Analysis of regulatory sequences in androgen-responsive genes

We have analysed the effect of androgens on the activity of promoters from MMTV, and the rat prostate C3(1) and mouse secretory protease inhibitor genes. MMTV promoter activity was stimulated by testosterone as well as progesterone and dexamethasone but not by oestradiol. Deletion analysis indicated that the three steroids acted through DNA sequences between nucleotides -201 and -69 upstream of the MMTV cap site. In contrast, the promoters for the C3(1) gene and the protease inhibitor gene were unaffected by testosterone in a number of cell types, including prostate cells, despite the fact that the MMTV promoter was stimulated in such cells.     

Physiological effects upon <Emphasis Type="Italic">Amblyomma americanum</Emphasis> (Acari: Ixodidae) infected with <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (Ascomycota: Hypocreales)

Unfed adult Amblyomma americanum were exposed to the entomopathogenic fungus Beauveria bassiana. Ticks exposed to the fungus exhibited reduced survival and increased water loss as indicated by change in weight. Treated ticks survived 7.2 ± 0.22 days (mean ± SE) and controls survived 17.9 ± 0.73 days (P = 0.01; df = 57). At death, ticks exposed to the fungus had lost 25.2 ± 0.84% of their starting weight; control ticks had lost 14.1 ± 0.85% of their starting weight (P = 0.01; df = 96). Water loss was highest immediately following inoculation, although losses continued to be higher than in uninoculated ticks. This suggests that fungal penetration causes sufficient cuticle damage to cause desiccation, although other water-loss avenues exist, including increased time of spiracular opening. Additionally this study did not eliminate the possibility of a negative impact on water vapor uptake. This is the first study to investigate the effect of an entomopathogenic fungus on the water balance of a tick.     

Automated multidimensional single molecule fluorescence microscopy feature detection and tracking

Characterisation of multi-protein interactions in cellular networks can be achieved by optical microscopy using multidimensional single molecule fluorescence imaging. Proteins of different species, individually labelled with a single fluorophore, can be imaged as isolated spots (features) of different colour light in different channels, and their diffusive behaviour in cells directly measured through time. Challenges in data analysis have, however, thus far hindered its application in biology. A set of methods for the automated analysis of multidimensional single molecule microscopy data from cells is presented, incorporating Bayesian segmentation-based feature detection, image registration and particle tracking. Single molecules of different colours can be simultaneously detected in noisy, high background data with an arbitrary number of channels, acquired simultaneously or time-multiplexed, and then tracked through time. The resulting traces can be further analysed, for example to detect intensity steps, count discrete intensity levels, measure fluorescence resonance energy transfer (FRET) or changes in polarisation. Examples are shown illustrating the use of the algorithms in investigations of the epidermal growth factor receptor (EGFR) signalling network, a key target for cancer therapeutics, and with simulated data.     

Characterizing genetic diversity of contemporary pacific chickens using mitochondrial DNA analyses

Background

Mitochondrial DNA (mtDNA) hypervariable region (HVR) sequences of prehistoric Polynesian chicken samples reflect dispersal of two haplogroups—D and E—by the settlers of the Pacific. The distribution of these chicken haplogroups has been used as an indicator of human movement. Recent analyses suggested similarities between prehistoric Pacific and South American chicken samples, perhaps reflecting prehistoric Polynesian introduction of the chicken into South America. These analyses have been heavily debated. The current distribution of the D and E lineages among contemporary chicken populations in the Western Pacific is unclear, but might ultimately help to inform debates about the movements of humans that carried them.

Objectives

We sought to characterize contemporary mtDNA diversity among chickens in two of the earliest settled archipelagoes of Remote Oceania, the Marianas and Vanuatu.

Methods

We generated HVR sequences for 43 chickens from four islands in Vanuatu, and for 5 chickens from Guam in the Marianas.

