Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 degrees C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled.     

Study of LH response to GnRH in the young male as a criterion of genetic merit for female reproduction in sheep

A high and a low response line in sheep were selected on the basis of the mean concentration of LH in 10-week-old Finn-Dorset ram lambs after an i.v. injection of 5 micrograms GnRH. After 8 male generations the mean LH response of the high line was more than 5-fold that of the low line and the heritability of the selected trait was estimated at 0.44 +/- 0.015. Highly significant line differences in mean LH response to GnRH were also found in males at 20 weeks of age and females at 10 and 20 weeks of age and the genetic correlations between the four LH response traits appear to be close to unity. Large line differences in the mean FSH response to GnRH were also found in both males and females at 10 and 20 weeks of age. Selection had little effect on the physical characteristics of lambs. High-response line ewes entering their first breeding season at about 7 months of age showed oestrus earlier in the season and had higher ovulation rates and numbers of lambs born per ewe lambing than did low-response line ewes. In the second breeding season, at about 19 months of age, the only line difference was a higher ovulation rate early in the breeding season in high-line ewes. It is suggested that these changes may be mediated by a more rapid response in high-line ewes to increased GnRH stimulation at puberty or at the beginning of the breeding season.     

Nonlinear magic: multiphoton microscopy in the biosciences

Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.     

Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms

Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated from the surface of the marine alga Ulva australis, were screened for synergistic interactions within biofilms when present together in different combinations. Four isolates, Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and Acinetobacter lwoffii, were found to interact synergistically in biofilms formed in 96-well microtiter plates: biofilm biomass was observed to increase by >167% in biofilms formed by the four strains compared to biofilms composed of single strains. When exposed to the antibacterial agent hydrogen peroxide or tetracycline, the relative activity (exposed versus nonexposed biofilms) of the four-species biofilm was markedly higher than that in any of the single-species biofilms. Moreover, in biofilms established on glass surfaces in flow cells and subjected to invasion by the antibacterial protein-producing Pseudoalteromonas tunicata, the four-species biofilms resisted invasion to a greater extent than did the biofilms formed by the single species. Replacement of each strain by its cell-free culture supernatant suggested that synergy was dependent both on species-specific physical interactions between cells and on extracellular secreted factors or less specific interactions. In summary, our data strongly indicate that synergistic effects promote biofilm biomass and resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms.     

Genetic Connectivity among Populations of an Endangered Snake Species from Southeastern Australia (Hoplocephalus bungaroides, Elapidae)

For endangered species that persist as apparently isolated populations within a previously more extensive range, the degree of genetic exchange between those populations is critical to conservation and management. A lack of gene flow can exacerbate impacts of threatening processes and delay or prevent colonization of sites after local extirpation. The broad-headed snake, Hoplocephalus bungaroides, is a small venomous species restricted to a handful of disjunct reserves near Sydney, Australia. Mark-recapture studies have indicated low vagility for this ambush predator, suggesting that gene flow also may be low. However, our analyses of 11 microsatellite loci from 163 snakes collected in Morton National Park, from six sites within a 10-km diameter, suggest relatively high rates of gene flow among sites. Most populations exchange genes with each other, with one large population serving as a source area and smaller populations apparently acting as sinks. About half of the juvenile snakes, for which we could reliably infer parentage, were collected from populations other than those in which we collected their putative parents. As expected from the snakes' reliance on rocky outcrops during cooler months of the year, most gene flow appears to be along sandstone plateaux rather than across the densely forested valleys that separate plateaux. The unexpectedly high rates of gene flow on a landscape scale are encouraging for future conservation of this endangered taxon. For example, wildlife managers could conserve broad-headed snakes by restoring habitats near extant source populations in areas predicted to be least affected by future climate change.     

Is tilting behaviour at low swimming speeds unique to negatively buoyant fish? Observations on steelhead trout,Oncorhynchus mykiss,and bluegill,Lepomis macrochirus

When swimming at low speeds, steelhead trout and bluegill sunfish tilted the body at an angle to the mean swimming direction. Trout swam using continuous body/caudal fin undulation, with a positive (head-up) tilt angle ( 0 , degrees) that decreased with swimming speed ( u , cm s⁻¹) according to: 0 =(164±96).u^{(−1.14±0.41)} (regression coefficients; mean±2 s.e. ). Bluegill swimming gaits were more diverse and negative (head down) tilt angles were usual. Tilt angle was −3·0 ± 0.9° in pectoral fin swimming at speeds of approximately 0.2–1.7 body length s⁻¹ (Ls⁻¹; 3–24 cm s⁻¹), −4.5 ±2.6° during pectoral fin plus body/caudal fin swimming at 1·2–1·7 L s⁻¹ (17–24cm s⁻¹), and −5.0± 1.0° during continuous body/caudal fin swimming at 1.6 and 2.5 L s⁻¹ (22 and 35cm s⁻¹). At higher speeds, bluegill used burst-and-coast swimming for which the tilt angle was 0.1±0.6°. These observations suggest that tilting is a general phenomenon of low speed swimming at which stabilizers lose their effectiveness. Tilting is interpreted as an active compensatory mechanism associated with increased drag and concomitant increased propulsor velocities to provide better stabilizing forces. Increased drag associated with trimming also explains the well-known observation that the relationship between tail-beat frequency and swimming speed does not pass through the origin. Energy dissipated because of the drag increases at low swimming speeds is presumably smaller than that which would occur with unstable swimming.     

Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules.

