effect. Interestingly, we show that ADAM12 endogenously expressed in tumor-associated stroma in the PyMT model does not influence tumor progression, but that ADAM12 expression by tumor cells is necessary for tumor progression in these mice. This finding is consistent with our observation that in human breast carcinoma, ADAM12 is almost exclusively located in tumor cells and, only rarely, seen in the tumor-associated stroma. We hypothesized, however, that the tumor-associated stroma may stimulate ADAM12 expression in tumor cells, on the basis of the fact that TGF-β1 stimulates ADAM12 expression and is a well-known growth factor released from tumor-associated stroma. TGF-β1 stimulation of ADAM12-negative Lewis lung tumor cells induced ADAM12 synthesis, and growth of these cells in vivo induced more than 200-fold increase in ADAM12 expression. Our observation that ADAM12 expression is significantly higher in the terminal duct lobular units (TDLU) adjacent to human breast carcinoma compared with TDLUs found in normal breast tissue supports our hypothesis that tumor-associated stroma triggers ADAM12 expression.