Cleavage and secretion is not required for Four-jointed function in Drosophila patterning

Four-jointed (fj) is required for proximodistal growth and planar polarity in Drosophila tissues. It encodes a predicted type II transmembrane protein with putative signal peptidase sites in its transmembrane domain, and its C terminus is secreted. Fj has therefore been proposed to act as a secreted signalling molecule. We show that Fj protein has a graded distribution in eye and wing imaginal discs, and is largely localised to the Golgi in vivo and in transfected cells. Forms of Fj that are constitutively secreted or anchored in the Golgi were assayed for function in vivo. We find that cleavage and secretion of Fj is not necessary for activity, and that Golgi-anchored Fj has increased activity over wild type. fj has similar phenotypes to those caused by mutations in the cadherin-encoding genes fat (ft) and dachsous (ds). We show that fj interacts genetically with ft and ds in planar polarity and proximodistal patterning. We propose that Fj may act in the Golgi to regulate the activity of Ft and Ds.     

Disruption of masking by hypothalamic lesions in Syrian hamsters

Negative masking of locomotor activity by light in nocturnal rodents is mediated by a non-image-forming irradiance-detection system in the retina. Structures receiving input from this system potentially contribute to the masking response. The suprachiasmatic nucleus (SCN) regulates locomotor activity and receives dense innervation from the irradiance-detection system via the retinohypothalamic tract, but its role in masking is unclear. We studied masking in adult Syrian hamsters (Mesocricetus auratus) with electrolytic lesions directed at the SCN. Hamsters were exposed to a 3.5:3.5 ultradian light/dark cycle and their wheel-running activity was monitored. Intact hamsters showed robust masking, expressing less than 20% of their activity in the light even though light and dark occurred equally during their active times. In contrast, hamsters with lesions showed, on average, as much activity in the light as in the dark. Tracing of retinal projections using cholera toxin subunit showed that the lesions damaged retinal projections to the SCN and to the adjacent subparaventricular zone. Retinal innervation outside the hypothalamus was not obviously affected by the lesions. Our results indicate that retinohypothalamic projections, and the targets of these projections, to the SCN and/or adjacent hypothalamic areas play an important role in masking.     

Solving a sticky problem: new genetic approaches to host cell adhesion by the Lyme disease spirochete

The Lyme disease spirochetes, comprised of at least three closely related species, Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii, are fascinating and enigmatic bacterial pathogens. They are maintained by tick-mediated transmission between mammalian hosts, usually small rodents. The ability of these bacteria, which have relatively small genomes, to survive and disseminate in both an immunocompetent mammal and in an arthropod vector suggests that they have evolved elegant and indispensable strategies for interacting with their hosts. Recognition of specific mammalian and tick tissues is likely to be essential for successful completion of the enzootic life cycle but, given the historical difficulties in genetic manipulation of these organisms, characterization of factors promoting cell adhesion has until recently largely been confined to either the manipulation of host cells or the analysis of potential bacterial ligands in the form of recombinant proteins. These studies have led to the identification of several mammalian receptors for Lyme disease spirochetes, including glycosaminoglycans, decorin, fibronectin and integrins, as well as a tick receptor for the bacterium, and also candidate cognate bacterial ligands. Recent advances in our ability to genetically manipulate Lyme disease spirochetes, particularly B. burgdorferi, are now providing us with firm evidence that these ligands indeed do promote bacterial adherence to host cells, and with new insights into the roles of these multifacted Borrelia-host cell interactions during mammalian and arthropod infection.     

Antioxidant properties of proanthocyanidins of Uncaria tomentosa bark decoction: a mechanism for anti-inflammatory activity