We have developed a technique to detect, recognize, and track each individual low density lipoprotein receptor (LDL-R) molecule and small receptor clusters on the surface of human skin fibroblasts. Molecular recognition and high precision (30 nm) simultaneous automatic tracking of all of the individual receptors in the cell surface population utilize quantitative time-lapse low light level digital video fluorescence microscopy analyzed by purpose-designed algorithms executed on an image processing work station. The LDL-Rs are labeled with the biologically active, fluorescent LDL derivative dil-LDL. Individual LDL-Rs and unresolved small clusters are identified by measuring the fluorescence power radiated by the sub-resolution fluorescent spots in the image; identification of single particles is ascertained by four independent techniques. An automated tracking routine was developed to track simultaneously, and without user intervention, a multitude of fluorescent particles through a sequence of hundreds of time-lapse image frames. The limitations on tracking precision were found to depend on the signal-to-noise ratio of the tracked particle image and mechanical drift of the microscope system. We describe the methods involved in (i) time-lapse acquisition of the low-light level images, (ii) simultaneous automated tracking of the fluorescent diffraction limited punctate images, (iii) localizing particles with high precision and limitations, and (iv) detecting and identifying single and clustered LDL-Rs. These methods are generally applicable and provide a powerful tool to visualize and measure dynamics and interactions of individual integral membrane proteins on living cell surfaces.     

ABA-regulated promoter activity in stomatal guard cells

CDeT6-19 is an ABA-regulated gene which has been isolated from Craterostigma plantagineum . The CDeT6-19 gene promoter has been fused to the β- glucuronidase reporter gene ( GUS ) and used to stably transform Arabidopsis thaliana and Nicotiana tabacum . This construct has been shown to be expressed in stomatal guard cells and often in the adjacent epidermal cells of both species in response to both exogenous ABA and drought stress. These results indicate that the stomatal guard cell is competent to relay an ABA signal to the nucleus. In contrast GUS expression directed by the promoter from a predominantly seed-specific, ABA-regulated gene, Em , or the promoter from the ABA-regulated CDeT27-45 gene is not detectable in the epidermal or guard cells of tobacco or Arabidopsis in response to ABA. The fact that not all ABA-regulated gene promoters are active in stomatal guard cells suggests that effective transduction of the signal is dependent upon particular regions within the gene promoter or that guard cells lack all or part of the specific transduction apparatus required to couple the ABA signal to these promoters. This suggests that there are multiple ABA stimulus response coupling pathways. The identification of a regulatory sequence from an ABA-induced gene which is expressed in stomatal guard cells creates the possibility of examining the role of Ca²⁺ and other second messengers in ABA-induced gene expression.     

Last glacial maximum biomes reconstructed from pollen and plant macrofossil data from northern Eurasia

Pollen and plant macrofossil data from northern Eurasia were used to reconstruct the vegetation of the last glacial maximum (LGM: 18,000 ± 2000 ¹⁴C yr bp ) using an objective quantitative method for interpreting pollen data in terms of the biomes they represent ( Prentice et al., 1996 ). The results confirm previous qualitative vegetation reconstructions at the LGM but provide a more comprehensive analysis of the data. Tundra dominated a large area of northern Eurasia (north of 57°N) to the west, south and east of the Scandinavian ice sheet at the LGM. Steppe‐like vegetation was reconstructed in the latitudinal band from western Ukraine, where temperate deciduous forests grow today, to western Siberia, where taiga and cold deciduous forests grow today. The reconstruction shows that steppe graded into tundra in Siberia, which is not the case today. Taiga grew on the northern coast of the Sea of Azov, about 1500 km south of its present limit in European Russia. In contrast, taiga was reconstructed only slightly south of its southern limit today in south‐western Siberia. Broadleaved trees were confined to small refuges, e.g. on the eastern coast of the Black Sea, where cool mixed forest was reconstructed from the LGM data. Cool conifer forests in western Georgia were reconstructed as growing more than 1000 m lower than they grow today. The few scattered sites with LGM data from the Tien‐Shan Mountains and from northern Mongolia yielded biome reconstructions of steppe and taiga, which are the biomes growing there today.     

Extensive gaps and biases in our knowledge of a well‐known fauna: implications for integrating biological traits into macroecology

Aim Ecologists seeking to describe patterns at ever larger scales require compilations of data on the global abundance and distribution of species. Comparable compilations of biological data are needed to elucidate the mechanisms behind these patterns, but have received far less attention. We assess the availability of biological data across an entire assemblage: the well‐documented demersal marine fauna of the United Kingdom. We also test whether data availability for a species depends on its taxonomic group, maximum body size, the number of times it has been recorded in a global biogeographic database, or its commercial and conservation importance. Location Seas of the United Kingdom. Methods We defined a demersal marine fauna of 973 species from 15 phyla and 40 classes using five extensive surveys around the British Isles. We then quantified the availability of data on eight key biological traits (termed biological knowledge) for each species from online databases. Relationships between biological knowledge and our predictors were tested with generalized linear models. Results Full data on eight fundamental biological traits exist for only 9% (n= 88) of the UK demersal marine fauna, and 20% of species completely lack data. Clear trends in our knowledge exist: fish (median biological knowledge score = six traits) are much better known than invertebrates (one trait). Biological knowledge increases with biogeographic knowledge and (to a lesser extent) with body size, and is greater in species that are commercially exploited or of conservation concern. Main conclusions Our analysis reveals deep ignorance of the basic biology of a well‐studied fauna, highlighting the need for far greater efforts to compile biological trait data. Clear biases in our knowledge, relating to how well sampled or ‘important’ species are suggests that caution is required in extrapolating small subsets of biologically well‐known species to ecosystem‐level studies.