PECAM-directed delivery of catalase to endothelium protects against pulmonary vascular oxidative stress

Targeted delivery of drugs to vascular endothelium promises more effective and specific therapies in many disease conditions, including acute lung injury (ALI). This study evaluates the therapeutic effect of drug targeting to PECAM (platelet/endothelial cell adhesion molecule-1) in vivo in the context of pulmonary oxidative stress. Endothelial injury by reactive oxygen species (e.g., H2O2) is involved in many disease conditions, including ALI/acute respiratory distress syndrome and ischemia-reperfusion. To optimize delivery of antioxidant therapeutics, we conjugated catalase with PECAM antibodies and tested properties of anti-PECAM/catalase conjugates in cell culture and mice. Anti-PECAM/catalase, but not an IgG/catalase counterpart, bound specifically to PECAM-expressing cells, augmented their H2O2-degrading capacity, and protected them against H2O2 toxicity. Anti-PECAM/catalase, but not IgG/catalase, rapidly accumulated in the lungs after intravenous injection in mice, where it was confined to the pulmonary endothelium. To test its protective effect, we employed a murine model of oxidative lung injury induced by glucose oxidase coupled with thrombomodulin antibody (anti-TM/GOX). After intravenous injection in mice, anti-TM/GOX binds to pulmonary endothelium and produces H2O2, which causes lung injury and 100% lethality within 7 h. Coinjection of anti-PECAM/catalase protected against anti-TM/GOX-induced pulmonary oxidative stress, injury, and lethality, whereas polyethylene glycol catalase or IgG/catalase conjugates afforded only marginal protective effects. This result validates vascular immunotargeting as a prospective strategy for therapeutic interventions aimed at immediate protective effects, e.g., for augmentation of antioxidant defense in the pulmonary endothelium and treatment of ALI.     

Laser-assisted microdissection and real-time PCR detect anti-inflammatory effect of perfluorocarbon

The aim of this study was to identify cell types involved in the anti-inflammatory effect of ventilation with perfluorocarbon in vivo. Fifteen anesthetized, surfactant-depleted piglets received either aerosolized perfluorocarbon (Aerosol-PFC), partial liquid ventilation (rLV) at functional residual capacity (FRC) volume (FRC-PLV), or intermittent mandatory ventilation (control). After laser-assisted microdissection of different lung cell types, mRNA expression of IL-8 and ICAM-1 was determined using TaqMan real-time PCR normalized to hypoxanthine phosphoribosyltransferase (HPRT). IL-8 mRNA expression (means +/- SE; control vs. Aerosol-PFC) was 356 +/- 142 copies IL-8 mRNA/copy HPRT mRNA vs. 3.5 +/- 1.8 in alveolar macrophages (P <0.01); 208 +/- 108 vs. 2.7 +/- 0.8 in bronchiolar epithelial cells (P <0.05); 26 +/- 11 vs. 0.7 +/- 0.2 in alveolar septum cells (P <0.01); 2.8 +/- 1.0 vs. 0.8 +/- 0.4 in bronchiolar smooth muscle cells (P <0.05); and 1.1 +/- 0.4 vs. 0.2 +/- 0.05 in vascular smooth muscle cells (P <0.05). With FRC-PLV, IL-8/HPRT mRNA expression was significantly lower in macrophages, bronchiolar epithelial, and vascular smooth muscle cells. ICAM-1 mRNA expression in vascular endothelial cells remained unchanged. Predominantly, alveolar macrophages and bronchiolar epithelial cells were involved in the inflammatory pulmonary process. The anti-inflammatory effect of Aerosol-PFC was most pronounced.     