Decoctions prepared from the bark of Uncaria tomentosa (cat's claw) are widely used in the traditional Peruvian medicine for the treatment of several diseases, in particular as a potent anti-inflammatory agent. Therefore, the main purpose of this study was to determine if the well-known anti-inflammatory activity of cat's claw decoction was related with its reactivity with the oxidant species generated in the inflammatory process and to establish a relationship between such antioxidant ability and its phenolic composition. We observed that the decoction prepared according to the traditional Peruvian medicine presented a potent radical scavenger activity, as suggested by its high capacity to reduce the free radical diphenylpicrylhydrazyl, and by its reaction with superoxide anion, peroxyl and hydroxyl radicals as well as with the oxidant species, hydrogen peroxide and hypochlorous acid. It also protected membrane lipids against peroxidation induced by the iron/ascorbate system, as evaluated by the formation of thiobarbituric acid-reactive substances (TBARs). The decoction phenolic profile was established by chromatographic analysis (HPLC/DAD and TLC) revealing essentially the presence of proanthocyanidins (oligomeric procyanidins) and phenolic acids, mainly caffeic acid. Thus, our results provide evidence for an antioxidant mechanism underlying the anti-inflammatory activity of cat's claw and support some of the biological effects of proanthocyanidins, more exactly its antioxidant and radical scavenging activities.     

Antibiosis and antixenosis to Aphis gossypii (Homoptera: Aphididac) in Colocasia esculenta

Fifty cultivars of taro, Colocasia esculenta (L.) Schott (Araceae), collected from islands in Micronesia and Polynesia, eight cultivars from the University of Hawaii's taro germplasm collection, and a closely related aroid, Xanthosorna sagittifolium (L.) (Araceae), were screened for antibiosis and antixenosis to Aphis gossypii Clover. Life history data for A. gossypii were collected by assessing survivorship and fecundity of aphids caged on taro leaves in the field. Significant differences in aphid reproductive rate and longevity were observed among the taro cultivars, and cultivars were ranked from most resistant to most susceptible. Antixenosis was assayed in the laboratory in a multiround choice test where A. gossypii were offered four leaf discs excised from different taro cultivars. Additionally, field observations of aphid abundance on taro cultivars were made to corroborate clip cage studies and laboratory experiments. 'Iliuaua','Rumung Mary','Maria', 'Ketan 36', and'Agaga' were the most resistant in terms of reducing aphid fecundity and survivorship, whereas the Iliuana,'Purple', 'TC-83001', and 'Putih 24' were least preferred in aphid choice tests. X. sagittifolium consistently exhibited strong aphid resistance. Resistant cultivars identified in this study may form the basis of breeding programs seeking to combine aphid resistance with other desirable agronomic traits in taro.     

Improved liquid chromatographic method for mitoxantrone quantification in mouse plasma and tissues to study the pharmacokinetics of a liposome entrapped mitoxantrone formulation

A simple, rapid HPLC method for quantification of mitoxantrone in mouse plasma and tissue homogenates in the presence of a liposome entrapped mitoxantrone formulation (LEM-ETU) is described. Sample preparation is achieved by protein precipitation of 100 microl plasma or 200 microl tissue homogenate with an equal volume of methanol containing 0.5 M hydrochloric acid:acetonitrile (90:10, v/v). Ametantrone is used as the internal standard (i.s.). Mitoxantrone and i.s. are separated on a C18 reversed phase HPLC column, and quantified by their absorbance at 655 nm. In plasma, the standard curve is linear from 5 to 1000 ng/ml, and the precision (%CV) and accuracy (percentage of nominal concentration) are within 10%. In mouse tissue (heart, kidney, liver, lung, and spleen) homogenates (5%, w/v), the standard curve is linear from 25 to 2000 ng/ml, with acceptable precision and accuracy. The method was used to successfully quantify mitoxantrone in mouse plasma and tissue samples to support a pharmacokinetic study of LEM-ETU in mice.     

Enolase from Streptococcus sobrinus is an immunosuppressive protein

A strategy of Streptococcus sobrinus, a major agent of dental caries, to survive and colonize the host consists of the production of a protein that suppresses the specific antibody responses. We have cloned the gene coding for a protein with immunosuppressive activity. It contains an open reading frame of 1302 base pairs encoding a polypeptide with 434 amino acid residues and a molecular mass of 46910 Da. The gene product is homologous to enolases from several organisms. The polypeptide was expressed in Escherichia coli as a hexahistidine-tagged protein and purified in a fluoride-sensitive enzymatically active form. Pretreatment of mice with the S. sobrinus recombinant enolase suppresses a primary immune response against T-cell dependent antigens. This immunosuppressive effect is specific to the antigen used in the immunization, as it is not observed when the immune response against other antigens is analysed. Furthermore, the S. sobrinus recombinant enolase stimulates an early production of interleukin-10, an anti-inflammatory cytokine, and not the pro-inflammatory cytokine IFN-gamma. These observations indicate that enolase acts in the suppression of the specific host immune response against S. sobrinus infection.     

