Antagonistic effects of two novel GIP analogs, (Hyp3)GIP and (Hyp3)GIPLys16PAL, on the biological actions of GIP and longer-term effects in diabetic ob/ob mice

This study examines the actions of the novel enzyme-resistant, NH2-terminally modified GIP analog (Hyp(3))GIP and its fatty acid-derivatized analog (Hyp(3))GIPLys(16)PAL. Acute effects are compared with the established GIP receptor antagonist (Pro(3))GIP. All three peptides exhibited DPP IV resistance, and significantly inhibited GIP stimulated cAMP formation and insulin secretion in GIP receptor-transfected fibroblasts and in clonal pancreatic BRIN-BD11 cells, respectively. Likewise, in obese diabetic ob/ob mice, intraperitoneal administration of GIP analogs significantly inhibited the acute antihyperglycemic and insulin-releasing effects of native GIP. Administration of once daily injections of (Hyp(3))GIP or (Hyp(3))GIPLys(16)PAL for 14 days resulted in significantly lower plasma glucose levels (P < 0.05) after (Hyp(3))GIP on days 12 and 14 and enhanced glucose tolerance (P < 0.05) and insulin sensitivity (P < 0.05 to P < 0.001) in both groups by day 14. Both (Hyp(3))GIP and (Hyp(3))GIPLys(16)PAL treatment also reduced pancreatic insulin (P < 0.05 to P < 0.01) without affecting islet number. These data indicate that (Hyp(3))GIP and (Hyp(3))GIPLys(16)PAL function as GIP receptor antagonists with potential for ameliorating obesity-related diabetes. Acylation of (Hyp(3))GIP to extend bioactivity does not appear to be of any additional benefit.     

Novel regulators of planar cell polarity: a genetic analysis in Drosophila

Planar cell polarity (PCP) is a common feature of many epithelia and epithelial organs. Although progress has been made in the dissection of molecular mechanisms regulating PCP, many questions remain. Here we describe a screen to identify novel PCP regulators in Drosophila. We employed mild gain-of-function (GOF) phenotypes of two cytoplasmic Frizzled (Fz)/PCP core components, Diego (Dgo) and Prickle (Pk), and screened these against the DrosDel genome-wide deficiency collection for dominant modifiers. Positive genomic regions were rescreened and narrowed down with smaller overlapping deficiencies from the Exelixis collection and RNAi-mediated knockdown applied to individual genes. This approach isolated new regulators of PCP, which were confirmed with loss-of-function analyses displaying PCP defects in the eye and/or wing. Furthermore, knockdown of a subset was also sensitive to dgo dosage or dominantly modified a dishevelled (dsh) GOF phenotype, supporting a role in Fz/PCP-mediated polarity establishment. Among the new "PCP" genes we identified several kinases, enzymes required for lipid modification, scaffolding proteins, and genes involved in substrate modification and/or degradation. Interestingly, one of them is a member of the Meckel-Gruber syndrome factors, associated with human ciliopathies, suggesting an important role for cell polarity in nonciliated cells.     

Sequential colonization by river periphyton analysed by microscopy and molecular fingerprinting

1. An artificial glass substratum was incubated in the River Danube for a period of 28 days in order to detect the sequential colonization of microorganisms.
2. Light and fluorescent microscopy showed that microalgae and the picoalgal fraction on the slides increased rapidly over the first 2 weeks of colonization. Diatoms were numerically the most abundant component of the periphyton and their species richness and diversity increased rapidly in the early phase of colonization whereas diversity subsequently increased moderately.
3. Evenness of the diatom community was initially high, lower in the intermediate phase and again higher later on. Succession involving early, intermediate and late colonizer species was observed. Community composition during the first 5 days of colonization was very different from later stages whereas there were only minor changes subsequently.
4. Molecular community analysis by means of terminal restriction fragment length polymorphism analysis of PCR amplified 16S rRNA and 18S rRNA genes pointed to even larger differences between the composition of samples obtained early and late in the period.
5. The number of 18S rRNA and 16S rRNA terminal restriction fragments (T-RF-s) was variable over the colonization period and the fragment patterns of both the bacterial and eukaryotic portion of the microbial community were variable, with most T-RF-s unique to a single sample, suggesting a wide diversity and dynamic properties of periphytic organisms.     

Biological control of the fish louse in a rainbow trout fishery

Novel egg-laying boards were found to be effective in the biological control of the freshwater fish louse Argulus foliaceus in a 12·9 ha rainbow trout Oncorhynchus mykiss fishery which had a high prevalence and intensity of infection of juvenile parasites in the early spring of 1999. Approximately 228 000 egg clutches were harvested during an extensive 14 week period of egg laying which peaked in June 1999. In contrast, only 1566 clutches were harvested in 2000, when egg laying activity showed a bi-modal distribution, peaking in May and again in July and August. Egg laying activity decreased 145-fold compared to 1999. Argulus foliaceus prevalence and mean intensity also decreased nine-fold and six-fold, respectively. The ratio of female to male A. foliaceus on rainbow trout in consecutive years was 2·9: 1 and 2·1: 1. Estimates of the size of the female A. foliaceus population based on egg-laying activity in 1999 exceeded that derived from measurements of prevalence and intensity of infection, whereas in 2000, this was more in balance. A minimum temperature of 10° C was identified for egg laying, which occurred continuously from May to October in a broadly synchronous manner. This produced almost two generations each year, with juveniles, adults and eggs undergoing anabiosis during winter.     

