Substrates induce conformational changes in human anion exchanger 1 (hAE1) as observed by fluorescence resonance energy transfer

The one-for-one exchange of Cl(-) and HCO(3)(-) ions is catalyzed by human erythrocyte anion exchanger 1 (hAE1) through a ping-pong mechanism whereby the protein exists in two main conformations, with the single anion-binding site exposed at either the cytoplasmic (inner) side (E(i)) or the extracellular side (E(o)), with interconversion between the two states being possible only after anion binding. Steady-state and time-resolved resonance energy transfer (FRET) techniques were used to determine the distance of the binding site for diTBA (bis-(1,3-diethylthiobarbituric acid)trimethine oxonol), a high affinity fluorescent oxonol inhibitor of hAE1, from a benchmark site (probably Lys-430) labeled by external fluorescein maleimide (FM). Using red cell ghost membranes, energy transfer distances were measured in media containing different anions between FM as the donor, covalently attached to one monomer, and diTBA as the acceptor, reversibly bound to the adjacent monomer of a hAE1 dimer. Energy transfer increased significantly in chloride or bicarbonate buffers relative to conditions where no transportable anions were present, that is, in citrate buffer. These differences in transfer efficiencies were interpreted in light of the conformational distributions of hAE1 in various buffers and the possible effects of diTBA itself on the distribution. The analysis indicates that the diTBA binding site comes closer to the FM site by approximately 7 A in chloride buffer as compared to that in citrate (or equivalent changes in diTBA orientation occur) because of the effects of anion binding. This provides the first direct physical evidence for structural changes in hAE1 induced by substrates.     

Binding of calcium to anticoagulant protein S: role of the fourth EGF module

Protein S is an anticoagulant protein containing a Gla (enclosing gamma-carboxyglutamic acids) module, a TSR (thrombin sensitive region) module, four EGF (epidermal growth factor)-like modules, and a SHBG (sex hormone binding globulin)-like region. Protein S is a cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa but also has APC-independent activities. The function of the fourth EGF module (EGF4) in protein S has so far not been clear. We have now investigated this module through studies of recombinant wild-type protein S and a naturally occurring mutant (Asn217Ser). The mutant has essentially normal APC anticoagulant activity and a previously reported secretion defect. In the wild-type protein, Asn217 is normally beta-hydroxylated. The binding of calcium to wild-type protein S is characterized by four high-affinity binding sites with K(D) values ranging from 10(-)(7) to 10(-)(9) M. Three of these binding sites are located in EGF modules. Using surface plasmon resonance, competition with a calcium chelator, and antibody-based methods, we found that one high-affinity binding site for calcium was lost in protein S Asn217Ser but that the mutation also affected the calcium-dependent conformation of EGF1. We conclude that binding of calcium to EGF4 of protein S, involving Asn217, is important for the maintenance of the structure of protein S. Also, the abolition of binding of calcium to EGF4, related to Asn217, impairs both the structure and function of EGF1.     

Survival of Yogurt Bacteria in the Human Gut

Whether Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus can be recovered after passage through the human gut was tested by feeding 20 healthy volunteers commercial yogurt. Yogurt bacteria were found in human feces, suggesting that they can survive transit in the gastrointestinal tract.     

A Constitutively Expressed, Truncated umuDC Operon Regulates the recA-Dependent DNA Damage Induction of a Gene in Acinetobacter baylyi Strain ADP1

In response to environmentally caused DNA damage, SOS genes are up-regulated due to RecA-mediated relief of LexA repression. In Escherichia coli, the SOS umuDC operon is required for DNA damage checkpoint functions and for replicating damaged DNA in the error-prone process called SOS mutagenesis. In the model soil bacterium Acinetobacter baylyi strain ADP1, however, the content, regulation, and function of the umuDC operon are unusual. The umuC gene is incomplete, and a remnant of an ISEhe3-like transposase has replaced the middle 57% of the umuC coding region. The umuD open reading frame is intact, but it is 1.5 times the size of other umuD genes and has an extra 5′ region that lacks homology to known umuD genes. Analysis of a umuD::lacZ fusion showed that umuD was expressed at very high levels in both the absence and presence of mitomycin C and that this expression was not affected in a recA-deficient background. The umuD mutation did not affect the growth rate or survival after UV-induced DNA damage. However, the UmuD-like protein found in ADP1 (UmuDAb) was required for induction of an adjacent DNA damage-inducible gene, ddrR. The umuD mutation specifically reduced the DNA damage induction of the RecA-dependent DNA damage-inducible ddrR locus by 83% (from 12.9-fold to 2.3-fold induction), but it did not affect the 33.9-fold induction of benA, an unrelated benzoate degradation gene. These data suggest that the response of the ADP1 umuDC operon to DNA damage is unusual and that UmuDAb specifically regulates the expression of at least one DNA damage-inducible gene.     

