Zinc-dependent interaction between dishevelled and the Drosophila Wnt antagonist naked cuticle

During Drosophila development, the naked cuticle (nkd) gene attenuates wingless/Wnt signaling through a negative feedback loop mechanism. Fly and vertebrate Nkd proteins contain a putative calcium-binding EF-hand motif, the EFX domain, that interacts with the basic/PDZ region of the Wnt signal transducer, dishevelled (Dsh). Here we show that Dsh binding by Drosophila Nkd in vitro is mediated by the EFX domain as well as an adjacent C-terminal sequence. In vivo data suggest that both of these regions contribute to the ability of Nkd to antagonize Wnt signaling. Mutations in the Nkd EF-hand designed to eliminate potential ion binding affected Nkd-Dsh interactions in the yeast two-hybrid assay but not in the glutathione S-transferase pull-down assay. Addition of the chelating agent EDTA abolished the in vitro Nkd-Dsh interaction. Surprisingly zinc, but not calcium, was able to restore Nkd-Dsh binding, suggesting a zinc-mediated interaction. Calcium 45- and zinc 65-blotting experiments show that Nkd is a zinc-binding metalloprotein. The results further clarify how Nkd may antagonize Wnt signaling via interaction with Dsh, and identify a novel zinc-binding domain in Drosophila Nkd that collaborates with the conserved EFX domain to bind Dsh.     

RNA recognition via the SAM domain of Smaug

The Nanos protein gradient in Drosophila, required for proper abdominal segmentation, is generated in part via translational repression of its mRNA by Smaug. We report here the crystal structure of the Smaug RNA binding domain, which shows no sequence homology to any previously characterized RNA binding motif. The structure reveals an unusual makeup in which a SAM domain, a common protein-protein interaction module, is affixed to a pseudo-HEAT repeat analogous topology (PHAT) domain. Unexpectedly, we find through a combination of structural and genetic analysis that it is primarily the SAM domain that interacts specifically with the appropriate nanos mRNA regulatory sequence. Therefore, in addition to their previously characterized roles in protein-protein interactions, some SAM domains play crucial roles in RNA binding.     

Water relations during desiccation of cysts of the potato-cyst nematode Globodera rostochiensis

The loss during desiccation of osmotically active water (OAW), which freezes during cooling to -45 degrees C, and osmotically inactive water (OIW), which remains unfrozen, from the cysts of the potato cyst nematode, Globodera rostochiensis, was determined using differential scanning calorimetry. Exotherms and endotherms associated with non-egg compartments were not detected after 5 min desiccation at 50% relative humidity and 20 degrees C. The pattern of water loss from the cysts indicates that water is lost from compartments outside the eggs first, that nearly all the non-egg water is OAW and that the OIW content of the cyst is contained within the eggs. Water is lost from the eggs only after the OAW content outside the eggs falls below that within the eggs. Both OAW and OIW are lost from the eggs during desiccation but the eggs retain a small amount of OIW. Other animals which survive some desiccation but which are not anhydrobiotic will tolerate the loss of OAW but not the loss of their OIW. Anhydrobiotic animals can survive the loss of both their OAW and a substantial proportion of their OIW.     

Effects of Hormone Therapy on Cognition and Mood in Recently Postmenopausal Women: Findings from the Randomized,Controlled KEEPS–Cognitive and Affective Study

Background

Menopausal hormone therapy (MHT) reportedly increases the risk of cognitive decline in women over age 65 y. It is unknown whether similar risks exist for recently postmenopausal women, and whether MHT affects mood in younger women. The ancillary Cognitive and Affective Study (KEEPS-Cog) of the Kronos Early Estrogen Prevention Study (KEEPS) examined the effects of up to 4 y of MHT on cognition and mood in recently postmenopausal women.

