Novel binding sites of 15-deoxy-Delta12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.     

Human Fc epsilon RIalpha-specific human single-chain Fv (scFv) antibody with antagonistic activity toward IgE/Fc epsilon RIalpha-binding

The alpha-chain of Fc epsilon RI (Fc epsilon RIalpha) plays a critical role in the binding of IgE to Fc epsilon RI. A fully human antibody interfering with this interaction may be useful for the prevention of IgE-mediated allergic diseases. Here, we describe the successful isolation of a human single-chain Fv antibody specific to human Fc epsilon RIalpha using human antibody phage display libraries. Using the non-immune phage antibody libraries constructed from peripheral blood lymphocyte cDNA from 20 healthy subjects, we isolated three phage clones (designated as FcR epsilon 27, FcR epsilon 51, and FcR epsilon 70) through two rounds of biopanning selection. The purified soluble scFv, FcR epsilon 51, inhibited the binding of IgE to recombinant Fc epsilon RIalpha, although both FcR epsilon 27 and FcR epsilon 70 showed fine binding specificity to Fc epsilon RIalpha. Since FcR epsilon 51 was determined to be a monomer by HPLC, BIAcore analysis was performed. The dissociation constant of FcR epsilon 51 to Fc epsilon RIalpha was estimated to be 20 nM, i.e., fortyfold lower than that of IgE binding to Fc epsilon RIalpha (K(d) = 0.5 nM). With these characteristics, FcR epsilon 51 exhibited inhibitory activity on the release of histamine from passively sensitized human peripheral blood mononuclear cells.     

Thermodynamic analysis of the increased stability of major histocompatibility complex class II molecule I-Ek complexed with an antigenic peptide at an acidic pH

The differential scanning calorimetry analysis of the murine major histocompatibility complex class II molecule, I-E(k), in complex with an antigenic peptide derived from mouse hemoglobin, showed that the thermal stability at the mildly acidic pH is higher than that at the neutral pH. Although the thermal unfolding of I-E(k)-hemoglobin was irreversible, we extracted the equilibrium thermodynamic parameters from the kinetically controlled heat capacity curves. Both the denaturation temperatures and the enthalpy changes were almost independent of the heating rate over 1 degrees C per min. The linear relation between the denaturation temperature and the calorimetric enthalpy change provided the heat capacity changes, which are classified into one for the mildly acidic pH region and another for the neutral pH region. The equilibrium thermodynamic parameters showed that the increased stability at the mildly acidic pH is because of the entropic effect. These thermodynamic data provided new insight into the current structural model of a transition to an open conformation at the mildly acidic pH, which is critical for the peptide exchange function of major histocompatibility complex class II in the endosome.     

An application of optimal diving models to diving behaviour of Brünnich's guillemots

Although theoretical models predict that the quality of foraging patches has little effect on optimal dive time with increasing depth, many empirical studies show that dive time at a given depth may vary. We developed a model that incorporated patch quality as a parameter of energy intake as a nonlinear function of time, and applied it to the diving behaviour of Brünnich's guillemots, Uria lomvia. The model indicated that optimal dive time can vary widely depending on the parameter. It also explained the convergence of observed dive times with travel time. Assuming the birds dived optimally, this parameter can be estimated from travel time and dive time for each dive. Foraging patches with larger estimated parameter values were favoured by the birds, suggesting that the parameter indicated patch quality. We used this parameter to test an optimal patch use model in divers. The results indicate that Brünnich's guillemots adjust their diving behaviour adaptively depending on patch quality, and that the optimal diving model is valid for prediction of observed dive patterns if patch quality is incorporated appropriately. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy

Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.     

Free energy calculations for the relative binding affinity between DNA and lambda-repressor

The change in the binding free energy between DNA and lambda-repressor resulting from a base substitution, thymine (T)-->deoxyuracil (abbreviated as U), was evaluated by the free energy perturbation method on the basis of molecular dynamics simulations for the DNA-lambda-repressor complex in water with all degrees of freedom and including long-range Coulomb interactions. The binding free energy change that we calculated (1.47 +/- 0.40 kcal/mol) was in good agreement with an experimental value (1.8 kcal/mol). We clarified why the small difference between T and U (CH(3) in T is replaced with H in U) caused such a significant change in the binding free energy: The substitution of CH(3) in T with H in U lowered the dissociated-state free energy level due to the gain of the hydration free energy. Furthermore, the T-->U substitution raised the free energy level in the associated state due to the loss of the favored van der Waals (vdW) interactions with the lambda-repressor amino acid residues. In other words, the amino acid residues of lambda-repressor can recognize the CH(3) in T through the vdW interactions with the CH(3). This recognition is enhanced in a water environment, because the hydrophobic CH(3) prefers the amino acid residues of lambda-repressor to water molecules.     