Results

Forty samples from Vanuatu and three from Guam were assigned to haplogroup D, supporting this as a Pacific chicken haplogroup that persists in the Western Pacific. Two haplogroup E lineages were observed in Guam and two in Vanuatu. Of the E lineages in Vanuatu, one was identical to prehistoric Vanuatu and Polynesian samples and the other differed by one polymorphism. Contrary to our expectations, we observed few globally distributed domesticate lineages not associated with Pacific chicken dispersal. This might suggest less European introgression of chickens into Vanuatu than expected. If so, the E lineages might represent lineages maintained from ancient Pacific chicken introductions. The Vanuatu sample might thus provide an opportunity to distinguish between maintained ancestral Pacific chicken lineages and replacement by global domesticates through genomic analyses, which could resolve questions of contemporary haplogroup E chicken relationships and inform interpretations of debated sequences from archaeological samples.     

Temporal variability and coherence of euphotic zone bacterial communities over a decade in the Southern California Bight

Time-series are critical to understanding long-term natural variability in the oceans. Bacterial communities in the euphotic zone were investigated for over a decade at the San Pedro Ocean Time-series station (SPOT) off southern California. Community composition was assessed by Automated Ribosomal Intergenic Spacer Analysis (ARISA) and coupled with measurements of oceanographic parameters for the surface ocean (0–5 m) and deep chlorophyll maximum (DCM, average depth ∼30 m). SAR11 and cyanobacterial ecotypes comprised typically more than one-third of the measured community; diversity within both was temporally variable, although a few operational taxonomic units (OTUs) were consistently more abundant. Persistent OTUs, mostly Alphaproteobacteria (SAR11 clade), Actinobacteria and Flavobacteria, tended to be abundant, in contrast to many rarer yet intermittent and ephemeral OTUs. Association networks revealed potential niches for key OTUs from SAR11, cyanobacteria, SAR86 and other common clades on the basis of robust correlations. Resilience was evident by the average communities drifting only slightly as years passed. Average Bray-Curtis similarity between any pair of dates was ∼40%, with a slight decrease over the decade and obvious near-surface seasonality; communities 8–10 years apart were slightly more different than those 1–4 years apart with the highest rate of change at 0–5 m between communities <4 years apart. The surface exhibited more pronounced seasonality than the DCM. Inter-depth Bray-Curtis similarities repeatedly decreased as the water column stratified each summer. Environmental factors were better predictors of shifts in community composition than months or elapsed time alone; yet, the best predictor was community composition at the other depth (that is, 0–5 m versus DCM).     

Behavioral risk factors for obesity during health transition in Vanuatu,South Pacific

The South Pacific archipelago of Vanuatu, like many developing countries, is currently experiencing a shift in disease burdens from infectious to chronic diseases with economic development. A rapid increase in obesity prevalence represents one component of this “health transition.” Objective: To identify behaviors associated with measures of obesity in Vanuatu. Design and Methods: Five hundred and thirty four adults from three islands varying in level of economic development were surveyed. Height, weight, waist, and hip circumferences; triceps, subscapular and suprailiac skinfolds; and percent body fat (%BF) by bioelectrical impedance were measured. Diet through 24‐h dietary recall and physical activity patterns using a survey were assessed. We analyzed prevalence of obesity and central obesity based on multiple indicators (body mass index, %BF, waist circumference, and waist‐to‐height ratio), and analyzed differences among islands and associations with behavioral patterns. Results: Obesity prevalence was lowest among rural and highest among suburban participants. Prevalence of central obesity was particularly high among women (up to 73.9%), even in rural areas (ranging from 14.7 to 41.2% depending on the measure used). Heavier reliance on animal protein and incorporation of Western foods in the diet—specifically, tinned fish and instant noodles—was significantly associated with increased obesity risk. Conclusions: Even in rural areas where diets and lifestyles remain largely traditional, modest incorporation of Western foods in the diet can contribute to increased risk of obesity. Early prevention efforts are thus particularly important during health transition. Where public health resources are limited, education about dietary change could be the best target for prevention.     

