Balancing the intermolecular forces in peptide amphiphiles for controlling self-assembly transitions.

While the influence of alkyl chain length and headgroup size on self-assembly behaviour has been well-established for simple surfactants, the rational control over the pH- and concentration-dependent self-assembly behaviour in stimuli responsive peptides remains an elusive goal. Here, we show that different amphiphilic peptides can have similar self-assembly phase diagrams, providing the relative strengths of the attractive and repulsive forces are balanced. Using palmitoyl-YYAAEEEEK(DO3A:Gd)-NH2 and palmitoyl-YAAEEEEK(DO3A:Gd)-NH2 as controls, we show that reducing hydrophobic attractive forces through fewer methylene groups in the alkyl chain will lead to a similar self-assembly phase diagram as increasing the electrostatic repulsive forces via the addition of a glutamic acid residue. These changes allow creation of self-assembled MRI vehicles with slightly different micelle and nanofiber diameters but with minimal changes in the spin-lattice T1 relaxivity. These findings reveal a powerful strategy to design self-assembled vehicles with different sizes but with similar self-assembly profiles.

Sex and the migraine brain.

The brain responds differently to environmental and internal signals that relate to the stage of development of neural systems. While genetic and epigenetic factors contribute to a premorbid state, hormonal fluctuations in women may alter the set point of migraine. The cyclic surges of gonadal hormones may directly alter neuronal, glial and astrocyte function throughout the brain. Estrogen is mainly excitatory and progesterone inhibitory on brain neuronal systems. These changes contribute to the allostatic load of the migraine condition that most notably starts at puberty in girls.

Dissemination of the entomopathogenic fungus verticillium lecanii (Zimmermann) Viégas (Hyphomycetales: Moniliaceae) in a population of Frankliniella occidentalis (Pergande, 1895) (Thysanoptera: Thripidae).

Tests were carried out to investigate the dissemination of the entomopathogenic fungus Verticillium lecanii (Zimmermann) Viégas in a population of Frankliniella occidentalis. The tested factors, which influence the efficacy of the fungus against the pest insect, have been the population density of the thrips at the application moment as well as the temperature. The population density influenced the dissemination of the fungal spores in the population. The higher the density has been, the higher the insetting control effect has been as well. The temperature influenced the speed of the fungal effect, too. The higher the temperature has been, the earlier the control effect started. However, an increase of the natural mortality was found as well.

The role of selenocysteine 133 in catalysis by the human type 2 iodothyronine deiodinase.

Human type 2 iodothyronine deiodinase (hD2) catalyzes the activation of T4 to T3. D2, like types 1 and 3 deiodinases, contains selenocysteine (Sec) in the highly conserved active center at position 133. To evaluate the contribution of Sec133 to the catalytic properties of hD2, we generated mutants in which cysteine (Cys) or alanine (Ala) replaced Sec133. The Km (T4) of Cys133D2 was 2.1 microM, strikingly higher than that of native D2 (1.4 nM). In contrast, the relative turnover number was 10-fold lower for Cys133D2, illustrating the greater potency of Se than S in supporting catalysis. The AlaD2 mutant was inactive. Studies in intact cells transiently expressing the native or Cys133D2 enzyme exhibited saturation kinetics expected from the Km as measured under in vitro conditions, indicating rapid equilibration of extracellular and intracellular T4. Blockade of the NTCP, OATP1-3, and LST-1 transporters with 10 mM sodium taurocholate did not alter the deiodination rate of T4 by Cys133D2 transiently expressed in intact cells, suggesting that intracellular transport of T4 is not rate limiting. These results illustrate that selenium plays a critical role in deiodination catalyzed by hD2.

Substrate-induced down-regulation of human type 2 deiodinase (hD2) is mediated through proteasomal degradation and requires interaction with the enzyme's active center.

