Okadaic acid induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha in human neutrophils

Human neutrophils differ from other cells by containing high amount of IkappaBalpha in the nucleus, and this increased nuclear IkappaBalpha accumulation is associated with the inhibition of NFkappaB activity and increased apoptosis. However, the mechanisms regulating NFkappaB activation and IkappaBalpha degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFkappaB activity and IkappaBalpha degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-delta (PKCdelta) and IkappaB kinase (IKK) inhibit the OA-induced activation of NFkappaB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFkappaB is regulated by a continuous phosphorylation and degradation of the nuclear IkappaBalpha.     

Peroxisome proliferator-activated receptor gamma ligands attenuate immunological symptoms of experimental allergic asthma

Asthma is characterized by a predominant T(H)2 type immune response to airborne allergens. Controlling T(H)2 cell function has been proposed as therapy for this disease. We show here that ligands for the nuclear receptor peroxisome proliferator activated receptor (PPAR)gamma significantly reduced the immunological symptoms of allergic asthma in a murine model of this disease. A PPARgamma ligand, 15-deoxy-delta(12,14)-prostaglandin J(2), significantly inhibited production of the T(H)2 type cytokine IL-5 from T cells activated in vitro. More importantly, in a murine model of allergic asthma, mice treated orally with ciglitazone, a potent synthetic PPARgamma ligand, had significantly reduced lung inflammation and mucous production following induction of allergic asthma. T cells from these ciglitazone treated mice also produced less IFNgamma, IL-4, and IL-2 upon rechallenge in vitro with the model allergen. Our results suggest that ligands for PPARgamma may be effective treatments for asthmatic patients.     

Inhibition of human salivary alpha-amylase by glucopyranosylidene-spiro-thiohydantoin

This study is the first report on the effectiveness and specificity of glucopyranosylidene-spiro-thiohydantoin (G-TH) inhibitor on the 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosyl-maltoside (GalG(2)CNP) hydrolysis catalysed by human salivary alpha-amylase (HSA). The inhibition of hydrolysis is a mixed-noncompetitive type. In any case, only one molecule of inhibitor binds to HSA. Since our substrate and inhibitor are small molecules the long enough active site facilitates accommodating both of them simultaneously. However, the product formation can be excluded from enzyme-substrate-inhibitor complex (ESI) since Dixon plots are linear. Kinetic constants calculated from secondary plots and nonlinear regression are almost entirely equal, confirming the fidelity of the suggested model. Kinetic constants (K(1i)=7.3mM, L(1i)=2.84 mM) show that G-TH is not such a potent inhibitor of HSA as acarbose and indicate higher stability for ESI than for enzyme-inhibitor complex.     

Interleukin-2 is one of the targets of 1,25-dihydroxyvitamin D3 in the immune system

Interleukin (IL)-2 knockout (KO) mice, which spontaneously develop symptoms of inflammatory bowel disease similar to ulcerative colitis in humans, were made vitamin D deficient (D-) or vitamin D sufficient (D+) or were supplemented with 1,25-dihydroxyvitamin D(3) (1,25D3). 1,25-Dihydroxyvitamin D3 supplementation, but not vitamin D supplementation, reduced the early mortality of IL-2 KO mice. However, colitis severity was not different in D-, D+, or 1,25D3 IL-2 KO mice. Cells from D- IL-2 KO mice produced more interferon (IFN)-gamma than cells from all other mice. Con A-induced proliferation was upregulated in IL-2 KO mice and downregulated in wildtype (WT) mice fed 1,25D3. All other measured immune responses in cells from IL-2 KO mice were unchanged by vitamin D status. In vitro addition of 1,25-dihydroxyvitamin D3 significantly reduced the production of IL-10 and IFN-gamma in cells from D- and D+ WT mice. Conversely, IFN-gamma and IL-10 production in cells from IL-2 KO mice were refractory to in vitro 1,25-dihydroxyvitamin D3 treatments. In the absence of IL-2, vitamin D was ineffective for suppressing colitis and ineffective for the in vitro downregulation of IL-10 or IFN-gamma production. One target of 1,25-dihydroxyvitamin D3 in the immune system is the IL-2 gene.     