Dissolved and particulate nutrient flux from three adjacent agricultural watersheds: A five-year study

Fluxes of dissolved and particulate nitrogen (N) and phosphorus(P) from three adjacent watersheds were quantified with ahigh-resolution sampling program over a five-year period. The watershedsvary by an order of magnitude in area (12,875, 7968 and 1206 ha), and inall three watersheds intensive agriculture comprises > 90% ofland. Annual fluxes of dissolved N and P per unit watershed area (exportcoefficients) varied 2X among watersheds, and patterns were notdirectly related to watershed size. Over the five-year period, meanannual flux of soluble reactive P (SRP) was 0.583 kg P ·ha^–1 · yr^–1 from the smallestwatershed and 0.295 kg P · ha^–1 ·yr^–1 from the intermediate-sized watershed, which hadthe lowest SRP flux. Mean annual flux of nitrate was 20.53 kg N ·ha^–1 · yr^–1 in the smallestwatershed and 44.77 kg N · ha^–1 ·yr^–1 in the intermediate-sized watershed, which had thehighest nitrate flux. As a consequence, the export ratio of dissolvedinorganic N to SRP varied from 80 (molar) in the smallest watershed to335 in the intermediate-sized watershed. Because most N was exported asnitrate, differences among watersheds in total N flux were similar tothose for nitrate. Hence, the total N:P export ratio was 42(molar) for the smallest watershed and 109 for the intermediate-sizedwatershed. In contrast, there were no clear differences among watershedsin the export coefficients of particulate N, P, or carbon, even though> 50% of total P was exported as particulate P in allwatersheds. All nutrient fractions were exported at higher rates in wetyears than in dry years, but precipitation-driven variability in exportcoefficients was greater for particulate fractions than for dissolvedfractions.Examination of hydrological regimes showed that, for all nutrientfractions, most export occurred during stormflow. However, theproportion of nitrate flux exported as baseflow was much greater thanthe proportion of SRP flux exported as baseflow, for all threewatersheds (25–37% of nitrate exported as baseflow vs.3–13% of SRP exported as baseflow). In addition, baseflowcomprised a greater proportion of total discharge in theintermediate-sized watershed (43.7% of total discharge) than theother two watersheds (29.3 and 30.1%). Thus, higher nitrateexport coefficients in the intermediate-sized watershed may haveresulted from the greater contribution of baseflow in this watershed.Other factors potentially contributing to higher nitrate exportcoefficients in this watershed may be a thicker layer of loess soils anda lower proportion of riparian forest than the other watersheds. Theamong-watershed variability in SRP concentrations and exportcoefficients remains largely unexplained, and might represent theminimum expected variation among similar agriculturalwatersheds.     

Deuterium NMR Study of the Effect of Ergosterol on POPE Membranes

We study the effect of ergosterol on the physical properties of 1-[²H₃₁]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) multibilayers using deuterium nuclear magnetic resonance. NMR spectra were taken as a function of temperature and ergosterol concentration up to 70 mol %. The spectral first moments show that there is a dramatic difference in the ability of ergosterol to disorder the gel phase and to order the liquid-crystalline phase of POPE membranes, an unusual behavior among lipid/sterol systems studied up to now. Further investigation of the liquid-crystalline phase shows that ergosterol (erg) increases the chain order of POPE-d31, but that this effect saturates at 10 mol % ergosterol. This is in marked contrast to the effect of cholesterol (chol) on POPE membranes: the chain order of POPE increases with cholesterol to at least 45 mol %. Moreover, we found that at higher ergosterol concentrations (>40 mol %) ergosterol decreases the POPE-d31 chain order, which, to our knowledge, has not been directly observed in other lipid/sterol systems. The temperature-composition phase diagram is presented. Finally, at all ergosterol concentrations, the chain order of liquid-crystalline-phase POPE is much smaller than that of comparable POPE/chol membranes. This implies that there is no liquid-ordered phase behavior for POPE/erg membranes.