Medical students' attitudes toward genetic testing of minors

The purpose of this study was to examine attitudes of medical students at a single university toward genetic testing in minors, defining attitudes as willingness to offer testing, and reasons for offering or not offering testing. A survey was distributed to all University of Arizona medical students (n = 428) during the 2003-2004 academic year. The survey consisted of three clinical vignettes concerning genetic testing for Huntington's disease (HD), BRCA1 breast cancer predisposition mutation, and cystic fibrosis (CF) carrier status. For each vignette, students responded to whether they would provide testing for a 7-year-old, a 17-year-old, and their reasons for each age and condition. One hundred thirty-five students (31.5%) responded to the survey. Medical students were significantly more likely to test a 7-year-old for CF carrier status (57%), than they were for a BRCA1 mutation (47%), and an HD mutation (40%). Students were significantly more likely to test a 17-year-old than a 7-year-old in each clinical scenario. Students who had completed a genetics course in medical school were significantly less likely to test a 7-year-old for a BRCA1 mutation than those who had not completed a formal course. Medical students' willingness to perform genetic testing in a minor is influenced by the type of condition, the age of the minor being tested, and the amount of genetics education received in medical school.     

Recovery of hand function through mental practice: A study protocol

Background  

The study aims to assess the therapeutic benefits of motor imagery training in stroke patients with persistent motor weakness. There is evidence to suggest that mental rehearsal of movement can produce effects normally attributed to practising the actual movements. Imagining hand movements could stimulate the redistribution of brain activity, which accompanies recovery of hand function, thus resulting in a reduced motor deficit.     

Metabolism of dietary cetoleic acid (22:1n-11) in mink (Mustela vison) and gray seals (Halichoerus grypus) studied using radiolabeled fatty acids

Cetoleic acid (22:1n-11) is a good indicator of diet in marine predators and has proven to be an important fatty acid (FA) when using adipose tissue FA composition to study diet in marine mammals and seabirds. Feeding studies have shown that 22:1 isomers are predictably underrepresented in adipose tissue relative to diet, implying that metabolism within the predator strongly influences the relationship between the level of these FAs in diet and adipose tissue. Fully understanding such metabolic processes for individual FAs is important for the quantitative estimation of predator diets. We employed a dual-label radioisotope tracer technique to investigate the potential modification of 22:1n-11 and its recovery in the blubber of gray seals (Halichoerus grypus) and in the adipose tissue and liver of mink (Mustela vison), a smaller model carnivore also accustomed to fish-based diets. In both seals and mink, (3)H radioactivity was found in the chain-shortened products of 22:1n-11, with 18:1 being the dominant product. We also found (3)H radioactivity in saturated FAs. The distribution patterns of (3)H radioactivity across the FAs isolated from seal blubber and mink subcutaneous adipose tissue were comparable, indicating that mink are a good model for the investigation of lipid metabolism in marine carnivores.     

Identification of collagen-induced arthritis loci in aged multiparous female mice

Collagen-induced arthritis in mice is one of the most commonly used autoimmune experimental models, with many similarities to rheumatoid arthritis. Since collagen-induced arthritis is a complex polygenic disease there is a need for identification of several major disease-controlling genes. Because rheumatoid arthritis particularly affects aged women, we have in the present study identified new genetic regions critical for collagen-induced arthritis by studying aged female mice of a cross between NFR/N and B10.Q (H-2^q haplotype). The mice in the present study had different reproductive histories, which did not significantly affect the onset, incidence or severity of the disease. A total of 200 female mice were used in a total genome-wide screening with 125 microsatellite markers. We found one new significant quantitative trait locus affecting the arthritis incidence, severity and day of onset on chromosome 11 (denoted Cia40), which colocalizes with a locus controlling pregnancy failure. Furthermore, a quantitative trait locus of suggestive significance associated with the incidence, severity and day of onset was identified on chromosome 1. Finally, a suggestively significant quantitative trait locus associated with collagen type II antibody titers was identified on chromosome 13. This study indicates that several gene loci control arthritis in aged multiparous females, and that at least one of these loci coincides with pregnancy failure.     

Expansion and Accelerated Evolution of 9-Exon Odorant Receptors in Polistes Paper Wasps

Independent origins of sociality in bees and ants are associated with independent expansions of particular odorant receptor (OR) gene subfamilies. In ants, one clade within the OR gene family, the 9-exon subfamily, has dramatically expanded. These receptors detect cuticular hydrocarbons (CHCs), key social signaling molecules in insects. It is unclear to what extent 9-exon OR subfamily expansion is associated with the independent evolution of sociality across Hymenoptera, warranting studies of taxa with independently derived social behavior. Here, we describe OR gene family evolution in the northern paper wasp, Polistes fuscatus, and compare it to four additional paper wasp species spanning ∼40 million years of evolutionary divergence. We find 200 putatively functional OR genes in P. fuscatus, matching predictions from neuroanatomy, and more than half of these are in the 9-exon subfamily. Most OR gene expansions are tandemly arrayed at orthologous loci in Polistes genomes, and microsynteny analysis shows species-specific gain and loss of 9-exon ORs within tandem arrays. There is evidence of episodic positive diversifying selection shaping ORs in expanded subfamilies. Values of omega (d_N/d_S) are higher among 9-exon ORs compared to other OR subfamilies. Within the Polistes OR gene tree, branches in the 9-exon OR clade experience relaxed negative (relaxed purifying) selection relative to other branches in the tree. Patterns of OR evolution within Polistes are consistent with 9-exon OR function in CHC perception by combinatorial coding, with both natural selection and neutral drift contributing to interspecies differences in gene copy number and sequence.