Pheromone binding by polymorphic mouse major urinary proteins

Mouse major urinary proteins (MUPs) have been proposed to play a role in regulating the release and capture of pheromones. Here, we report affinity measurements of five recombinant urinary MUP isoforms (MUPs-I, II, VII, VIII, and IX) and one recombinant nasal isoform (MUP-IV) for each of three pheromonal ligands, (+/-)-2-sec-butyl-4,5-dihydrothiazole (SBT), 6-hydroxy-6-methyl-3-heptanone (HMH), and (+/-)dehydro-exo-brevicomin (DHB). Dissociation constants for all MUP-pheromone pairs were determined by isothermal titration calorimetry, and data for SBT were corroborated by measurements of intrinsic protein fluorescence. We also report the isolation of MUP-IV protein from mouse nasal extracts, in which MUP-IV mRNA has been observed previously. The affinity of each MUP isoform for SBT (K(d) approximately 0.04 to 0.9 micro M) is higher than that for DHB (K(d) approximately 26 to 58 micro M), which in turn is higher than that for HMH (K(d) approximately 50 to 200 micro M). Isoforms I, II, VIII, and IX show very similar affinities for each of the ligands. MUP-VII has approximately twofold higher affinity for SBT but approximately twofold lower affinity for the other pheromones, whereas MUP-IV has approximately 23-fold higher affinity for SBT and approximately fourfold lower affinity for the other pheromones. The variations in ligand affinities of the MUP isoforms are consistent with structural differences in the binding cavities of the isoforms. The data indicate that the concentrations of available pheromones in urine may be influenced by changes in the expression levels of urinary MUPs or the excretion levels of other MUP ligands. The variation in pheromone affinities of the urinary MUP isoforms provides only limited support for the proposal that MUP heterogeneity plays a role in regulating profiles of available pheromones. However, the binding data support the proposed role of nasal MUPs in sequestering pheromones and possibly transporting them to their receptors.     

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.     

The centrosomal protein TACC3 is essential for hematopoietic stem cell function and genetically interfaces with p53-regulated apoptosis

TACC3 is a centrosomal/mitotic spindle-associated protein that is highly expressed in a cell cycle-dependent manner in hematopoietic lineage cells. During embryonic development, TACC3 is expressed in a variety of tissues in addition to the hematopoietic lineages. TACC3 deficiency causes an embryonic lethality at mid- to late gestation involving several lineages of cells. Hematopoietic stem cells, while capable of terminal differentiation, are unable to be expanded in vitro or in vivo in reconstitution approaches. Although gross alterations in centrosome numbers and chromosomal segregation are not observed, TACC3 deficiency is associated with a high rate of apoptosis and expression of the p53 target gene, p21(Waf1/Cip1). Hematopoietic stem cell functions, as well as deficiencies in other cell lineages, can be rescued by combining the TACC3 deficiency with p53 deficiency. The results support the concept that TACC3 is a critical component of the centrosome/mitotic spindle apparatus and its absence triggers p53-mediated apoptosis.     

Refinement of d(GCGAAGC) hairpin structure using one- and two-bond residual dipolar couplings

The structure of the ¹³C,¹⁵N-labeled d(GCGAAGC) hairpin, as determined by NMR spectroscopy and refined using molecular dynamics with NOE-derived distances, torsion angles, and residual dipolar couplings (RDCs), is presented. Although the studied molecule is of small size, it is demonstrated that the incorporation of diminutive RDCs can significantly improve local structure determination of regions undefined by the conventional restraints. Very good correlation between the experimental and back-calculated small one- and two-bond ¹H-¹³C, ¹H-¹⁵N, ¹³C-¹³C and ¹³C-¹⁵N coupling constants has been attained. The final structures clearly show typical features of the miniloop architecture. The structure is discussed in context of the extraordinary stability of the d(GCGAAGC) hairpin, which originates from a complex interplay between the aromatic base stacking and hydrogen bonding interactions.     

Sphericity of the femoral head on anterior-posterior radiographs of dysplastic hips

We analyzed the sphericity of the femoral head of dysplastic hips. Using standard anterior-posterior radiographs of the hips, we assessed the femoral head's deviation from a spherical shape using a computer algorithm and via Severin grading. The method presented could serve as a useful tool to quantify differences in sphericity in cases where it is difficult to grade the hip radiologically.