Clinical implications of thermal therapy in lifestyle-related diseases

Systemic thermal therapy, such as taking a warm-water bath and sauna, induces systemic vasodilation. It was found that repeated sauna therapy (60 degrees C for 15 min) improved hemodynamic parameters, clinical symptoms, cardiac function, and vascular endothelial function in patients with congestive heart failure. Vascular endothelial function is impaired in subjects with lifestyle-related diseases, such as hypertension, hyperlipidemia, diabetes mellitus, obesity, and smoking. Sauna therapy also improved endothelial dysfunction in these subjects, suggesting a preventive role for atherosclerosis. In animal experiments, sauna therapy increases mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in aortas. In normal-weight patients with appetite loss, repeated sauna therapy increased plasma ghrelin concentrations and daily caloric intake and improved feeding behavior. In obese patients, the body weight and body fat significantly decreased after 2 weeks of sauna therapy without increase of plasma ghrelin concentrations. On the basis of these data, sauna therapy may be a promising therapy for patients with lifestyle-related diseases.     

Chimeric gene library construction by a simple and highly versatile method using recombination-dependent exponential amplification

A simple and efficient method for the construction of chimeric gene libraries termed RDA-PCR (recombination-dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction. A chimeric gene library is generated from homologous parental genes with additional primer-annealing sequences at their "heads" and "tails". Two primers ("skew primers") are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes. During the RDA-PCR, short annealing/extension periods facilitate homologous recombination. The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified. As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively). The crossover point profile of RDA-PCR clones was compared with those obtained by (modified) family shuffling. PCR restriction fragment polymorphism (PCR-RFLP) analysis of the RDA-PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences. PCR-RFLP analysis also indicated that the crossover points of RDA-PCR chimeras were distributed over the entire protein-coding region. Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA-PCR resulted in a higher diversity in crossover points than family shuffling.     

Expression and function of the Ror-family receptor tyrosine kinases during development: lessons from genetic analyses of nematodes,mice, and humans

Receptor tyrosine kinases (RTKs) play crucial roles in various developmental processes. Ror-family RTKs are characterized by the intracellular tyrosine kinase domains, highly related to those of the Trk-family RTKs, and by the extracellular Frizzled-like cysteine-rich domains (CRDs) and Kringle domains. Rors are evolutionally conserved among Caenorhabditis elegans, Aplysia, Drosophila melanogaster, Xenopus, mice, and humans. In D. melanogaster and mammals, pairs of structurally related Rors are found, while a single Ror protein is identified in C. elegans or Aplysia. In Aplysia and D. melanogaster, Rors are expressed exclusively in developing nervous systems. On the other hand, rather widespread expression of Rors was observed in C. elegans and mammals. Mutations in Ror of C. elegans cause inappropriate axon outgrowth as well as defects in cell migration and asymmetric cell division. It has also been reported that the nematode Ror possesses kinase-dependent and kinase-independent functions. Mouse Rors, Ror1, and Ror2, are expressed mainly in migrating neural crest cells and mesenchymal cells, and Ror2-deficient mice exhibit skeletal abnormalities and ventricular septal defects in the heart. Although Ror1-deficient mice exhibit no apparent skeletal or cardiac abnormalities, Ror1/Ror2 double mutant mice show markedly enhanced skeletal and cardiac abnormalities compared with Ror2 mutant mice, indicating genetic interaction of Ror1 and Ror2. In humans, mutations within Ror2 have been found in two genetic skeletal disorders, recessive Robinow syndrome and dominant Brachydactyly type B (BDB), further emphasizing critical functions of Ror2 during developmental morphogenesis. In this article, we also discuss the signaling machinery mediated by Ror-family RTKs with a particular emphasis on our recent structure-function analyses of Ror-family RTKs.