Phosphorylation of the WASP-VCA domain increases its affinity for the Arp2/3 complex and enhances actin polymerization by WASP

Wiskott-Aldrich syndrome protein (WASP) and neural (N)-WASP regulate dynamic actin structures through the ability of their VCA domains to bind to and stimulate the actin nucleating activity of the Arp2/3 complex. Here we identify two phosphorylation sites in the VCA domain of WASP at serines 483 and 484. S483 and S484 are substrates for casein kinase 2 in vitro and in vivo. Phosphorylation of these residues increases the affinity of the VCA domain for the Arp2/3 complex 7-fold and is required for efficient in vitro actin polymerization by the full-length WASP molecule. We propose that constitutive VCA domain phosphorylation is required for optimal stimulation of the Arp2/3 complex by WASP.     

SNP genotyping on a genome-wide amplified DOP-PCR template

With the increasing demand for higher throughput single nucleotide polymorphism (SNP) genotyping, the quantity of genomic DNA often falls short of the number of assays required. We investigated the use of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to generate a template for our SNP genotyping methodology of fluorescence polarization template-directed dye-terminator incorporation detection. DOP-PCR employs a degenerate primer (5′-CCGACTCGAGNNNNNNATGTGG-3′) to produce non-specific uniform amplification of DNA. This approach has been successfully applied to microsatellite genotyping. We compared genotyping of DOP-PCR-amplified genomic DNA to genomic DNA as a template. Results were analyzed with respect to feasibility, allele loss of alleles, genotyping accuracy and storage conditions in a high-throughput genotyping environment. DOP-PCR yielded overall satisfactory results, with a certain loss in accuracy and quality of the genotype assignments. Accuracy and quality of genotypes generated from the DOP-PCR template also depended on storage conditions. Adding carrier DNA to a final concentration of 10 ng/µl improved results. In conclusion, we have successfully used DOP-PCR to amplify our genomic DNA collection for subsequent SNP genotyping as a standard process.     

Sympathetic-renal interaction in chronic arterial pressure control

The effects of neonatal sympathectomy of donors or recipients on posttransplantation arterial pressure were investigated in spontaneously hypertensive rats (SHR) by renal transplantation experiments. Conscious mean arterial pressure (MAP) and renal vascular resistance were 136 +/- 1 mmHg and 15.5 +/- 1.2 mmHg x ml(-1) x min x g in sympathectomized SHR (n = 8) vs. 158 +/- 4 mmHg (P < 0.001) and 20.8 +/- 1.1 mmHg x ml(-1) x min x g (P < 0.05) in controls (n = 10). Seven weeks after transplantation of a kidney from neonatally sympathectomized SHR donors, MAP in SHR recipients (n = 10) was 20 mmHg lower than in controls transplanted with a kidney from hydralazine-treated SHR (n = 10) (P < 0.05) associated with reduced sodium sensitivity of MAP. Neonatal sympathectomy also lowered MAP in F1-hybrids (F1H; SHR x Wistar-Kyoto rats). Within 6 wk after transplantation, renal grafts from untreated SHR increased MAP by 20 mmHg in sympathectomized F1H (n = 10) and by 35 mmHg in sham-treated F1H (n = 8) (P < 0.05). Neonatal sympathectomy induces chronic changes in SHR kidney function leading to a MAP reduction even when extrarenal sympathetic tone is restored. Generalized reduction in sympathetic tone resets the kidney-fluid system to reduced MAP and blunts the extent of arterial pressure rise induced by an SHR kidney graft.     

Flotillin-1/reggie-2 traffics to surface raft domains via a novel golgi-independent pathway. Identification of a novel membrane targeting domain and a role for palmitoylation

Flotillins are lipid raft-associated proteins, which have been implicated in neuronal regeneration and insulin signaling. We now show that newly synthesized flotillin-1 reaches the plasma membrane via a Sar1-independent and brefeldin A-resistant targeting pathway. Consistent with post-translational membrane association of flotillin, protease sensitivity experiments suggest that flotillin-1 is not a transmembrane protein but is associated with the cytoplasmic face of the plasma membrane. The N terminus of flotillin contains a prohibitin-like domain (PHB), which shows homology to a number of proteins associated with raft domains including stomatin, podocin, and prohibitin. We show that the PHB domain of flotillin can efficiently target a heterologous protein, green fluorescent protein, to the plasma membrane. Another PHB-containing protein, stomatin, traffics to the plasma membrane via the conventional secretory pathway. Plasma membrane association of both full-length flotillin and the green fluorescent protein-tagged PHB domain of flotillin is dependent on palmitoylation and requires a conserved cysteine residue, Cys-34, in the PHB domain. The results identify a novel targeting mechanism for plasma membrane association of flotillin-1 involving a Golgi-independent trafficking pathway, the PHB domain, and palmitoylation.     