The discovery of long-acting saligenin β₂ adrenergic receptor agonists incorporating a urea group

A series of novel, potent and selective human β(2) adrenoceptor agonists incorporating a urea moiety on the terminal right-hand side phenyl ring of (R)-salmeterol is presented. Urea 9j had long duration of action in vitro on guinea pig trachea, and also in vivo similar to that of salmeterol. It had lower oral absorption and bioavailability than salmeterol in both rat and dog. It had a turnover ratio similar to salmeterol, with no evidence for formation of any aniline metabolites in human liver microsomes and hepatocytes. However no crystalline salts suitable for inhaled delivery were identified.     

Involvement of β3-adrenergic receptors in in vivo cardiovascular regulation in rainbow trout (Oncorhynchus mykiss)

Currently, we have little information concerning the involvement of β₃-adrenergic receptors (AR) in cardiovascular regulation in fishes. The goal of this study was to investigate the effect of β₃-AR ligands on in vivo cardiovascular function in larval and adult rainbow trout (Oncorhynchus mykiss). In adult fish, injection of BRL₃₇₃₄₄ (β₃-AR agonist) resulted in an increase in heart rate (f_H) (~ 31%) while stroke volume (Sv) was reduced (? 25.9%). Injection of SR_59230A (β₃-AR antagonist) and propranolol (β₁/β₂-AR antagonist) resulted in increases in dorsal aorta blood pressure (P_DA) with differing effect on cardiac variables (f_H and Sv). To confirm specificity of the results, BRL₃₇₃₄₄ was injected following sequential injections of phentolamine (α₁-AR antagonist), atropine (muscarinic antagonist), propranolol and SR_59230A. While phentolamine had no effect on BRL₃₇₃₄₄, atropine completely abolished the influence of BRL₃₇₃₄₄ on f_H, Sv and cardiac output (Q). In larval trout, BRL₃₇₃₄₄ (10 and 100 μM) induced a significant concentration-dependent increase in f_H while SR_59230A (1 and 10 μM) and propranolol (1 and 10 μM) separately caused a significant concentration-dependent decrease. These data suggest that β₃-ARs have an important role in regulation of cardiovascular function, and provide evidence for a potential interaction between muscarinic and adrenergic receptors in rainbow trout.     

Role of the membrane cortex in neutrophil deformation in small pipets.

The simplest model for a neutrophil in its "passive" state views the cell as consisting of a liquid-like cytoplasmic region surrounded by a membrane. The cell surface is in a state of isotropic contraction, which causes the cell to assume a spherical shape. This contraction is characterized by the cortical tension. The cortical tension shows a weak area dilation dependence, and it determines the elastic properties of the cell for small curvature deformations. At high curvature deformations in small pipets (with internal radii less than 1 micron), the measured critical suction pressure for cell flow into the pipet is larger than its estimate from the law of Laplace. A model is proposed where the region consisting of the cytoplasm membrane and the underlying cortex (having a finite thickness) is introduced at the cell surface. The mechanical properties of this region are characterized by the apparent cortical tension (defined as a free contraction energy per unit area) and the apparent bending modulus (introduced as a bending free energy per unit area) of its middle plane. The model predicts that for small curvature deformations (in pipets having radii larger than 1.2 microns) the role of the cortical thickness and the resistance for bending of the membrane-cortex complex is negligible. For high curvature deformations, they lead to elevated suction pressures above the values predicted from the law of Laplace. The existence of elevated suction pressures for pipets with radii from 1 micron down to 0.24 micron is found experimentally. The measured excess suction pressures cannot be explained only by the modified law of Laplace (for a cortex with finite thickness and negligible bending resistance), because it predicts unacceptable high cortical thicknesses (from 0.3 to 0.7 micron). It is concluded that the membrane-cortex complex has an apparent bending modulus from 1 x 10(-18) to 2 x 10(-18) J for a cortex with a thickness from 0.1 micron down to values much smaller than the radius of the smallest pipet (0.24 micron) used in this study.