Methods and Findings

KEEPS, a randomized, double-blinded, placebo-controlled clinical trial, was conducted at nine US academic centers. Of the 727 women enrolled in KEEPS, 693 (95.3%) participated in the ancillary KEEPS-Cog, with 220 women randomized to receive 4 y of 0.45 mg/d oral conjugated equine estrogens (o-CEE) plus 200 mg/d micronized progesterone (m-P) for the first 12 d of each month, 211 women randomized to receive 50 μg/d transdermal estradiol (t-E2) plus 200 mg/d m-P for the first 12 d of each month, and 262 women randomized to receive placebo pills and patches. Primary outcomes included the Modified Mini-Mental State examination; four cognitive factors: verbal learning/memory, auditory attention/working memory, visual attention/executive function, and speeded language/mental flexibility; and a mood measure, the Profile of Mood States (POMS). MHT effects were analyzed using linear mixed-effects (LME) models, which make full use of all available data from each participant, including those with missing data. Data from those with and without full data were compared to assess for potential biases resulting from missing observations. For statistically significant results, we calculated effect sizes (ESs) to evaluate the magnitude of changes.On average, participants were 52.6 y old, and 1.4 y past their last menstrual period. By month 48, 169 (24.4%) and 158 (22.8%) of the 693 women who consented for ancillary KEEPS-Cog were lost to follow-up for cognitive assessment (3MS and cognitive factors) and mood evaluations (POMS), respectively. However, because LME models make full use all available data, including data from women with missing data, 95.5% of participants were included in the final analysis (n = 662 in cognitive analyses, and n = 661 in mood analyses). To be included in analyses, women must have provided baseline data, and data from at least one post-baseline visit. The mean length of follow-up was 2.85 y (standard deviation [SD] = 0.49) for cognitive outcomes and 2.76 (SD = 0.57) for mood outcomes. No treatment-related benefits were found on cognitive outcomes. For mood, model estimates indicated that women treated with o-CEE showed improvements in depression and anxiety symptoms over the 48 mo of treatment, compared to women on placebo. The model estimate for the depression subscale was −5.36 × 10⁻² (95% CI, −8.27 × 10⁻² to −2.44 × 10^−2; ES = 0.49, p < 0.001) and for the anxiety subscale was −3.01 × 10⁻² (95% CI, −5.09 × 10⁻² to −9.34 × 10⁻³; ES = 0.26, p < 0.001). Mood outcomes for women randomized to t-E2 were similar to those for women on placebo. Importantly, the KEEPS-Cog results cannot be extrapolated to treatment longer than 4 y.

Conclusions

The KEEPS-Cog findings suggest that for recently postmenopausal women, MHT did not alter cognition as hypothesized. However, beneficial mood effects with small to medium ESs were noted with 4 y of o-CEE, but not with 4 y of t-E2. The generalizability of these findings is limited to recently postmenopausal women with low cardiovascular risk profiles.

Trial Registration

ClinicalTrials.gov NCT00154180 and NCT00623311     

Effect of chronic treatment with Rosiglitazone on Leydig cell steroidogenesis in rats: <Emphasis Type="Italic">In vivo</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> studies

Background  

The present study was designed to examine the effect of chronic treatment with rosiglitazone - thiazolidinedione used in the treatment of type 2 diabetes mellitus for its insulin sensitizing effects - on the Leydig cell steroidogenic capacity and expression of the steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage enzyme (P450scc) in normal adult rats.     

Kinetics of active and passive components of water exchange between the air and a mite, Dermatophagoides farinae

Female North American house dust mites were found to exchange water with the ambient air from two compartments. At humidities above the critical equilibrium activity (CEA), transpiration out of a single large compartment was observed using HTO as a tracer for water. Total sorption into this compartment was also observed by following changes in the specific radioactivity. The sorption data required that an active process or pump be present. The water in this pump is the second compartment above the CEA. Below the CEA the large compartment could be identified as a compartment characterized by a small transpiration rate constant. The pump below the CEA becomes a rapidly transpiring fast compartment. By separating the water pool into two compartments, it was possible to relate a_v to k and $\text{m}\text{?}{}_{\mathrm{<mtext>S</mtext>}}$. The major effect of a_v on k was related to its effect on the permeability of the cuticle. The influence of a_v on $\text{m}\text{?}{}_{\mathrm{<mtext>S</mtext>}}$ was different for active and passive sorption. Above the CEA the pump operated at full capacity and active $\text{m}\text{?}{}_{\mathrm{<mtext>S</mtext>}}$ was directly proportional to a_v. Passive sorption was influenced by a_v in two ways. The driving force for $\text{m}\text{?}{}_{\mathrm{<mtext>S</mtext>}}$ was further reduced below saturation by the effect of a_v on the permeability of the exchange surface.     

Plasticity of influenza haemagglutinin fusion peptides and their interaction with lipid bilayers

A detailed molecular dynamics study of the haemagglutinin fusion peptide (N-terminal 20 residues of the HA2 subunits) in a model bilayer has yielded useful information about the molecular interactions leading to insertion into the lipids. Simulations were performed on the native sequence, as well as a number of mutant sequences, which are either fusogenic or nonfusogenic. For the native sequence and fusogenic mutants, the N-terminal 11 residues of the fusion peptides are helical and insert with a tilt angle of approximately 30 degrees with respect to the membrane normal, in very good agreement with experimental data. The tilted insertion of the native sequence peptide leads to membrane bilayer thinning and the calculated order parameters show larger disorder of the alkyl chains. These results indicate that the lipid packing is perturbed by the fusion peptide and could be used to explain membrane fusion. For the nonfusogenic sequences investigated, it was found that most of them equilibrate parallel to the interface plane and do not adopt a tilted conformation. The presence of a charged residue at the beginning of the sequence (G1E mutant) resulted in a more difficult case, and the outcomes do not fall straightforwardly into the general picture. Sequence searches have revealed similarities of the fusion peptide of influenza haemagglutinin with peptide sequences such as segments of porin, amyloid alpha eta peptide, and a peptide from the prion sequence. These results confirm that the sequence can adopt different folds in different environments. The plasticity and the conformational dependence on the local environment could be used to better understand the function of fusion peptides.