Benzopyrans in <Emphasis Type="Italic">Hypericum polyanthemum</Emphasis> Klotzsch ex Reichardt cultured in vitro

Hypericum polyanthemum Klotzsch ex Reichardt, an endemic species of Southern Brazil, was micropropagated on MS medium supplemented with 1.78 μM BAP. Shoot proliferation and rooting was achieved on hormone-free medium and plantlets survived acclimatization. The bioactive compounds: 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran (HP1), 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2) and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3) were quantified in the leaves, stems and roots of propagated and acclimatized plantlets and compared with the field-grown plants. The HPLC analysis revealed that the three benzopyrans are accumulated in the aerial parts and the concentration varied with the age of the plant whereas the roots were capable of accumulating only HP3. Greatest yield of HP1 (7.12 mg/g DW) was quantified in the leaves of the acclimatized plantlets, whereas the flowers of the plants from natural habitat displayed higher amounts of HP2 (11.04 mg/g DW) and HP3 (13.99 mg/g DW).     

Low utilization of circulating glucose after food withdrawal in Snell dwarf mice

Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively.     

Human immunodeficiency virus-specific CD8+ T-cell activity is detectable from birth in the majority of in utero-infected infants

Human immunodeficiency virus (HIV)-infected infants in sub-Saharan Africa typically progress to AIDS or death by 2 years of life in the absence of antiretroviral therapy. This rapid progression to HIV disease has been related to immaturity of the adaptive immune response in infants. We screened 740 infants born to HIV-infected mothers and tracked development and specificity of HIV-specific CD8⁺ T-cell responses in 63 HIV-infected infants identified using gamma interferon enzyme-linked immunospot assays and intracellular cytokine staining. Forty-four in utero-infected and 19 intrapartum-infected infants were compared to 45 chronically infected children >2 years of age. Seventy percent (14 of 20) in utero-infected infants tested within the first week of life demonstrated HIV-specific CD8⁺ T-cell responses. Gag, Pol, and Nef were the principally targeted regions in chronic pediatric infection. However, Env dominated the overall response in one-third (12/36) of the acutely infected infants, compared to only 2/45 (4%) of chronically infected children (P = 0.00083). Gag-specific CD4⁺ T-cell responses were minimal to undetectable in the first 6 months of pediatric infection. These data indicate that failure to control HIV replication in in utero-infected infants is not due to an inability to induce responses but instead suggest secondary failure of adaptive immunity in containing this infection. Moreover, the detection of virus-specific CD8⁺ T-cell responses in the first days of life in most in utero-infected infants is encouraging for HIV vaccine interventions in infants.     

Multiplexing siRNAs to compress RNAi-based screen size in human cells

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (~800 siRNAs, ~400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.     

Factors influencing owner satisfaction with companion-dog-training facilities

The aim of this study was to survey people currently attending companion-dog-training facilities about their reasons for attending training, their expectations prior to training, their training experiences, and the factors contributing to their satisfaction with these experiences. The 178 participants indicated that they experienced few canine behavioral difficulties prior to attending training, with factors other than dog behavior influencing their decision to attend a particular facility. Features identified as important were characteristics of the instructors and the facility itself, opportunities for the dog caretaker (owner) to develop dog-training skills and knowledge, and specific canine behavior outcomes. Satisfaction was a multidimensional concept: satisfaction with the instructors and facility being dissociable from satisfaction with progress made by the dog and handler. Each of these represented different aspects of the training experience. Training facilities may need to address a wide range of issues to engage additional dog owners in training activities. Identification of these issues is an important first step in this process.     

Sprouty2 binds Grb2 at two different proline-rich regions, and the mechanism of ERK inhibition is independent of this interaction

Sprouty2 has been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. Sprouty2 directly interacts with the adapter protein Grb2, member of the receptor tyrosine kinase-induced signaling pathways. In considering the functional role of Grb2, we investigated whether the interaction with this protein was responsible for ERK pathway inhibition. We found that the binding between Sprouty2 and Grb2 is constitutive, independent of Sprouty2 tyrosine phosphorylation, although it is increased when fibroblast growth factor receptor is activated. This connection is mediated by the N-terminal SH3 domain of Grb2 and two Sprouty2 proline-rich stretches (residues 59-64 and 303-307). Most importantly, a double Sprouty2 mutant (hSpry2 P59AP304A), which is unable to bind Grb2, developed at a similar inhibition level of fibroblast growth factor receptor-ERK pathway than that which originated from Sprouty2 wt. These results are evidence that the Sprouty2 mechanism of ERK inhibition is independent of Grb2 binding.     