The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats

The P2X(2) subtype of purine receptor was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines of the guinea-pig, and nerve cells of submucosal ganglia in the intestine. Nerve cells with strong and with weak immunoreactivity could be distinguished. Immunoreactivity in both strongly and weakly immunoreactive neurons was absorbed with P2X(2) receptor peptide. In the myenteric plexus, strong immunoreactivity was in nitric oxide synthase (NOS)- and in calbindin-immunoreactive neurons. In all regions, over 90% of NOS-immunoreactive neurons were strongly P2X(2) receptor immunoreactive. The intensity of reaction varied in calbindin neurons; in the ileum, 90% were immunoreactive for the receptor, about one-third having a strong reaction. In the submucosal ganglia, all vasoactive intestinal peptide-immunoreactive neurons were P2X(2) receptor immunoreactive, but there was no receptor immunoreactivity of calretinin or neuropeptide Y neurons. Varicose nerve fibres with P2X(2) receptor immunoreactivity were found in the gastric myenteric ganglia. These fibres disappeared after vagus nerve section. It is concluded that the P2X(2) receptor is expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons, and that the receptor also occurs on the endings of vagal afferent fibres in the stomach.     

Structure and function of an early divergent form of laminin in hydra: a structurally conserved ECM component that is essential for epithelial morphogenesis

As a major component of the extracellular matrix (ECM), laminin has been found in many vertebrate and invertebrate organisms. Its molecular structure is very similar across species lines and its biological function in the ECM has been extensively studied. In an effort to study ECM structure and function in hydra, we have cloned a partial hydra laminin alpha chain and the full-length hydra laminin beta chain using ECM-enriched cDNA libraries. Analysis of deduced amino acid sequences indicated that both polypeptides have high sequence similarity to a number of invertebrate and vertebrate laminin alpha and beta subunits. Rotary shadow analysis of isolated hydra laminin indicates it has a heterotrimeric organization that is characteristic of vertebrate laminins. A putative integrin-class protein was also identified using a cell-binding peptide sequence from the laminin beta chain as an affinity probe, indicating that integrins are possible cell surface receptors in hydra. In agreement with previous results for the hydra laminin beta chain, in situ hybridization experiments revealed that hydra laminin alpha chain mRNA is restricted to endodermal cells. As with a number of other hydra ECM components, higher levels of laminin alpha chain mRNA are localized to regions where cell migration and differentiation are actively undertaken such as the base of tentacles, the peduncle region, buds, regenerating tentacles, and at the head end during regeneration. The role of laminin in morphogenesis was studied using an antisense approach and the results indicated that translation of the laminin alpha chain is required for head regeneration.     

Phosphorylation by protein kinase CK2: a signaling switch for the caspase-inhibiting protein ARC

Caspases play a central role in apoptosis, but their activity is under the control of caspase-inhibiting proteins. A characteristic of caspase-inhibiting proteins is direct caspase binding. It is yet unknown how the localization of caspase-inhibiting proteins is regulated and whether there are upstream signals controlling their function. Here we report that the function of ARC is regulated by protein kinase CK2. ARC at threonine 149 is phosphorylated by CK2. This phosphorylation targets ARC to mitochondria. ARC is able to bind to caspase-8 only when it is localized to mitochondria but not to the cytoplasm. Our results reveal a molecular mechanism by which a caspase-inhibiting protein requires phosphorylation in order to prevent apoptosis.