Type 2 iodothyronine deiodinase (D2) catalyzes the first step in thyroid hormone action, the deiodination of T4 to T3. Endogenous D2 activity is posttranslationally regulated by substrate that accelerates its degradation through the ubiquitin-proteasome pathway. To understand how D2 activity correlates with D2 protein during its normal decay and rT3-induced down-regulation, HEK-293 cells, transiently expressing human D2, were labeled with Na75SeO3 and then treated with 100 microM cycloheximide (CX), 30 nM rT3, and/or 10 microM MG132, a specific proteasome inhibitor, for 2-4 h. D2 protein and enzyme activity changed in parallel, disappearing with a half-life of 2 h in the presence of CX, or 1 h when CX + rT3 were combined. Treatment with MG132 blocked these effects. We created selenocysteine (Sec) 133 to cysteine (Cys) or alanine (Ala) D2 mutants, without changing Sec 266. The CysD2 activity and protein levels were also parallel, with a similar half-life of approximately 2 h, whereas the rT3-induced D2 down-regulation required approximately 1000-fold higher rT3 concentration (30 microM) due to a proportionally higher Michaelis constant of CysD2. In similar experiments, the AlaD2 mutant retained the short half-life but was not catalytically active and not susceptible to rT3-accelerated degradation. We conclude that substrate-induced loss of D2 activity is due to proteasomal degradation of the enzyme and requires interaction with the catalytic center of the protein.

Characterization of the thyroxine-binding site of thyroxine-binding globulin by site-directed mutagenesis.

The principal transport protein for T4 in human blood, thyroxine-binding globulin (TBG), binds T4 with an exceptionally high affinity (Ka = 10(10) M(-1)). Its homology to the superfamily of the serpins has recently been used in the design of chimeric proteins, providing experimental evidence that an eight-stranded beta-barrel domain encompasses the ligand-binding site. We have now characterized the T4 binding site by site-directed mutagenesis. Sequence alignment of TBG from several species revealed a phylogenetically highly conserved stretch of amino acids comprising strands 2B and 3B of the beta-barrel motif. Mutations within this region (Val228Glu, Cys234Trp, Thr235Trp, Thr235Gln, Lys253Ala, and Lys253Asp), designed to impose steric hindrance or restriction of its mobility, had no significant influence on T4 binding. However, binding affinity was 20-fold reduced by introduction of an N-linked glycosylation site at the turn between strands 2B and 3B (Leu246Thr) without compromising the proper folding of this mutant as assessed by immunological methods. In most other serpins, this glycosylation site is highly conserved and has been shown to be crucial for cortisol binding of corticosteroid-binding globulin, the only other member of the serpins with a transport function. The ligand-binding site could thus be located to a highly aromatic environment deep within the beta-barrel. The importance of the binding site's aromatic character was investigated by exchanging phenylalanines with alanines. Indeed, these experiments revealed that substitution of Phe249 in the middle of strand 3B completely abolished T4 binding, while the substitution of several other phenylalanines had no effect.

The Caenorhabditis elegans homologue of thioredoxin reductase contains a selenocysteine insertion sequence (SECIS) element that differs from mammalian SECIS elements but directs selenocysteine incorporation.

Thioredoxin reductases (TRR) serve critical roles in maintaining cellular redox states. Two isoforms of TRR have been identified in mammals: both contain a penultimate selenocysteine residue that is essential for catalytic activity. A search of the genome of the invertebrate, Caenorhabditis elegans, reveals a gene highly homologous to mammalian TRR, with a TGA selenocysteine codon at the corresponding position. A selenocysteyl-tRNA was identified in this organism several years ago, but no selenoproteins have been identified experimentally. Herein we report the first identification of a C. elegans selenoprotein. By (75)Se labeling of C. elegans, one major band was identified, which migrated with the predicted mobility of the C. elegans TRR homologue. Western analysis with an antibody against human TRR provides strong evidence for identification of the C. elegans selenoprotein as a member of the TRR family. The 3'-untranslated region of this gene contains a selenocysteine insertion sequence (SECIS) element that deviates at one position from the previously invariant consensus "AUGA." Nonetheless, this element functions to direct selenocysteine incorporation in mammalian cells, suggesting conservation of the factors recognizing SECIS elements from worm to man.

Modularity of serpins. A bifunctional chimera possessing alpha1-proteinase inhibitor and thyroxine-binding globulin properties.