Selective cytotoxicity of Pancratistatin-related natural <Emphasis Type="Italic">Amaryllidaceae</Emphasis> alkaloids: evaluation of the activity of two new compounds

Background  

Pancratistatin (PST), a compound extracted from an Amaryllidaceae (AMD) family plant, has been shown to specifically induce apoptosis in cancer cells with no/minimal toxic effect on normal cells. A systematic synthetic approach has indicated that the minimum cytotoxic pharmacophore comprises the trans-fused b/c-ring system containing the 2, 3, 4-triol unit in the C-ring. To further explore the structure-activity relationship of this group of compounds we have investigated the anti-cancer efficacy and specificity of two PST-related natural compounds, AMD4 and AMD5. Both of these compounds lack the polyhydroxylated lycorane element of PST instead having a methoxy-substuituted crinane skeleton.     

Evolution of opsin expression in birds driven by sexual selection and habitat

Theories of sexual and natural selection predict coevolution of visual perception with conspecific colour and/or the light environment animals occupy. One way to test these theories is to focus on the visual system, which can be achieved by studying the opsin-based visual pigments that mediate vision. Birds vary greatly in colour, but opsin gene coding sequences and associated visual pigment spectral sensitivities are known to be rather invariant across birds. Here, I studied expression of the four cone opsin genes (Lws, Rh2, Sws2 and Sws1) in 16 species of New World warblers (Parulidae). I found levels of opsin expression vary both across species and between the sexes. Across species, female, but not male Sws2 expression is associated with an index of sexual selection, plumage dichromatism. This fits predictions of classic sexual selection models, in which the sensory system changes in females, presumably impacting female preference, and co-evolves with male plumage. Expression of the opsins at the extremes of the light spectrum, Lws and Uvs, correlates with the inferred light environment occupied by the different species. Unlike opsin spectral tuning, regulation of opsin gene expression allows for fast adaptive evolution of the visual system in response to natural and sexual selection, and in particular, sex-specific selection pressures.     

Evolutionary dynamics of Rh2 opsins in birds demonstrate an episode of accelerated evolution in the New World warblers (Setophaga)

Low rates of sequence evolution associated with purifying selection can be interrupted by episodic changes in selective regimes. Visual pigments are a unique system in which we can investigate the functional consequences of genetic changes, therefore connecting genotype to phenotype in the context of natural and sexual selection pressures. We study the RH2 and RH1 visual pigments (opsins) across 22 bird species belonging to two ecologically convergent clades, the New World warblers (Parulidae) and Old World warblers (Phylloscopidae) and evaluate rates of evolution in these clades along with data from 21 additional species. We demonstrate generally slow evolution of these opsins: both Rh1 and Rh2 are highly conserved across Old World and New World warblers. However, Rh2 underwent a burst of evolution within the New World genus Setophaga, where it accumulated substitutions at 6 amino acid sites across the species we studied. Evolutionary analyses revealed a significant increase in d_N/d_S in Setophaga, implying relatively strong selective pressures to overcome long‐standing purifying selection. We studied the effects of each substitution on spectral tuning and found they do not cause large spectral shifts. Thus, substitutions may reflect other aspects of opsin function, such as those affecting photosensitivity and/or dark–light adaptation. Although it is unclear what these alterations mean for colour perception, we suggest that rapid evolution is linked to sexual selection, given the exceptional plumage colour diversification in Setophaga.     

SWS2 visual pigment evolution as a test of historically contingent patterns of plumage color evolution in warblers

Distantly related clades that occupy similar environments may differ due to the lasting imprint of their ancestors—historical contingency. The New World warblers (Parulidae) and Old World warblers (Phylloscopidae) are ecologically similar clades that differ strikingly in plumage coloration. We studied genetic and functional evolution of the short‐wavelength‐sensitive visual pigments (SWS2 and SWS1) to ask if altered color perception could contribute to the plumage color differences between clades. We show SWS2 is short‐wavelength shifted in birds that occupy open environments, such as finches, compared to those in closed environments, including warblers. Phylogenetic reconstructions indicate New World warblers were derived from a finch‐like form that colonized from the Old World 15–20 Ma. During this process, the SWS2 gene accumulated six substitutions in branches leading to New World warblers, inviting the hypothesis that passage through a finch‐like ancestor resulted in SWS2 evolution. In fact, we show spectral tuning remained similar across warblers as well as the finch ancestor. Results reject the hypothesis of historical contingency based on opsin spectral tuning, but point to evolution of other aspects of visual pigment function. Using the approach outlined here, historical contingency becomes a generally testable theory in systems where genotype and phenotype can be connected.