Regulation of CC ligand 5/RANTES by acid sphingomyelinase and acid ceramidase

Acid sphingomyelinase (aSMase) generates the bioactive lipid ceramide (Cer) from hydrolysis of sphingomyelin (SM). However, its precise roles in regulating specific sphingolipid-mediated biological processes remain ill defined. Interestingly, the aSMase gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase) via alternative trafficking of a shared protein precursor. Previously, our laboratory identified Ser(508) as a crucial residue for the constitutive and regulated secretion of S-SMase in response to inflammatory cytokines, and demonstrated a role for S-SMase in formation of select cellular Cer species (Jenkins, R. W., Canals, D., Idkowiak-Baldys, J., Simbari, F., Roddy, P., Perry, D. M., Kitatani, K., Luberto, C., and Hannun, Y. A. (2010) J. Biol. Chem. 285, 35706-35718). In the present study using a chemokine/cytokine screen, we identified the chemokine CCL5 (formerly known as RANTES) as a candidate-specific downstream target for aSMase. Regulation of CCL5 by aSMase was subsequently validated using both loss-of-function and gain-of-function models indicating that aSMase is both necessary and sufficient for CCL5 production. Interestingly, cells deficient in acid ceramidase (aCDase) also exhibited defects in CCL5 induction, whereas cells deficient in sphingosine kinase-1 and -2 exhibited higher levels of CCL5, suggesting that sphingosine and not sphingosine 1-phosphate (S1P) is responsible for the positive signal to CCL5. Consistent with this, co-expression of aSMase and aCDase was sufficient to strongly induce CCL5. Taken together, these data identify a novel role for aSMase (particularly S-SMase) in chemokine elaboration by pro-inflammatory cytokines and highlight a novel and shared function for aSMase and aCDase.     

Factors regulating the contributions of fixed nitrogen by pasture and crop legumes to different farming systems of eastern Australia

On-farm and experimental measures of the proportion (%Ndfa) and amounts of N₂ fixed were undertaken for 158 pastures either based on annual legume species (annual medics, clovers or vetch), or lucerne (alfalfa), and 170 winter pulse crops (chickpea, faba bean, field pea, lentil, lupin) over a 1200 km north-south transect of eastern Australia. The average annual amounts of N₂ fixed ranged from 30 to 160 kg shoot N fixed ha^–1 yr^–1 for annual pasture species, 37–128 kg N ha^–1 yr^–1 for lucerne, and 14 to 160 kg N ha^–1 yr^–1 by pulses. These data have provided new insights into differences in factors controlling N₂ fixation in the main agricultural systems. Mean levels of %Ndfa were uniformly high (65–94%) for legumes growing at different locations under dryland (rainfed) conditions in the winter-dominant rainfall areas of the cereal-livestock belt of Victoria and southern New South Wales, and under irrigation in the main cotton-growing areas of northern New South Wales. Consequently N₂ fixation was primarily regulated by biomass production in these areas and both pasture and crop legumes fixed between 20 and 25 kg shoot N for every tonne of shoot dry matter (DM) produced. Nitrogen fixation by legumes in the dryland systems of the summer-dominant rainfall regions of central and northern New South Wales on the other hand was greatly influenced by large variations in %Ndfa (0–81%) caused by yearly fluctuations in growing season (April–October) rainfall and common farmer practice which resulted in a build up of soil mineral-N prior to sowing. The net result was a lower average reliance of legumes upon N₂ fixation for growth (19–74%) and more variable relationships between N₂ fixation and DM accumulation (9–16 kg shoot N fixed/t legume DM). Although pulses often fixed more N than pastures, legume-dominant pastures provided greater net inputs of fixed N, since a much larger fraction of the total plant N was removed when pulses were harvested for grain than was estimated to be removed or lost from grazed pastures. Conclusions about the relative size of the contributions of fixed N to the N-economies of the different farming systems depended upon the inclusion or omission of an estimate of fixed N associated with the nodulated roots. The net amounts of fixed N remaining after each year of either legume-based pasture or pulse crop were calculated to be sufficient to balance the N removed by at least one subsequent non-legume crop only when below-ground N components were included. This has important implications for the interpretation of the results of previous N₂ fixation studies undertaken in Australia and elsewhere in the world, which have either ignored or underestimated the N present in the nodulated root when evaluating the contributions of fixed N to rotations.     

Concerted transpositions of mobile genetic elements coupled with fitness changes in Drosophila melanogaster

In an inbred low-activity (LA) strain of Drosophila melanogaster with a low level of fitness and a complex of inadaptive characters, in situ hybridization reveals an invariant pattern of distribution of three copia-like elements (mdg-1, mdg-3, and copia). Rare, spontaneous, multiple transpositions of mobile elements in the LA strain were shown to be coupled with a drastic increase of fitness. A changed pattern of various types of mobile elements was also observed on selecting the LA strain for higher fitness. High-fitness strains show transpositions of mobile elements to definite chromosomal sites ("hot spots"). Concerted changes in the location of three different mobile elements were found to be coupled with an increase of fitness. The mdg-1 distribution patterns were also examined in two low-fitness strains independently selected from the high-fitness ones. Fitness decrease was accompanied by mdg-1 excision from the hot spots of their location usually detected in the high-fitness strains. The results suggest the existence of a system of adaptive transpositions of mobile elements that takes part in fitness control.