Assays for measuring in vitro basophil activation induced by recombinant allergens

The diagnosis of type I allergy is essentially based on clinical data, skin tests, and measurements of allergen-specific IgE. However, the determination of specific IgE per se does not permit a definitive conclusion concerning the response of effector cells to the respective allergen(s) and consecutive clinical symptoms in all patients. In an attempt to overcome this problem, a number of basophil-activation assays have been developed during the last few years. Today, allergen-induced activation of blood basophils can be employed as a specific and reliable measure of IgE-dependent responses in sensitized individuals. Using recombinant allergens and basophil-specific markers, these novel assays appear to serve as simple and useful tests in component-resolved diagnosis of type I allergies. In the current article, the biochemical, functional, and technical background of these basophil tests is discussed.     

Epidermolysis bullosa simplex-type mutations alter the dynamics of the keratin cytoskeleton and reveal a contribution of actin to the transport of keratin subunits

Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.     

Mal de Meleda (MDM) caused by mutations in the gene for SLURP-1 in patients from Germany,Turkey, Palestine,and the United Arab Emirates

Mal de Meleda (MDM) or keratosis palmoplantaris transgrediens of Siemens is an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma (PPK) and transgressive keratosis with an onset in early infancy. There is no associated involvement of other organs; however, a spectrum of clinical presentations with optional and variable features has been described. Mutations in the ARS (component B)-81/s gene ( LY6LS) on chromosome 8q24-qter, which encodes SLURP-1, have recently been identified in patients with MDM. Here, we have analyzed four MDM families for mutations in SLURP-1. In a large Palestinian pedigree with multiple consanguinity, patients are homozygous for a new mutation that substitutes an arginine for a conserved glycine residue at position 86. A different mutation in Turkish patients results in the same amino acid exchange. Some remarkable similarities are seen in the clinical picture of patients from both families. Patients of an Emirati Bedouin family have a homozygous alteration of the translation initiation codon. In a German family with no known consanguinity, we have shown pseudodominant inheritance. Three affected children and their affected mother are homozygous for the missense mutation W15R. Our findings indicate that the MDM type of transgressive PPK is caused by SLURP-1 mutations in patients from various origins and demonstrate allelic heterogeneity for mutations in SLURP-1.     

WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains

WW domains mediate protein-protein interactions in a number of different cellular functions by recognizing proline-containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty-two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/phi)P(p,g)PPpR, (p/phi)PPRgpPp, PPLPp, (p/xi)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001). In addition to these distinct binding motifs, group-specific WW domain consensus sequences were identified. For PPxY-recognizing domains, phospho-tyrosine binding was also observed. Based on the sequences of the PPx(Y/poY)-specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)-recognition activity for WW domains, which were not assayed. PPx(Y/poY)-binding was found to be a common property of NEDD4-like ubiquitin ligases.     

Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.     

Dichotomy of autoreactive Th1 and Th2 cell responses to desmoglein 3 in patients with pemphigus vulgaris (PV) and healthy carriers of PV-associated HLA class II alleles

Pemphigus vulgaris (PV) is the most severe autoimmune bullous skin disorder and is primarily associated with circulating autoantibodies (autoAb) against desmoglein 3 (Dsg3). In light of recent evidence that autoreactive T cells are critical for the induction and regulation of Ab production, the goal of this study was to characterize and quantitate autoreactive T cells in patients with PV and healthy controls. Peripheral Dsg3-reactive Th cells from 28 patients with acute-onset, chronic active, and remittent PV were quantitated by MACS secretion assay. Dsg3-reactive Th2 cells were detected at similar frequencies in all studied PV patients, while the number of autoreactive Th1 cells exceeded those of Th2 cells in chronic active PV. In contrast, healthy carriers of the PV-associated HLA class II alleles, DRB1*0402 and DQB1*0503, exhibited exclusively Dsg3-reactive Th1 cell responses, while healthy carriers of other HLA class II alleles did not. Moreover, the presence of IgG1 and IgG4 against Dsg3 was directly related to the ratio of Dsg3-reactive Th1/Th2 cells. T cell recognition of Dsg3 was restricted by HLA-DRB1*0402 and DQB1*0503 in PV patients and Dsg3-responsive healthy donors. These observations strongly suggest 1) that the appearance of Dsg3-reactive Th2 cells is restricted to patients with PV; 2) that specific HLA class II alleles that are prevalent in PV are critical for T cell recognition of Dsg3 in PV patients and Dsg3-responsive healthy donors; and 3) that autoAb production is associated with both Th1 and Th2 cells.     