An exciting application of protein engineering is the creation of proteins with novel functions by the retrofitting of native proteins. Such attempts might be facilitated by the idea of a mosaic architecture of proteins out of structural units. Even though numerous theoretical concepts deal with the delineation of structural "modules," their potential in the design of proteins has not yet been sufficiently exploited. To address this question we used a gain of function approach by designing modular chimeric molecules out of two structurally homologous but functionally diverse members of the superfamily of serine-proteinase inhibitors, alpha1-proteinase inhibitor and thyroxine-binding globulin. Substitution of two of four alpha1-proteinase inhibitor modules (Lys222 to Leu288 and Pro362 to Lys394, respectively), identified by alpha-backbone distance analysis, with their thyroxine-binding globulin homologues resulted in a bifunctional chimera with inhibition of human leukocyte elastase and high affinity thyroxine binding. To our knowledge, this is the first report on a bifunctional chimera engineered from modules of homologous globular proteins. Our results demonstrate how a modular concept can facilitate the design of new functional proteins by swapping structural units chosen from members of a protein superfamily.

The 3'-untranslated region of human type 2 iodothyronine deiodinase mRNA contains a functional selenocysteine insertion sequence element.

Type 2 deiodinase (D2) catalyzes the 5'-deiodination of thyroxine to form 3,5,3'-triiodothyronine. Two mammalian D2 cDNAs have been identified containing 2 kilobases (kb) of the 7-kb mRNA including the complete coding sequence. Both contain in-frame TGA codons, which should serve as selenocysteine codons. However, the selenocysteine insertion sequence (SECIS) elements required for the decoding of UGA as a selenocysteine in the 3'-untranslated region (UTR) of the mRNA are not present. We have identified two overlapping expressed sequence tag clones, which contain the missing 4.4-kb 3'-UTR of the human D2 (hD2) cDNA. Computer analysis predicts a stem loop structure 280 base pairs 5' to the polyadenylation site, which has potent SECIS activity. A fragment containing these sequences hybridizes to D2 mRNA in human thyroid. A G to A mutation in the essential AUGA motif of this element abolished its function. Transfection of the hD2 coding region plus the 3'-UTR results in the expression of D2, and its in vitro transcribed mRNA programs D2 activity in Xenopus oocytes. This is the first identification of a SECIS element in a mammalian D2 cDNA and establishes that hD2 is a bona fide selenoprotein.

The team physician's bag.

This article discusses the choices to be made by the team physician before venturing out on the road with the team, including an analysis of hardware from the actual travel kit to the supplies needed. Typical problems encountered are reviewed in context to preparing the medications that will be carried on the road. Organization and periodic review of needs and supplies is vital to a successful trip.

Thallium-201 scintigraphy in complete left bundle branch block.

Nineteen symptomatic patients with left bundle branch block (LBBB) were examined by thallium-201 (TI-201) exercise scintigraphy and selective coronary arteriography. All elicited significant anteroseptal perfusion defects in the exercise scintigrams, but in only 4 was coronary artery disease (CAD) involving the left anterior descending coronary artery present. To further elucidate the effect of LBBB on septal TI-201 uptake in the absence of CAD, TI-201 scintigrams combined with regional myocardial blood flow measurements using radioactive microspheres were carried out in 7 dogs during right atrial and right ventricular pacing (LBBB in the ECG) at similar heart rates. During right atrial pacing, TI-201 uptake was homogeneous in the entire left ventricle, as were tissue flows. During right ventricular pacing, TI-201 activity was reduced to 69% of maximal TI-201 activity within the septum, whereas it averaged 90% in the lateral wall (p less than 0.05) in 6 dogs. Correspondingly, regional myocardial blood flow was lower within the septum as compared with that in the lateral wall, averaging 89 and 120 ml/min/100 g, respectively (p less than 0.005). In 1 dog, normal TI-201 distribution and tissue flows were found in both studies. Thus, symptomatic patients with LBBB may elicit abnormal TI-201 exercise scintigrams, suggesting anteroseptal ischemia despite normal coronary arteries. The electrical induction of LBBB in dogs results, in most instances, in a comparable reduction in septal TI-201 uptake associated with diminished septal blood flow. Therefore, exercise-induced septal perfusion defects in the presence of LBBB do not necessarily indicate CAD even in symptomatic patients, but may reflect functional ischemia due to asynchronous septal contraction.