Amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS) are neurodegenerative conditions that affect large motor neurons of the central nervous system. We have identified a familial juvenile PLS (JPLS) locus overlapping the previously identified ALS2 locus on chromosome 2q33. We report two deletion mutations in a new gene that are found both in individuals with ALS2 and those with JPLS, indicating that these conditions have a common genetic origin. The predicted sequence of the protein (alsin) may indicate a mechanism for motor-neuron degeneration, as it may include several cell-signaling motifs with known functions, including three associated with guanine-nucleotide exchange factors for GTPases (GEFs).

Prostanoids generated by COX‐2 are involved in the regulation of inflammation but their exact role in the innate immune response has not been defined. We investigated whether COX‐2 is involved in host defense against Pseudomonas aeruginosa pneumonia. In vitro studies, in a macrophage cell line, showed that cytotoxic strain of P aeruginosa (PA103) induced significant COX‐2 protein expression and enzymatic function. In vivo data showed that infection with PA103 increased COX‐2 protein production in whole lung tissue compared to mice that were infected with mutant bacteria that lack ExoU (ΔU) or ExoU and ExoT (ΔUT). COX‐2–/– mice had accentuated clearance of cytotoxic P. aeruginosa from the lungs. We further tested the effects of COX‐2 products such as prostaglandin E2 on the function of phagocytic cells. Our studies indicate that prostaglandin E2 may be involved through interacting with the EP2 receptors in modulating the host response because treatment of macrophages with prostaglandin E2 suppressed production of reactive oxygen species. Furthermore there was enhanced bacterial clearance in EP2 receptor–/– mice compared to the wild‐type controls. Thus it is possible that inhibition of COX‐2 or EP2 receptors could be an effective adjunctive treatment for severe or resistant P. aeruginosa pneumonia.

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently discovered molecule that is expressed on the cell surface of monocytes and neutrophils. Engagement of TREM-1 triggers synthesis of proinflammatory cytokines in response to microbes, but the extent and mechanism by which TREM-1 modulates the inflammatory response is poorly defined. In the present study, we investigated the functional effects of blocking TREM-1 on the Toll-like receptor (TLR)4-mediated signaling pathway in macrophages. By transfecting cells with small hairpin interfering RNA molecules to TREM-1 (shRNA), we confirmed that TREM-1 mRNA and protein expression was greatly attenuated in RAW cells in response to treatment with LPS. PCR array for genes related to or activated by the TLR pathway revealed that although the expression of TLR4 itself was not significantly altered by silencing of TREM-1, expression of several genes, including MyD88, CD14, IkappaBalpha, IL-1beta, MCP-1, and IL-10 was significantly attenuated in the TREM-1 knockdown cells in response to treatment with LPS. These data indicate that expression of TREM-1 modulates the TLR signaling in macrophages by altering the expression of both adaptor and effector proteins that are critical to the endotoxin response.

We report an improved LC-MS-MS assay that accurately measures prostaglandins D 2 (PGD 2 ) and E 2 (PGE 2 ) in cell culture supernatants and other biological fluids. The limit of detection for each prostaglandin was 20 pg/mL (0.20 pg; 0.55 fmol on-column), and the inter-day and intra-day coefficients of variation were less than 5%. Both d 4 -PGE 2 and d 4 -PGD 2 were used as surrogate standards to control for differential loss and degradation of the analytes. Stability studies indicated that sample preparation time should be less than 8 h to measure PGD 2 accurately, whereas preparation time did not affect PGE 2 measurement due to its greater stability in biological samples. As an application of the method, PGD 2 and PGE 2 were measured in culture supernatants from A549 cells and RAW 264.7 cells. The human lung alveolar cell line A549 was found to produce PGE 2 but no PGD 2 while the murine macrophage cell line RAW 264.7 produced PGD 2 and only trace amounts of PGE 2 . This direct comparison showed that COX-2 gene expression can lead to differential production of PGD 2 and PGE 2 by epithelial cells and macrophages. Since PGE 2 is anti-asthmatic and PGD 2 is pro-asthmatic, we speculate that the balance of production of these eicosanoids by epithelial cells and macropahges in the lung contributes to the pathogenesis of COPD, bronchiectasis, asthma, and lung cancer.