Heterogeneous perfusion is a consequence of uniform shear stress in optimized arterial tree models

Using optimized computer models of arterial trees we demonstrate that flow heterogeneity is a necessary consequence of a uniform shear stress distribution. Model trees are generated and optimized under different modes of boundary conditions. In one mode flow is delivered to the tissue as homogeneously as possible. Although this primary goal can be achieved, resulting shear stresses between blood and the vessel walls show very large spread. In a second mode, models are optimized under the condition of uniform shear stress in all segments which in turn renders flow distribution heterogeneous. Both homogeneous perfusion and uniform shear stress are desirable goals in real arterial trees but each of these goals can only be approached at the expense of the other. While the present paper refers only to optimized models, we assume that this dual relation between the heterogeneities in flow and shear stress may represent a more general principle of vascular systems.     

Transgenic mice expressing lipoprotein lipase in adipose tissue. Absence of the proximal 3'-untranslated region causes translational upregulation

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein and adipocyte metabolism. Defects in LPL can lead to hypertriglyceridemia and the subsequent development of atherosclerosis. The mechanisms of regulation of this enzyme are complex and may occur at multiple levels of gene expression. Because the 3'-untranslated region (UTR) is involved in LPL translational regulation, transgenic mice were generated with adipose tissue expression of an LPL construct either with or without the proximal 3'-UTR and driven by the aP2 promoter. Both transgenic mouse colonies were viable and expressed the transgene, resulting in a 2-fold increase in LPL activity in white adipose tissue. Neither mouse colony exhibited any obvious phenotype in terms of body weight, plasma lipids, glucose, and non-esterified fatty acid levels. In the mice expressing hLPL with an intact 3'-UTR, hLPL mRNA expression approximately paralleled hLPL activity. However in the mice without the proximal 3'-UTR, hLPL mRNA was low in the setting of large amounts of hLPL protein and LPL activity. In previous studies, the 3'-UTR of LPL was critical for the inhibitory effects of constitutively expressed hormones, such as thyroid hormone and catecholamines. Therefore, these data suggest that the absence of the 3'-UTR results in a translationally unrepressed LPL, resulting in a moderate overexpression of adipose LPL activity.     

Evidence for the presence of the Kennedy and Bremer- Greenberg pathways in Caenorhabditis elegans

Nematodes were found to synthesize phosphorylcholine-containing molecules not present in higher organisms, i.e. phosphorylcholine-substituted glycosphingolipids and (glyco)proteins. Investigations on the biosynthesis of these structures provided first biochemical evidence for the presence of the Kennedy and Bremer-Greenberg pathways in the model organism Caenorhabditis elegans.     

Redox-triggered FTIR difference spectra of FAD in aqueous solution and bound to flavoproteins

Flavin adenine dinucleotide (FAD) and three different flavoproteins in aqueous solution were subjected to redox-triggered Fourier transform infrared difference spectroscopy. The acquired vibrational spectra show a great number of positive and negative peaks, pertaining to the oxidized and reduced state of the molecule, respectively. Density functional theory calculations on the B3LYP/6-31G(d) level were employed to assign several of the observed bands to vibrational modes of the isoalloxazine moiety of the flavin cofactor in both its oxidized and, for the first time, its reduced state. Prominent modes measured for oxidized FAD include nu(C(4)=O) and nu(C(2)=O) at 1716 and 1674 cm(-1), respectively, nu(C(4a)=N(5)) at 1580 cm(-1), and nu(C(10a)=N(1)) at 1548 cm(-1). Measured modes of the reduced form of FAD include nu(C(2)=O) at 1692 cm(-1), nu(C(4)=O) at 1634 cm(-1), and nu(C(4a)=C(10a)) at 1600 cm(-1). While the overall shape of the enzyme spectra is similar to the shape of the spectrum of free FAD, there are numerous differences in detail. In particular, the nu(C=N) modes of the flavin exhibit frequency shifts in the protein-bound form, most prominently for pyruvate oxidase where nu(C(10a)=N(1)) downshifts by 14 cm(-1) to 1534 cm(-1). The significance of this shift and a possible explanation in connection with the bent conformation of the flavin cofactor in this enzyme are discussed.