Morphological Variation in Populations of <Emphasis Type="Italic">Eulemur albocollaris</Emphasis> and <Emphasis Type="Italic">E. fulvus rufus</Emphasis>

Sexual dimorphism in body size and canine weaponry is commonly associated with high levels of male-male competition. When group living species do not rely heavily on male-male competition for access to females, sperm competition may represent a viable alternative strategy. Unlike most haplorhine primates, lemurs are typically monomorphic in body weight and canine height. We assessed variability of body mass dimorphism and canine size dimorphism in brown lemurs using morphometric data from 3 populations in southeastern Madagascar: Eulemur fulvus rufus, E. albocollaris, and hybrids of the species. We found significant male-biased canine dimorphism in E. albocollaris in conjunction with body-size monomorphism. We observed similar patterns in the hybrids, but E. fulvus rufus exhibited significant female-biased size dimorphism and canine monomorphism. Testes volume was relatively high across study populations. Thus, sperm competition appears to be strong in brown lemurs. E. albocollaris males combine sperm competition with large canines, but not higher body mass, indicating a difference in sexual strategy from most lemurs. Patterns of body mass and canine size dimorphism are not uniform across brown lemur populations, indicating that future work on these populations can explicitly test models that predict relationships between size dimorphism and various types of competition.     

Feeding efficiency of the larval ctenophore Mnemiopsis leidyi A. Agassiz (Ctenophora, Lobata)

Larval stages of the ctenophore Mnemiopsis leidyi rely on metazoanprey, such as Acartia tonsa nauplii and copepodites, to supporthigh growth rates. However, M. leidyi larvae <0.5 mm (totallength) had low retention efficiencies (REs) (proportion ofencountered prey actually ingested), 5.78 ± 2.6% (mean± SE), of nauplii and were often damaged by their encounters.REs of nauplii rapidly increased, 38.94 ± 3.73%, as larvaegrew to a size of     

Characterization of the linker 2 region in human vimentin using site-directed spin labeling and electron paramagnetic resonance

Site-directed spin labeling and electron paramagnetic resonance were used to probe residues 281-304 of human vimentin, a region that has been predicted to be a non-alpha-helical linker and the beginning of coiled-coil domain 2B. Though no direct test of linker structure has ever been made, this region has been hypothesized to be flexible with the polypeptide chains looping away from one another. EPR analysis of spin-labeled mutants indicates that (a) several residues reside in close proximity, suggesting that adjacent linker regions in a dimer run in parallel, and that (b) the polypeptide backbone is relatively rigid and inflexible in this region. However, this region does not show the characteristics of a coiled-coil as has been identified elsewhere in the molecule. Within this region, spectra from positions 283 and 291 are unique from all others thus far examined. These positions, predicted to be in a noncoiled-coil structure, display a significantly stronger interaction than the a-d contact positions of coiled-coil regions. Analysis of the early stages of assembly by dialysis from 8 M urea and progressive thermal denaturation shows the close apposition and structural rigidity at residues 283 and 291 occurs very early in assembly and with a relatively sudden onset, well before coiled-coil formation in other parts of the molecule. These features are inconsistent with hypotheses that envision the linkers as flexible regions, or as looping away from one another, and raise the possibility that the linker may be the site at which dimer alignment and/or formation is initiated. Spin labels placed further downstream yield spectra suggesting that the first regular heptad of rod domain 2 begins at position 302. In conjunction with our previous characterization of region 305-336 and the solved structure of rod 2B from 328-405, the full extent of coiled-coil domain in rod 2B is now known, spanning from vimentin positions 302-405.     

Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery

The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compound libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temperature (Delta Tm) obtained in the presence of ligands relative to that obtained in the absence of ligands. In this article, we report that the thermal shift assay can be conducted in an inexpensive, commercially available device for temperature control and fluorescence detection. The binding affinities obtained from thermal shift assays are compared with the binding affinities measured by isothermal titration calorimetry and with the IC(50) values from enzymatic assays. The potential pitfalls in the data analysis of thermal shift assays are also discussed.     

Pterin-based ornamental coloration predicts yolk antioxidant levels in female striped plateau lizards (Sceloporus virgatus)

1. Maternal investment in egg quality can have important consequences for offspring fitness. For example, yolk antioxidants can affect embryonic development as well as juvenile and adult phenotype. Thus, females may be selected to advertise their yolk antioxidant deposition to discriminatory males via ornamental signals, perhaps depending on the reproductive costs associated with signal production. 2. Female striped plateau lizards (Sceloporus virgatus) develop pterin-based orange colour patches during the reproductive season that influence male behaviour and that are positively associated with the phenotypic quality of the female and her offspring. Here, we assessed one potential developmental mechanism underlying the relationship between offspring quality and female ornamentation in S. virgatus, by examining the relationship between ornament expression and yolk antioxidant levels. 3. As expected, concentrations of the yolk antioxidants vitamin A, vitamin E and carotenoids (lutein and zeaxanthin) were strongly positively intercorrelated. Eggs from larger clutches had fewer antioxidants than eggs from smaller clutches, suggesting that females may be limited in antioxidant availability or use. Fertilized and unfertilized eggs did not differ in yolk antioxidant levels. 4. The size of a female's ornament was positively related to both the concentration and total amount of yolk antioxidants, and ornament colour was positively related to yolk antioxidant concentration. Thus, in S. virgatus, female ornaments may advertise egg quality. In addition, these data suggest that more ornamented females may produce higher-quality offspring, in part because their eggs contain more antioxidants. As the colour ornament of interest is derived from pterins, not carotenoids, direct resource trade-offs between ornaments and eggs may be eliminated, reducing reproductive costs associated with signalling. 5. This is the first example of a positive relationship between female ornamentation and yolk antioxidants in reptiles and may indicate the general importance of these patterns in oviparous vertebrates.     

Glutathione Reductase-null Malaria Parasites Have Normal Blood Stage Growth but Arrest during Development in the Mosquito

Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a γ-glutamylcysteine synthetase (γ-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for γ-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and γ-GCS by simultaneous disruption of gr and γ-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism.     

Regulation of PKR by HCV IRES RNA: Importance of Domain II and NS5A

Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2α, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5′-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of ∼ 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.     

Design and synthesis of orally bioavailable serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitors

The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.