Dematin is an actin-bundling protein of the erythroid membrane skeleton and is abundantly expressed in human brain, heart, skeletal, muscle, kidney, and lung. The 48-kDa subunit of dematin contains a headpiece domain which was originally identified in villin, and actin-binding protein of the brush-border cytoskeleton. The head-piece domain of villin is essential for its morphogenic function in vivo. Here we report the primary structure of 52-kDa subunit of dematin which differs from the 48-kDa subunit by a 22-amino-acid insertion within its headpiece domain. A unique feature of the insertion sequence of the 52-kDa subunit is its homology to erythrocyte protein 4.2. The insertion sequence also includes a cysteine residue which may explain the formation of sulfhydryl-linked trimers of dematin. Actin binding measurements using recombinant fusion proteins revealed that each monomer of dematin contains two F-actin binding sites: one in the headpiece domain and the other in the undefined N-terminal domain. Although the actin bundling activity of intact dematin was abolished by phosphorylation, no effect of phosphorylation was observed on the actin binding activity of fusion proteins. Using somatic cell hybrid panels and fluorescence in situ hybridization, the dematin gene was localized on the short arm of chromosome 8. The dematin locus, 8p21.1, is distal to the known locus of human erythroid ankyrin (8p11.2) and may contribute to the etiology of hemolytic anemia in a subset of patients with severe hereditary spherocytosis.

Autosomal dominant hereditary spastic paraplegia is genetically heterogeneous, with at least five loci identified by linkage analysis. Recently, mutations in spastin were identified in SPG4, the most common locus for dominant hereditary spastic paraplegia that was previously mapped to chromosome 2p22. We identified five novel mutations in the spastin gene in five families with SPG4 mutations from North America and Tunisia and showed the absence of correlation between the predicted mutant spastin protein and age at onset of symptoms.

Stimulation of platelet PAR-1 receptors results in the rapid (10 to 30 seconds) and extensive (30% to 40% of total) guanosine triphosphate (GTP) charging of endogenous platelet rac, previously identified as a possible key intermediate in the signal pathway between PAR-1 and actin filament barbed-end uncapping, leading to actin assembly. During PAR-1–mediated platelet activation, rac distributes from the cell interior to the cell periphery, and this reorganization is resistant to the inhibition of PI-3-kinase activity. Rac, in resting or activated platelets, is Triton X-100 soluble, suggesting that it does not form tight complexes with actin cytoskeletal proteins, though its retention in octyl-glucoside-treated platelets and ultrastructural observations of activated platelets implies that rac binds to plasma membranes, where it can interact with phosphoinositide kinases implicated in actin assembly reactions. PAR-1 stimulation also rapidly and extensively activates cdc42, though, in contrast to rac, some cdc42 associates with the actin cytoskeleton in resting platelets, and the bound fraction increases during stimulation. The differences in subcellular distribution and previous evidence showing quantitatively divergent effects of rac and cdc42 on actin nucleation in permeabilized platelets indicate different signaling roles for these GTPases.

Grebe-type chondrodysplasia exhibits a severe form of limb shortening and appendicular bone dysmorphogenesis. Here we report a family with seven males and six females who inherited the disorder in an autosomal recessive fashion. While the carrier parents did not exhibit any apparent skeletal abnormalities, all affected patients had a similar phenotype with unaffected axial and craniofacial bones. Since mutations in the cartilage-derived morphogenetic protein 1 (CDMP1) gene have been reported in similar acromesomelic chondrodysplasias, we examined genomic DNA from affected and normal subjects for possible mutations in CDMP1. In affected subjects, an insertion of a C at nucleotide 297 of the coding sequence was discovered. This insertion produced a shift in the reading frame at amino acid residue 99, causing premature termination of the polypeptide six amino acids downstream. DNA samples from 41 control subjects did not show this mutation. The truncated CDMP1 protein in these subjects is predicted to cause a total loss of its signaling function. The present report confirms that CDMP1 plays an important role in the regulation of axial bone growth during development and suggests that its absence does not impair other developmental processes.