SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation

Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions remain undefined. Here we report the efficient reconstitution of ATP-, GTP-, cytosol- and dynamin-dependent formation of clathrin-coated vesicles in permeabilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndapin I and amphiphysin II inhibit coated-vesicle formation in vitro through interactions with membrane-associated proteins. Most of the SH3 domains tested selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interactions between SH3 domains and their partners function sequentially in endocytic coated-vesicle formation.     

Re-Examining the Association between Vitamin D and Childhood Caries

Background

Previous studies have reported an inverse association between vitamin D and childhood dental caries, but whether this is causal is unclear.

Objective

To determine the causal effect of circulating 25-hydroxyvitamin D concentration on dental caries experience, early caries onset and the requirement for a dental general anesthetic.

Design

A Mendelian randomization study was undertaken, using genetic variants known to be associated with circulating 25-hydroxyvitamin D concentrations in 5,545 European origin children from the South West of England. Data on caries and related characteristics were obtained from parental and child completed questionnaires between 38 and 91 months and clinical assessments in a random 10% sample at 31, 44 and 61 months.

Results

In multivariable confounder adjusted analyses no strong evidence for an association of 25-hydroxyvitamin D with caries experience or severity was found but there was evidence for an association with early caries onset, or having a general anesthetic for dental problems. In Mendelian randomization analysis the odds ratio for caries experience per 10 nmol/L increase in 25-hydroxyvitamin D was 0.93 (95% confidence interval: 0.83, 1.05; P = 0.26) and the odds ratio for dental general anaesthetic per 10 nmol/L increase in 25-hydroxyvitamin D was 0.96 (95% confidence interval: 0.75, 1.22; P = 0.72).

Conclusions

This Mendelian randomization study provides little evidence to support an inverse causal effect of 25-hydroxyvitamin D on dental caries. However, the estimates are imprecise and a larger study is required to refine these analyses.     

Transgenic Mice Expressing Human Immunodeficiency Virus Type 1 in Immune Cells Develop a Severe AIDS-Like Disease

We have constructed transgenic (Tg) mice expressing the entire human immunodeficiency virus type 1 (HIV-1) coding sequences in cells targeted by HIV-1 infection in humans. These Tg mice developed a severe AIDS-like disease leading to early death (<1 month). They developed muscle wasting, severe atrophy and fibrosis of lymphoid organs, tubulointerstitial nephritis, and lymphoid interstitial pneumonitis. In addition the expression of RANTES was increased in various tissues of these Tg mice relative to that in the normal controls. Disease appearance was correlated with the levels of transgene expression. The numerous pathologies observed in these mice are remarkably similar to those observed in human AIDS and, more specifically, in pediatric AIDS.     

Native faunal communities depend on habitat from non-native plants in novel but not in natural ecosystems

Invasive non-native plants are a major driver of native biodiversity loss, yet native biodiversity can sometimes benefit from non-native species. Depending on habitat context, even the same non-native species can have positive and negative effects on biodiversity. Blackberry (Rubus fruticosus aggregate) is a useful model organism to better understand a non-native plant with conflicting impacts on biodiversity. We used a replicated capture-mark-recapture study across 11 consecutive seasons to examine the response of small mammal diversity and abundance to vegetation structure and density associated with non-native blackberry (R. anglocandicans) in native, hybrid and blackberry-dominated novel ecosystems in Australia. Across the three habitat types, increasing blackberry dominance had a positive influence on mammal diversity, while the strength and direction of this influence varied for abundance. At a microhabitat scale within hybrid and native habitat there were no significant differences in diversity, or the abundance of most species, between microhabitats where blackberry was absent versus dominant. In contrast, in novel ecosystems diversity and abundances were very low without blackberry, yet high (comparable to native ecosystems) within blackberry as it provided functionally-analogous vegetation structure and density to the lost native understory. Our results indicate the ecological functions of non-native plant species vary depending on habitat and need to be considered for management. Comparative studies such as ours that apply a standardized approach across a broad range of conditions at the landscape and habitat scale are crucial for guiding land managers on control options for non-native species (remove, reduce or retain and contain) that are context-sensitive and scale-dependent.     

Autoshaping in the rat: conditioned licking response to a stimulus that signals sucrose reinforcement

The present experiments were designed to determine if repeated presentations of an empty sipper tube (the conditioned stimulus or CS) with the response-independent delivery of a sucrose solution (the unconditioned stimulus or US) from a second spout results in the development of Pavlovian conditioned responding. In Experiment 1, rats in the experimental condition received paired CS-US presentations whereas subjects in the control condition were exposed to random presentations of CS and US. In Experiment 2, an additional control condition (CS alone) was included and, to encourage generalized responding between the US and CS, the CS tube was filled with water for all groups. The results of both experiments indicate that the CS-directed responding in the paired CS-US condition was Pavlovian in nature. Thus, the present procedure serves as an autoshaping task in which conditioned licking is generated.     

High-throughput expression,purification, and characterization of recombinant Caenorhabditis elegans proteins

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more difficult as functional proteomics efforts focus on the proteins from higher organisms, since issues of correctly identifying intron-exon boundaries and efficiently expressing and solubilizing the (often) multi-domain proteins from higher eukaryotes are challenging. Recently, 12,000 open-reading-frame (ORF) sequences from Caenorhabditis elegans have become available for functional proteomics studies [Nat. Gen. 34 (2003) 35]. We have implemented a high-throughput screening procedure to express, purify, and analyze by mass spectrometry hexa-histidine-tagged C. elegans ORFs in Escherichia coli using metal affinity ZipTips. We find that over 65% of the expressed proteins are of the correct mass as analyzed by matrix-assisted laser desorption MS. Many of the remaining proteins indicated to be "incorrect" can be explained by high-throughput cloning or genome database annotation errors. This provides a general understanding of the expected error rates in such high-throughput cloning projects. The ZipTip purified proteins can be further analyzed under both native and denaturing conditions for functional proteomics efforts.     

Structure and dynamics of model pore insertion into a membrane

A cylindrical transmembrane molecule is constructed by linking hydrophobic sites selected from a coarse grain model. The resulting hollow tube assembly serves as a representation of a transmembrane channel, pore, or a carbon nanotube. The interactions of a coarse grain di-myristoyl-phosphatidyl-choline hydrated bilayer with both a purely hydrophobic tube and a tube with hydrophilic caps are studied. The hydrophobic tube rotates in the membrane and becomes blocked by lipid tails after a few tens of nanoseconds. The hydrophilic sites of the capped tube stabilize it by anchoring the tube in the lipid headgroup/water interfacial region of each membrane leaflet. The capped tube remains free of lipid tails. The capped tube spontaneously conducts coarse grain water sites; the free-energy profile of this process is calculated using three different methods and is compared to the barrier for water permeation through the lipid bilayer. Spontaneous tube insertion into an undisturbed lipid bilayer is also studied, which we reported briefly in a previous publication. The hydrophobic tube submerges into the membrane core in a carpetlike manner. The capped tube laterally fuses with the closest leaflet, and then, after plunging into the membrane interior, rotates to assume a transbilayer orientation. Two lipids become trapped at the end of the tube as it penetrates the membrane. The hydrophilic headgroups of these lipids associate with the lower tube cap and assist the tube in crossing the interior of the membrane. When the rotation is complete these lipids detach from the tube caps and fuse with the lower leaflet lipids.